tom's thesis submitted copy

Design, Synthesis and Application of Enzyme Responsive Hydrogel

Particles using Peptide Actuators

A thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy in the Faculty of Engineering and Physical

Sciences

2009

Tom O. McDonald

School of Materials

Enzyme Responsive Hydrogel Particles using Peptide Actuators Tom O. McDonald

Contents

DECLARATION........................................................................................................14

ABSTRACT..............................................................................................................15

ACKNOWLEDGEMENTS...........................................................................................16

1 INTRODUCTION...........................................................................................18

1.1. MOTIVATION OF THE PROJECT...................................................................19

1.2. LAYOUT OF THE THESIS..............................................................................19

2 LITERATURE REVIEW...............................................................................22

2.1. INTRODUCTION...........................................................................................22

2.2. STIMULI RESPONSIVE MATERIALS..............................................................22

2.2.1. Chemical hydrogels...............................................................................22

2.3. BIORESPONSIVE HYDROGELS.....................................................................23

2.3.1. Introduction...........................................................................................23

2.3.2. Actuation based on changes in crosslinking density.............................24

2.3.2.1. Systems incorporating peptide crosslinkers...................................24

2.3.2.2. Non-covalent crosslinking interactions.........................................29

2.3.3. Actuation based on electrostatic interactions........................................34

2.3.3.1. Electrostatic interactions between charges on the polymer

network..........................................................................................................35

2.3.3.2. Electrostatic interactions between charges present in pendant

actuators .......................................................................................................39

2.3.4. Actuation based on conformational changes.........................................42

2.3.5. Summary................................................................................................45

2.4. PEGA.........................................................................................................46

2.4.1. The history of PEGA..............................................................................46

2.4.2. Enzyme catalysed synthesis on PEGA...................................................53

2.4.3. Summary................................................................................................56

2.5. MICROFLUIDIC POLYMERISATION OF PARTICLES.......................................57

2.5.1. Summary................................................................................................67

2.6. AIMS OF THESIS..........................................................................................69

2


3 PEGA POLYMERISATION, CHARACTERISATION AND ENZYME

RESPONSIVE SWELLING THROUGH FUNCTIONALISATION WITH

PEPTIDE ACTUATORS........................................................................................70

3.1. ABSTRACT..................................................................................................70

3.2. INTRODUCTION...........................................................................................70

3.3. EXPERIMENTAL..........................................................................................74

3.3.1. Materials................................................................................................74

3.3.2. Inverse suspension polymerisation........................................................74

3.3.3. Charged PEGA polymerisation.............................................................74

3.3.4. Microscopy and particle size analysis...................................................75

3.3.5. Solid phase peptide synthesis.................................................................76

3.3.5.1. Solid phase synthesis of dipeptides and enzyme treatment...........77

3.3.5.2. Solid phase peptide synthesis of peptide actuators........................78

3.3.6. HPLC.....................................................................................................78

3.3.7. Two-photon microscopy.........................................................................78

3.3.8. Determining particle swelling...............................................................79

3.3.9. Assessing accessibility...........................................................................79

3.3.10. Entrapping payload...........................................................................79

3.3.11. Fluorimetry........................................................................................79

3.3.12. Microscopy and determination of swelling........................................80

3.4. RESULTS AND DISCUSSION.........................................................................81

3.4.1. Production of µPEGA800........................................................................81

3.4.1.1. Particle morphology and size characterisation of µPEGA............82

3.4.2. Production of µPEGA+ and µPEGA-.....................................................83

3.4.3. Further characterisation of µPEGA......................................................88

3.4.3.1. Amine characterisation by two-photon microscopy and enzyme

compatibility..................................................................................................88

3.4.3.2. Comparison of enzymatic hydrolysis within µPEGA800 and

macroparticles................................................................................................90

3.4.4. Functionalisation with peptide actuators..............................................91

3.4.4.1. Enzyme responsive increase in accessibility.................................92

3.4.4.2. Demonstration of the encapsulation of a payload..........................95

3.4.4.3. Enzyme specific release.................................................................96

3


3.4.4.4. Enzyme responsive increase in swelling; effect of ionic strength. 97

3.5. CONCLUSIONS............................................................................................98

4 DESIGNING NEW PEPTIDE ACTUATORS FOR IMPROVED

ENZYME RESPONSIVE BEHAVIOUR..............................................................99

4.1. ABSTRACT..................................................................................................99

4.2. INTRODUCTION...........................................................................................99

4.2.1. Branched peptide actuators...................................................................99

4.3. MATERIALS AND METHODS......................................................................102

4.3.1. Materials..............................................................................................102

4.3.2. Inverse suspension polymerisation and polymer characterisation......102

4.3.3. Solid phase peptide synthesis...............................................................103

4.3.4. Microscopy and determination of swelling..........................................103

4.3.5. Two-photon microscopy.......................................................................103

4.3.6. Entrapping payload.............................................................................104

4.3.7. Release measurements.........................................................................104

4.3.8. HPLC and LCMS.................................................................................104

4.4. RESULTS AND DISCUSSION.......................................................................106

4.4.1. Microparticle characterisation and peptide functionalisation............106

4.4.2. Actuator design and responsiveness of peptide functionalised particles. .

.............................................................................................................107

4.4.3. Characterisation of enzyme action on peptide actuators....................111

4.4.4. Enzyme triggered release.....................................................................113

4.5. CONCLUSIONS..........................................................................................115

5 MICROFLUIDIC PREPARATION OF LOW POLYDISPERSITY PEGA

PARTICLES...........................................................................................................116

5.1. ABSTRACT................................................................................................116

5.2. INTRODUCTION.........................................................................................116

5.3. MATERIALS AND METHODS......................................................................119

5.3.1. Microfluidic system..............................................................................119

5.3.2. Flow focussing setup............................................................................119

5.3.3. Monomer and continuous phase preparation......................................120

5.3.4. Particle size measurement...................................................................120

4


5.4. RESULTS AND DISCUSSION.......................................................................121

5.4.1. Microfluidic system..............................................................................121

5.4.1.1. Variation of particle size with flow rate ratios and surfactant

concentration................................................................................................121

5.4.1.2. Effect of increasing total flow rate on particle size.....................126

5.4.1.3. Effect of flow ratios and surfactant concentration on polydispersity

.....................................................................................................127

5.4.1.4. Variation of particle production rates with the conditions assessed. .

.....................................................................................................127

5.4.2. Using a simplified microfluidic flow-focussing device to produce

polymer particles.............................................................................................129

5.4.2.1. Orientation of device...................................................................129

5.4.2.2. Optimising conditions with the FF device...................................131

5.4.2.3. Effect of changing oil phase........................................................133

5.4.3. Optimum conditions for particle production.......................................134

5.4.3.1. Particle size distribution...............................................................134

5.4.4. Morphology of particles.......................................................................135

5.5. CONCLUSIONS..........................................................................................136

6 CONCLUSIONS............................................................................................137

7 FUTURE WORK...........................................................................................139

8 APPENDICES................................................................................................141

8.1. BACKGROUND..........................................................................................141

8.1.1. Polymers..............................................................................................141

8.1.1.1. Peptides and proteins...................................................................141

8.1.2. Two-photon microscopy.......................................................................143

8.1.3. Fluorescence resonance energy transfer.............................................144

8.2. SUPPLEMENTARY DATA............................................................................145

8.2.1. HPLC solvent gradient........................................................................145

8.2.2. Synthesis of activated disulfide-methacrylamide monomer.................145

9 REFERENCES...............................................................................................147

5


List of figures

Figure 1-1. The inverse suspension polymerisation of µPEGAs...............................20

Figure 1-2. Enzyme responsive µPEGA....................................................................20

Figure 1-3. Enzyme responsive µPEGA through the incorporation of branched

peptide actuators........................................................................................................21

Figure 1-4. Production of PEGA particles by microfluidic polymerisation..............21

Figure 2-1. Schematic representation of the different categories of bioresponsive

hydrogels....................................................................................................................24

Figure 2-2. Cell responsive synthetic hydrogels........................................................26

Figure 2-3. Antigen responsive hydrogel..................................................................31

Figure 2-4. Displacement-Induced Switching Rates of Bioresponsive Hydrogel

Microlenses................................................................................................................32

Figure 2-5. Novel synthesis of HPMA copolymers containing peptide grafts and

their self assembly into hybrid hydrogels..................................................................34

Figure 2-6. The enzymes and the reactions they catalyse used in glucose responsive

hydrogels....................................................................................................................36

Figure 2-7. Characterization of glucose-sensitive insulin release systems in

simulated in vivo conditions......................................................................................37

Figure 2-8. Enzyme-responsive hydrogel particles for the controlled release of

proteins: Designing peptide actuators to match payload...........................................41

Figure 2-9. Peptide actuator designed for the release of negatively charged protein

molecules...................................................................................................................42

Figure 2-10. Ligand responsive hydrogel that relies on conformational changes.....44

Figure 2-11. Chemical structure of PEGA.................................................................46

Figure 2-12. Inhibition of cruzipain visualized in a fluorescence quenched solid-

phase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin B and

cathepsin L.................................................................................................................48

Figure 2-13. Using two photon microscopy to quantify enzymatic reaction rates on

polymer beads............................................................................................................50

Figure 2-14. A micropatterned hydrogel platform for chemical Synthesis and

biological analysis......................................................................................................52

6


Figure 2-15. Real-time imaging of protease action on substrates covalently

immobilised to polymer supports..............................................................................53

Figure 2-16. Biomimetic synthesis and optimization of cyclic peptide antibiotics.......

...................................................................................................................................55

Figure 2-17. Formation of dispersions using "flow focusing" in microchannels......58

Figure 2-18. An axisymmetric flow-focusing microfluidic device (AFFD).............60

Figure 2-19. Interfacial Polymerization within a Simplified Microfluidic Device:

Capturing Capsules....................................................................................................61

Figure 2-20. Polymer particles with various shapes and morphologies produced in

continuous microfluidic reactors...............................................................................63

Figure 2-21. Continuous microfluidic reactors for polymer particles.......................65

Figure 2-22. A micro-reactor for preparing uniform molecularly imprinted polymer

beads..........................................................................................................................66

Figure 2-23. A predictive approach of the influence of the operating parameters on

the size of polymer particles synthesized in a simplified microfluidic system.........67

Figure 3-1. Schematic of the enzyme responsive swelling of PEGA particles

functionalised with linear peptide actuators. ...........................................................73

Figure 3-2. Monomers used in the synthesis of PEGA and its charged variants.......75

Figure 3-3. Solid phase peptide synthesis scheme.....................................................76

Figure 3-4. Chemical reactions involved in peptide synthesis..................................77

Figure 3-5. Polymerisation of PEGA particles by inverse suspension polymerisation.

...................................................................................................................................82

Figure 3-6. Optical micrograph of microparticles is water........................................83

Figure 3-7. Chemical structure of PEGA and its charged variants.72........................84

Figure 3-8. PEGA+ microparticles.............................................................................85

Figure 3-9. Optical micrographs PEGA-,...................................................................86

Figure 3-10. The uptake of water by the three different types of dry PEGA

microparticles and the effect of ionic strength on the swelling of the three different

types of PEGA microparticles...................................................................................90

Figure 3-12. Comparison of time dependence of enzyme reactions on µPEGA and

commercially available macroparticles.....................................................................91

7


Figure 3-13. A schematic of enzyme responsive microparticles illustrating both

successful and unsuccessful cleavage of the ECP resulting in a change of

accessibility to a 40 kDa FITC labelled dextran........................................................95

Figure 3-14. Confocal microscopy images of representative microparticles in an

aqueous solution of 40 kDa fluorescently labelled dextran.......................................95

Figure 3-15. The pH responsive loading of the 40 kDa FITC labelled dextran (1

mg/ml) into the PEGA particles, gain of images varied............................................97

Figure 4-1. Schematic of enzyme responsive branched peptide actuator................101

Figure 4-2. HPLC quantification of Fmoc removed after each coupling step for

linear and branched peptide actuators......................................................................106

Figure 4-3. pH responsive swelling behaviour of peptide actuators on PEGA

microparticles...........................................................................................................107

Figure 4-4. Enzyme responsive swelling behaviour of peptide actuator

functionalised µPEGA at pH 7................................................................................109

Figure 4-5. HPLC and MS analysis of enzyme hydrolysis of branched peptide

actuators...................................................................................................................110

Figure 4-6. Effect of ionic strength on the maximal enzyme responsive swelling of

branched peptide actuator functionalised µPEGA at pH 7......................................111

Figure 4-7. Comparison of thermolysin action on both linear and branched peptide

actuators on µPEGA................................................................................................112

Figure 4-8. µPEGA particles functionalised with the branched peptide actuator at pH

7...............................................................................................................................114

Figure 5-1. Schematic of microfluidic setup for the synthesis of controlled-size

polymer particles......................................................................................................118

Figure 5-2. Effect of Qc/Qd on mean particle diameter with no surfactant in the

continuous phase,.....................................................................................................122

Figure 5-3. Effect of surfactant concentration on particle size................................124

Figure 5-4. Effect of total flow rate (at constant Qc/Qd) on mean particle diameter.

.................................................................................................................................126

Figure 5-5. Effect of Qc/Qd and surfactant concentration on polydispersity...........128

Figure 5-6. Effect of Qc/Qd and surfactant concentration on particle production rate.

.................................................................................................................................128

Figure 5-7. Schematic representation of the ‘flow-focussing’ device.....................129

8


Figure 5-8. Effect device orientation on droplet and resulting particle formation.. 131

Figure 5-9. Development of flow-focussing setup..................................................132

Figure 5-10. Optical micrographs of PEGA particles produced using FF device with

silicone oil at a variety of conditions......................................................................133

Figure 5-11. PEGA Particles produced under the optimum conditions..................135

Figure 5-12. The typically slightly asymmetric shape of particles produced by

microfluidics each image is of particles produced under different conditions........136

Figure 8-1. The structures of the twenty DNA encoded amino acids. The single letter

abbreviation is indicated with the brackets..............................................................143

Figure 8-2. Jablonski energy diagram showing a comparison of the excitation of a

fluorophore with a single photon (confocal microscopy) and two photons (two-

photon microscopy).................................................................................................144

Figure 8-3. HPLC solvent gradient used in analytical runs.....................................145

Figure 8-4. Synthesis of PDTEMA.130.....................................................................146

Figure 8-5. Characterisation of PDTEMA by NMR................................................146

9


List of tables

Table 2-1. Selected studies of bioresponsive hydrogels based on peptide

crosslinkers................................................................................................................25

Table 2-2. Selected studies of bioresponsive hydrogels based on non-covalent

crosslinking interactions............................................................................................30

Table 2-3. Selected studies of bioresponsive hydrogels based on electrostatic

interactions.................................................................................................................35

Table 3-2. Specificity of the enzymes used towards amino acids (AA) in the

substrate and their molecular weight.........................................................................93

Table 3-1. Values for HPLC relative enzyme cleavage for each ECP......................94

10


List of abbreviations

α-CD α-cyclodextrin

ABP Aminobenzophenone

AFFD Axisymmetric flow-focusing microfluidic device

APS Ammonium persulfate

APTMS 3-aminopropyltrimethoxysilane

BDDA 1,4-butanediol diacrylate

CaM Calmodulin

CDDP Cisplatin

CPGs Controlled pore glasses

CV Coefficient of variation

DCM Dichloromethane

DEAEMA Diethylaminoethyl methacrylate

DIC Differential interference contrast

DIPEA N,N-Diisopropylethylamine

DMAEMA N,N-dimethylaminoethyl methacrylate

DMSO Dimethyl sulfoxide

DOPA L-3,4-dihydroxylphenylalanine

DTT Dithiothreitol

ECM Extracellular matrix

ECP Enzyme cleavable peptide

EG Ethylene glycol

EGDMA Ethyleneglycol dimethacrylate

ESEM Environmental scanning electron microscopy

FF Flow-focussing

FITC Fluorescein isothiocyanate

Fmoc 9-fluorenylmethoxycarbonyl

FRET Fluorescence quenched peptide substrate

GOx Glucose oxidase

HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate

HOBt Hydroxybenzotriazole

HPLC High-performance liquid chromatography

11


HPMC Hydroxypropyl methyl cellulose ether

HPMA N-(2-hydroxypropyl)methacrylamide methacryoyl

ID Internal diameter

IVD Intervertebral discs

LbL Layer-by-layer assembly

LCST Lower-critical solution temperature

µPEGA Microparticular PEGA

MA Methacrylate

MAA Methacrylic acid

MALDI Matrix-assisted laser desorption/ionization

MBAA N,N’-methylenebisacrylamide

MFFD Microfluidic flow-focusing device

MMA Methyl methacrylate

MMP Matrix metalloproteinase

MS Mass spectrometry

NSA N-succinimidylacrylate

PAAc Poly(acrylic acid)

PCP Phosphotpanteheine

PDMA Poly(dimethylsiloxane)

PDTEMA N-[2-(2-pyridyldithio)]ethyl methacrylamide

PEG Poly(ethylene glycol)

PEGA Poly(ethylene glycol)-co-acrylamide

PEGMA Poly(ethylene glycol) monomethacylate

PETA-3 Pentaerythritol triacryalte

PGA Penicillin G amidase

PNIPAAm Poly(N-isopropylacrylamide)

poly(HEMA) poly(2-hydroxyethyl methacrylate)

PLE Porcine liver esterase

PU Polyurethane

PVDF polyvinylidene fluoride

Qc Flow rate of the continuous phase

Qd Flow rate of the dispersed phase

SEM Scanning electron micrscopy

12


Semi-IPN Semi-interpenetrating network

Span 20 Sorbitan monolaurate

SPPS Solid-phase peptide synthesis

TEGDMA Tetraethylene glycol dimethacrylate

TEMED N,N,N′,N′- tetramethylethylenediamine

TFA Trifluoroacetic acid

TFP Trifluoperazine ligand

TG Transglutaminase

THF Tetrahydrofuran

TPGDA Tripropyleneglycol diacrylate

TPM Two-photon microscopy

tTG Tissue transglutaminase

13


Declaration

No portion of this work has been submitted in support of an application for another degree or qualification of this or any other university or institute of learning.

Copyright

(i) The author of this thesis (including any appendices and/or schedules to this thesis) owns any copyright in it (the “Copyright”) and s/he has given The University of Manchester the right to use such Copyright for any administrative, promotional, educational and/or teaching purposes. (ii) Copies of this thesis, either in full or in extracts, may be made only in accordance with the regulations of the John Rylands University Library of Manchester. Details of these regulations may be obtained from the Librarian. This page must form part of any such copies made.

(iii) The ownership of any patents, designs, trade marks and any and all other intellectual property rights except for the Copyright (the “Intellectual Property Rights”) and any reproductions of copyright works, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property Rights and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property Rights and/or Reproductions.

(iv) Further information on the conditions under which disclosure, publication and exploitation of this thesis, the Copyright and any Intellectual Property Rights and/or Reproductions described in it may take place is available from the Head of the School of Materials.

14


Abstract

Stimuli responsive materials are well documented and function by translating a molecular

event into a macroscopic transition. Polymer hydrogels are a particular type of material that

offers excellent potential for applications within the field of biomaterials. In this thesis an

enzyme responsive polymer hydrogel has been demonstrated that functions through the

incorporation of designed peptide actuators. The experimental findings within this thesis are

separated into three separate chapters.

The first experimental chapter describes the synthesis of poly(ethylene glycol)-co-

acrylamide microparticles (μPEGA) by inverse suspension polymerisation. Particles with

neutral, positive or negative charge were prepared and characterised. Neutral µPEGA were

selected were chosen as the preferred polymer particles. These particles were then utilised as

the basis of the enzyme responsive system. Here linear peptide actuators were incorporated

into the particles. These particles demonstrated an enzyme specific increase in accessibility.

Additionally, functionalised particles displayed pH responsive behaviour allowing for the

physical entrapment of a payload. The release of this payload was only possible when the

functionalised particles were exposed to the target enzyme. However, at physiological ionic

strength no response of the particles was observed due to electrostatic screening.

In order to overcome electrostatic screening, branched peptide actuators were developed and

incorporated into μPEGA. These peptide actuators provided enhanced charge density and

the functionalised particles were able to respond through an increase in swelling to the target

enzyme at physiological ionic strength. Using this system it was possible to selectively

release a macromolecule at physiological ionic strength in response to the target enzyme.

The large size distribution of μPEGA was addressed in the final experimental chapter. Here,

PEGA particles were prepared by a simplified microfluidic setup assembled using a needle

and tubing. The size of the particles produced was determined by the surfactant

concentration and the relative flow rates of the dispersed and continuous phases.

15


Acknowledgements

The last three years of my PhD have been an invaluable time in my life, I feel I have

greatly improved a variety of my skills and abilities as well as maturing as a person.

I have to firstly thank Rein (now Professor Ulijn!) for all his time, comments,

feedback and assistance he has given me throughout my PhD. The opportunities I

have had and a great number of my achievements have only been possible due to

him. I was also very fortunate to have Brian Saunders as my second supervisor, he

has always been available to help me as well as offering me support whenever it

was needed. His constructive criticism regarding my thesis as been invaluable,

thank you Brian!

There were, of course, numerous other individuals who have been very influential in

the completion of this PhD. These people have taught me new skills along with

refining other aspects on my research for which I am very grateful: Paul Thornton,

my mentor at the start of my PhD, without whom I would have been lost. Rob Mart

for his continuous technical advice (no matter how many times I asked...). Last but

not least, Andrew Hirst for his exceptionally useful advice with my writing, along

with his thorough proof reading of this thesis.

All my friends I have made during my studies have made the last three years very

enjoyable and memorable: Richard (our trips to Fab Café), Simon (gym visits and

trading sporting notes), Grace (discussing world politics), Rumana (the cat ‘jokes’),

Alison (all the conference trips), Claire (teaching me so many ‘useful’ French

phrases), Kapil, Riaz (no you can’t take the eighth decimal place into account!)

Louise, Andy T, Bobby T (stay off the T120s...) Nurguse, Andrew, Apurba and

Kate it wouldn’t have been the same without you.

Two technicians have been particularly helpful with my experimental work: Robert

Fernandez for his continuous help with confocal and TPM. Also, Andy Wallwork

for his time and assistance in my attempts to use stereolithography to produce

microfluidic devices.

16


The support I have received from my family (including the regular questioning

asking when I would get a job!) has been very important. I am very grateful for the

encouragement that my parents gave me throughout my time at university.

Finally I have to say many thanks to my girlfriend Jenny Hayes, she has put up with

my last seven years at university! She has also given me enormous moral and

financial support. Hopefully I can repay this in kind during her upcoming teacher

training.

17


1 Introduction

Biomaterials science is an interdisciplinary field that encompasses medicine,

biology, chemistry and materials science. Biomaterials1 have been defined as

substances other than food or drugs used in therapeutic or diagnostic systems.2 This

area of science has been applied to the fabrication of medical devices. These include

metallic hip replacements,3 ceramic dental crowns,4 polymeric drug delivery

systems5 and hydrogel based contact lenses.6 Overall, the development of

biomaterials has allowed a great number of diseases to be treated more effectively

and improve the quality of life for patients.7,8 In the majority of cases current

biomaterials are inert i.e. they do not interact with their surroundings. This

characteristic is somewhat limiting when the material is used to replace a function in

the dynamic and changing environment of biological systems. For this reason, there

has been great interest in designed biomaterials that can respond (e.g. swelling,9

payload release10 and dissolution11) to an environmental stimulus. A particularly

interesting choice of stimulus would be enzymes, nature’s catalysts, because of their

vital importance to living systems. Enzymes drive and control almost every process

that occurs in the human body from respiration to growth.12 Of special interest is the

role enzymes have within disease processes, these include: the metastasis of

tumours,13 chronic wounds14 and arthritis.15

A common strategy to incorporate responsiveness into a biomaterial is based on

hydrogels. These are water-containing three-dimensional structures composed of

either hydrophilic polymer networks (chemical hydrogels)16 or self-assembled

(macro) molecules.17-19 The former, chemical hydrogels are an area of focus in this

thesis. These crosslinked (either physically of chemically) hydrophilic polymers

give polymer networks that can contain between ten to many thousand times their

dry mass in water.16 This highly hydrated nature of hydrogels presents similar

characteristics to biological tissue. Additionally, the properties of these polymer

18


networks can be altered by changing the polymer’s interactions with neighbouring

chains or the water molecules surrounding the polymer chains.

This thesis consists of two main areas of research, (i) the development of a hydrogel

system amenable to chemical modification thereby incorporating enzyme

responsiveness and (ii), the design and development of enzyme responsive

functionalities.

1.1. Motivation of the project

The motivation of this research was to develop enzyme responsive hydrogel

particles based on peptide actuators. This proof-of-concept research would

demonstrate the potential application of peptide actuators in the enzyme specific

release of a payload. In the future systems such as these might serve a role in the

triggered delivery of drugs.

1.2. Layout of the thesis

Within this thesis the experimental chapters are presented separately, each with its

own introduction, results, discussion, and conclusion sections. By separating the

chapters in this manner it is intended to give the reader a clear view to each section

of research.

The first experimental chapter, Chapter 3 investigates the synthesis and

characterisation of poly(ethylene glycol)-co-acrylamide (PEGA) microparticles

(µPEGA) by way of inverse suspension polymerisation (Figure 1-1). The resulting

microparticles were the functionalisation with peptide actuators. Finally, the

responsive behaviour of these particles was then determined and exploited for

enzyme specific release of a payload Figure 1-2.

19


Figure 1-1. The inverse suspension polymerisation of µPEGAs.

Figure 1-2. Enzyme responsive µPEGA. Scheme of enzyme responsiveness of linear peptide

actuators.

Chapter 4 goes on to tackle the limitations of the linear peptide actuators with the

design and development of branched peptide actuators. µPEGA functionalised with

these peptide actuators demonstrate enzyme specific swelling at physiological ionic

strength (Figure 1-3).

20


Figure 1-3. Enzyme responsive µPEGA through the incorporation of branched peptide

actuators. Schematic of enzyme responsive branched peptide actuator.

The final experimental chapter explores the application of microfluidic devices in

the production of PEGA particles, with the aim of addressing the high polydispersity

of the particles when made by inverse suspension polymerisation (Figure 1-4).

Figure 1-4. Production of PEGA particles by microfluidic polymerisation. Schematic

representation of microfluidic setup.

21


2 Literature review

2.1. Introduction

This chapter aims to give the reader a good understanding of the literature

predominately from the last ten years in three separate fields: (i) Bioresponsive

hydrogels. (ii) The previous research undertaken on the polymer poly(ethylene

glycol)-co-acrylamide (PEGA), on which most of the experimental work is based.

(iii) The use of microfluidic devices to produce polymer particles.

2.2. Stimuli responsive materials

Stimuli responsive materials are very well documented,20-25 these materials function

by translating a molecular event into a macroscopic transition. Polymer (chemical)

hydrogels are a particular type of responsive material that offers excellent potential

for applications (e.g. sensing,26 drug delivery27 or tissue engineering28) within the

field of biomaterials.23,29

2.2.1. Chemical hydrogels

Chemical hydrogels are hydrophilic polymers that are highly crosslinked resulting in

insoluble 3-D networks. The first synthetic hydrogels were developed in 1960 by

Wichterle and Lim30 when they produced poly(2-hydroxyethyl methacrylate)

poly(HEMA). They showed that these new hydrophilic networks overcame many

problems associated with polymers that were previously being used in medical

applications. The generation of materials available at that time had poor

biocompatibility and upon implantation caused severe local inflammation. These

chemical hydrogels offered completely new properties due to their high water

content; which was similar to surrounding tissues, gave mechanical properties

comparable to biological materials and allowed the diffusion of metabolites through

the material. At the time this was published Wichterle and Lim suggested that these

polymers could be used to manufacture contact lenses and arteries.30 Subsequently

22


there has been great interest in exploiting hydrogels within the field of biomedical

applications. Currently the main application of these biomaterials in medicine is as

contact lenses. Indeed, contact lenses are now used by approximately 125 million

people worldwide to augment eye sight.31

Stimuli responsive hydrogels have been shown to respond to numerous stimuli

including pH,32 temperature,33 electric fields,34 ionic strength35 and small

molecules.36,37 It is beyond the scope of this thesis to effectively cover the field of

stimuli responsive hydrogels therefore, further details of these materials can be

found in a number of excellent review articles.21,24,32,38,39

2.3. Bioresponsive Hydrogels*

Bioresponsive hydrogels allow for the potential treatment of diseases by responding

directly to a biological marker for the disease. In the future these systems may

provide more effective medical treatment through highly targeted specific responses.

2.3.1. Introduction

There is a huge range of biological stimuli, these generally fall into two main

categories: small molar mass substances (such as glucose or calcium ions) or

macromolecules (including enzymes and proteins). Through the incorporation of a

moiety capable of recognising a specific biological stimulus and utilising this

recognition event to produce a response it is possible to make a material

‘bioresponsive’. In this thesis the term ‘bioresponsive’ is defined as stimuli

responsive materials that change properties in response to a biological molecular

recognition event.40 These molecular interactions are then translated through

molecular actuation into macroscopic changes in the properties of the material.

This section will focus on different methods used to actuate macroscopic responses

of chemical hydrogels to a biological stimulus. The preparation of these devices will

be covered in detail along with how these systems have been used in the

development of biosensor, drug delivery and tissue scaffold systems. The methods

* Short parts of this section have been published by the author in: T.O. McDonald, R.J. Williams,

A.G. Patrick, B.G. Cousins and R.V. Ulijn, Bio-responsive chemically cross-linked and physical

hydrogels. Biomedical applications of electroactive polymer actuators. Wiley. 2009.

23


of actuation within bioresponsive materials can generally be divided into three

categories (Figure 2-5), changes in: (i) cross-linking density, (ii) electrostatic

interactions and (iii) molecular conformation.

Figure 2-5. Schematic representation of the different categories of bioresponsive hydrogels.

2.3.2. Actuation based on changes in crosslinking density

Systems based on actuation through changes in cross-linking density can be

separated into two main categories: covalent crosslinking of polymers by peptides

which are modified by enzymes, or, non-covalent interactions between crosslinking

moieties in which these interactions can be disrupted by competition between free

moieties and those responsible for crosslinking.

2.3.2.1. Systems incorporating peptide crosslinkers

The incorporation of peptide crosslinkers into the hydrogel offers the potential for

enzymes that hydrolyse peptide bonds (proteases) to specifically degrade the

polymer network. There are a number of different approaches by which these

peptides have been integrated into the various polymers to prepare responsive

systems (Table 2-1).

24


25


Table 2-1. Selected studies of bioresponsive hydrogels based on peptide crosslinkers.

# Polymer Stimulus Biorecognition Response Ref

1 PEG MMP-1 Enzyme hydrolysis of

peptide crosslinks

Gel

dissolution

41

2 Poly(NIPAM-

co-AAC)

MMP-13 Enzyme hydrolysis of

peptide crosslinks

Gel

dissolution

42

3 PEG TG Enzymatic

crosslinking of

peptides

Hydrogel

formation

43

4 Polyacrylamide Chymotrypsin Enzyme hydrolysis of

peptide crosslinks

Hydrogel

dissolution

11

5 PEG Factor XIIIa

& MMP

Enzymatic

crosslinking of

peptides & Enzyme

hydrolysis of peptide

crosslinks

Hydrogel

formation or

hydrogel

dissolution

44

6 Poly(HPMA) Calcium ions Enzymatic degradation

of polymer

Release of

payload

45

2.3.2.1.1 Enzyme catalysed changes in peptide crosslinker density

The innovative development of systems based on enzyme catalysed changes in

peptide crosslinker density (Table 2-1, entries 1-5) was by Hubbell and co-

workers.41 In the designing of a cell-responsive hydrogel, in which the hydrated

environment was remodelled by cell-secreted enzymes. The main structural

component of the hydrogel was polyethylene glycol (PEG), offering a hydrophilic

polymer network that resists protein adsorption. Other biological functionalities

were introduced in the form of peptides (Figure 2-6 A). Four-armed PEG molecules

with vinyl sulfone end groups were first reacted with a low stoichiometric ratio of

mono-cysteine peptide based on RGD (the tripeptide determined to be the cell-

attachment domain in fibronectin).46 This partially derivatised PEG was then

crosslinked by reacting with a bis-cysteine peptide (Ac-GCRD-GPQG↓IWGQ-

DRCG-NH2) (arrow indicates enzyme cleavage site). The flanking of the cysteine

residues with the RD sequence provides water solubility and an optimal pKa for the

26


formation of cysteine thiolate. This system formed elastic hydrogels under

physiological conditions. Dynamic rheometry (in which the material is subjected to

an oscillating strain)47 was used to observe gel formation showing that at higher pH

the gel point occurred more rapidly (Figure 2-6B).

Figure 2-6. Cell responsive synthetic hydrogels. A:(1) A Michael type addition reaction

between vinyl-sulfone functionalised multiarm PEGs and mono-cysteine adhesion peptides (2)

and bis-cysteine MMP substrate peptides was used to form gels from aqueous solutions in the

presence of cells. These hydrogel networks were designed to respond to local protease activity

at the cell surface (3). B: Dynamic rheometry of a typical gelation reaction, showing the elastic

modulus (G’) and loss modulus (G’’) and the gel point’s sensitivity to pH. C: The swelling and

degradation of the gel network in responsive to incubation with MMP-1. 41

Addition of matrix metalloproteinase (MMP)-1 solution to the gel induced a volume

increase until network dissolution was attained as a result of enzyme catalysed

peptide hydrolysis. Conversely, a hydrogel formed using a peptide crosslinker with a

sequence that the protease does not have specificity for did not show any change in

volume upon MMP-1 treatment (Figure 2-6 C). MMP sensitive hydrogels allowed

27


cells to migrate with the hydrogel and in vivo studies showed that extensive

vascularisation only occurred with the MMP sensitive peptide crosslinker.41

Furthermore, Healy and co-workers demonstrated a similar system incorporating

protease cleavable crosslinks.42 This system was based NIPAAm and acrylic acid

(AAc) monomers. The peptide crosslinker (QPQG-LAK-NH2) was synthesised

through the acrylation of the amine groups of the lysine residues and the (N-terminal

amine) of glutamine with acryloyl chloride. The resulting hydrogels (by free-radical

polymerisation) were injected through a 2 mm aperture without demonstrating

appreciable macroscopic fracture. Enzyme degradation of the gels occurred when

exposed to MMP-13 with degradation time dependant on cross-linking density. In

this way, degradation times could be modulated, ranging from approximately 100

hours for the lowest crosslinking density up to 300 hours with the highest degree of

crosslinking.42

In another study a system that responded to enzymes by hydrogel formation rather

than degradation was shown by Messersmith and co-workers.43 The protein

crosslinking enzyme transglutaminase (TG) was used to catalyse reactions between

short peptides conjugated to PEG. This enzyme catalysed an acyl-transfer reaction

between the γ-carboxamide group of protein-bound glutaminyl residues and the ε-

amino group of Lys residues, resulting in the formation of ε-(γ-glutamyl)lysine

isopeptide sidechain bridges. Through the rational design of peptide substrates for

TG rapid formation of a hydrogel was observed when multifunctional PEG

molecules with conjugated peptides were exposed to the enzyme. The incorporation

of the adhesive amino acid L-3,4-dihydroxylphenylalanine (DOPA) was also

demonstrated for the first time with the best substrates for TG crosslinking found to

be DOPA-FKG-NH2 and DOPA-GQQQLG-NH2.43

In 2004 Moore and co-workers published an enzyme responsive hydrogel utilising a

novel conjugation technique.11 Earlier methods based on reacting peptide amine

groups with acryloyl chloride or the Michael addition of cysteines to vinyl sulfones

described by Hubbell had clear limitations. The acrylol chloride reactions lacked

selectivity while Michael additions required a basic residue to be near to the cysteine

28


(in order to lower the pKa of the thiol giving increased rates of reaction). In the work

presented by Moore and co-workers a scheme that allowed for the selective coupling

of acrylamides to peptide sequences was demonstrated. The disulphide exchange

between an active thiol containing monomer and thiol on a cysteine side chain

occurred under acidic conditions and was selective for only thiols. The acidic

environment firstly protonated the pyridyl group providing a good leaving group and

secondly protonated the lysine residues preventing them from performing Michael

addition to the methacrylamide functionality. The activated disulphide monomers

were conjugated to the peptide and these peptide crosslinkers was then

copolymerised with acrylamide by way of UV initiators. With this methodology

poly(acrylamide) hydrogels crosslinked with peptides (CYKC) were prepared that

would dissolve when subjected to chymotrypsin solution. Chymotrypsin solutions

were flowed through microchannels containing hydrogel disks while the sizes of

these disks were monitored. The control sample (containing a peptide for which

chymotrypsin does not have specificity, CSKC) remained unaffected by the enzyme

solution while the test sample containing the enzyme cleavable peptide shrank until

complete dissolution occurred at 20 minutes. 11

More recently, Lutolf and co-workers have further developed the concept of cell

responsive hydrogels with the aim of further mimicking the dynamic remodelling of

the ECM that occurs in vivo.44 This includes the formation of new bonds as well as

degradation. Here, much like Messersmith and co-workers, they made use of a type

of transglutaminase; the crosslinking enzyme factor XIIIa. This enzyme plays a key

role in fibrin clot formation upon tissue damage by catalysing acyl-transfer between

the carboxamide group of a protein-bound glutamine (Q) residue and the ε amino

group in a lysine (K) residue. Multiarm PEG molecules were functionalised with

either Gln in the form of its XIIIa acceptor substrate (NQEQVSPL) or an MMP

cleavable peptide terminated at the C-terminus with Lys. A mixture of these

precursors formed a hydrogel in the presence of factor XIIIa with a gel point time of

about 750 seconds as determined by real-time shear rheometric measurements. The

bioactive binding ligand RGD could also be incorporated in the network by having

the XIIIa acceptor substrate at the C-terminus of RGD. Upon treatment of these

hydrogels with an MMP solution rapid dissolution was observed (within 10 minutes)

29


dependant on the peptide crosslinker sequence.44 In their most recent publication

they quantitatively incorporated growth factor proteins (via the same XIIIa

crosslinking method) that were released upon cell-derived proteolytic degradation of

the gels.28

Messersmith and co-workers again made use of a transglutaminase (TG) enzyme

this time to conjugate peptides to cartilage tissue. Cartilage had previously been

shown to contain a type of TG, tissue transglutaminase (tTG). When cartilage was

incubated with peptides covalently attached to biotin (via a diethylene glycol or PEG

spacer) those that were coupled to the cartilage by the tTG enzyme were detected by

staining with fluorescein anti-biotin antibody. Only peptides to which the enzyme

had specificity (-FKG-NH2 and –GQQQLG-NH2) were found to have coupled to the

cartilage. While peptides in which the asparagine and ornithine residues (analogues

carrying the same charge with different structure) were substituted for glutamine and

lysine respectively were not coupled to the cartilage.48

2.3.2.1.2 Enzyme hydrolysis of hydrogel network

A different approach to using enzymes in a responsive system based on hydrogel

degradation has been described by Kost and co-workers.49 In that work a calcium

responsive system was prepared through the incorporation of a non-active enzyme

into a polymer matrix consisting of the enzyme’s substrate. The enzyme used was

the starch hydrolysing, α-amylase, while the polymer matrix was a mixture of starch

and hydroxypropyl methyl cellulose ether (HPMC). α-amylase’s are known to

contain at least one atom of calcium firmly and specifically bound to the enzyme

molecule (protein) that is essential for its activity. Therefore, firstly de-activated α-

amylase was prepared by chelating the calcium ions without denaturating the

enzyme. Enzyme containing tablets were then prepared by wet granulation in which

all ingredients were ground, mixed then dried before being pressed into tablets.

Upon incubation of the tablets a concentration dependant release of the model drug

myoglobin was obtained with a constant rate of release over 30 hours.45

2.3.2.2. Non-covalent crosslinking interactions

Systems have also been described based on non-covalent crosslinking interactions.

These typically involve the functionalisation of the monomers with two separate

30


biological moieties that are able to specifically bind to one another through

molecular recognition. When these functional monomers are copolymerised, the

biological groups non-covalently bind to each other, thereby crosslinking the

polymer. The material therefore responds to the presence of one of the crosslinking

moieties in free solution through a reduction in crosslink density due to the

competition between the free moieties and those crosslinking the polymer (Table 2-

2).

Table 2-2. Selected studies of bioresponsive hydrogels based on non-covalent crosslinking

interactions


1 Polyacrylamide Antigen Antigen-antibody

bonding

Reversible

swelling

27

2 Poly(NIPAM-

co-AAC)

Antigen Biotin-Antibiotin

binding

Reversible

swelling

26

3 Poly(HPMA) Coiled-coil

forming

peptides

Self-assembly through

coiled-coil formation

Hydrogel

formation

50

An excellent early example of this type of system is the antigen responsive hydrogel

described by Uragami and co-workers.27 In this study a semi-interpenetrating

network (semi-IPN) hydrogel containing both grafted antigens and their

corresponding specific antibody was prepared. Vinyl functionality was introduced to

both of the biological moieties by reacting them with N-succinimidylacrylate (NSA)

(which reacts with the primary amines). The modified goat-anti-rabbit antibody

(GAR) IgG was then copolymerised with acrylamide to give a polymer solution.

Vinyl rabbit IgG (antigen) was mixed with the GAR IgG polymer solution,

acrylamide, a crosslinker N,N’-methylenebisacrylamide (MBAA) and polymerised

with free-radical redox initiators (Figure 2-7 A). Antigen-antibody binding then

caused the formation of further cross-links within the polymer reducing the swelling

(Figure 2-7 B). The presence of free antigens in the solution around the hydrogel

created competition between the grafted antigens leading to a decrease in cross-

linking density, thus an increase in swelling of the hydrogel (Figure 2-7 C). This

increase in swelling was reversible. By removing the hydrogel from the antigen

31


solution and washing, the polymer would return to approximately its original

volume. This response also corresponds to an increase in permeability of the

polymer and by using the polymer as a membrane, selective permeation of a protein

was shown in response to the specific antibody (Figure 2-7 D). More recently a

similar design was employed to produce an antigen-responsive membrane that has

gating properties (allow for the opening of closing of pores) for selective diffusion

in response to the presence of a free antigen.51

Figure 2-7. Antigen responsive hydrogel. A: Synthesis of the antigen-antibody semi-IPN

hydrogel. B: Diagram of a suggested mechanism for the swelling of an antigen-antibody semi-

IPN hydrogel in response to a free antigen. C: Antigen recognition by antigen-antibody semi-

IPN hydrogel. D: Reversible swelling changes and antigen-responsive permeation profiles. 27

Lyon and co-workers have demonstrated an antigen responsive hydrogel, 26 this

system consists of a co-polymer of pNIPAAm and acrylic acid with N,N’-

methylenebisacrylamide as the crosslinker prepared by free radical initiated

precipitation polymerisation. In this process the monomer(s) and initiator are soluble

in the solvent and initiation takes place in solution. The polymer chains then grow

until they reach a critical chain length where they exceed their solubility and

precipitate from solution.52 Lyon and co-workers covalently incorporated a biotin

moiety into the microgels by coupling biotin hydrazide to the carboxyl group of the

acrylic acid using a water-soluble carbodiimide (1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide). Aminobenzophenone (ABP) was then also

32


coupled to the polymer using N,N’-dicyclohexylcarbodiimide in dimethyl sulfoxide

(DMSO). Glass coverslips functionalised with the cationic silane 3-

aminopropyltrimethoxysilane (APTMS) were then placed in an aqueous solution of

biotin/ABP functionalised polymer particles which strongly attached to the glass

surface via Coulombic interactions. These ‘microlens’ covered surfaces were then

exposed to a solution of anti-biotin (antibody), leading to antigen-antibody binding

which was followed by photoligation of the antibody to the ABP. This treatment

effectively led to a crosslinking of the surface of the microlenses by antigen-

antibody binding. Exposure of these microlenses to biocytin (antigen) led to

competition in binding with anti-biotin resulting in a decrease in the crosslinking

density of the surface of the microlenses (Figure 2-8 A). This resulted in an increase

in swelling of the polymer and a corresponding decrease in the microlens focussing

power. This was determined using brightfield transmission and differential

interference contrast (DIC) optical microscopy (Figure 2-8 B). The response rate of

the hydrogel microlenses were found to be strongly coupled to analyte

concentration.26

Figure 2-8. Displacement-Induced Switching Rates of Bioresponsive Hydrogel Microlenses. A:

The microgels are functionalized with biotin and ABP (top left), are cross-linked by anti-biotin

(top centre) and exposed to a solution of biocytin (top right). B: Response of hydrogel

microlenses to incubation with anti-biotin antibodies. Microscopic images of hydrogel

33


microlenses (left element in each image) and biotin/ABP functionalized hydrogel microlenses

(right element in each image) are shown, (a) before and (b) after incubation with anti-biotin.

The scale bar is 2 µm.26

Kopeček and co-workers describe a hybrid hydrogel (a hydrogel system consisting

of at least two distinct classes of molecules connected either covalently or non-

covalently) in which the crosslinking was achieved through the self-assembly of

coiled-coil forming peptides (Figure 2-9 A). These peptides were grafted onto a

polymer backbone of N-(2-hydroxypropyl)methacrylamide (HPMA). Coiled-coils

are a native protein conformation that consists of two or more α-helices wound

together to form a super-helix.53 The attraction of incorporating these types of

peptides into a polymer is that the association and disassociation of these coiled-

coils is determined by the primary structure of the amino acid sequence. In this

system polymerisable functionality was imparted into the peptides by capping the N-

termini of the peptide with N-methacyloylglycyl-glycyltryptophan. This offered the

same functionality as HPMA ensuring the compatibility of the comonomers in free

radical copolymerisation. Examination of the self-assembly of these copolymers into

hydrogels was achieved using microrheology and dynamic light scattering. Dynamic

light scattering evaluated the size of nanoparticles formed as association of coiled-

coil forming peptides led to gelation. Analysis of the hydrodynamic radius, Rh

showed that particle size was independent of graft copolymer concentration at low

concentrations but increased dramatically as the concentration was increased above

5.52 mg/ml (Figure 2-9 B). These results indicated that the self-assembly process

was strongly governed by polymer concentration. Temperature also had an effect on

the self-assembly process, as temperature was increased (Figure 2-9 C) there is a

gradual increase in the Rh. The increased thermal energy of the system led to an

increase in the probably of a collision of the peptide grafts making the self-assembly

process more effective at higher temperature.50 This system demonstrates that

coiled-coil interactions can be used to induce the self-assembly of polymers, this

offers the potential to use this interaction to crosslink hydrogels and potentially

screen for coiled-coil forming peptides.

34


Figure 2-9. Novel synthesis of HPMA copolymers containing peptide grafts and their self

assembly into hybrid hydrogels. A: the self-assembly of copolymers into hydrogels. B:

Concentration dependence of the hydrodynamic radii Rh of polymer clusters. C: Temperature

dependence of the hydrodynamic radii Rh of polymer clusters.50

2.3.3. Actuation based on electrostatic interactions

A second mechanism to actuate a change in swelling is through electrostatic

repulsion/attraction within the polymeric network. These charges are typically

induced on the polymer chain but it has also been demonstrated that grafting specific

actuators on to the polymer is possible ().

35


Table 2-3. Selected studies of bioresponsive hydrogels based on electrostatic interactions


1 Poly(HEMA-co-

DMAEMA)

Glucose Conversion of glucose

to gluconic acid

Increased

swelling

36

2 Poly(PEGMA-co-

DEAEMA)


to gluconic acid

Increased

swelling

54,55

3 Poly(AAC) grafted

onto porous

membrane of PVDF


to gluconic acid

Increased

permeabilit

y of

membrane

56

4 PEGA Protease Enzymatic hydrolysis

of peptide actuators

Increased

swelling

57,58

2.3.3.1. Electrostatic interactions between charges on the polymer network

Actuation based on electrostatic interactions within the polymer network is widely

studied in the context of glucose responsive polymers for the treatment of

diabetes.36,54-56,59-61 Generally glucose oxidase (GOx) is immobilised in the hydrogel,

when glucose is present it is converted to gluconic acid, which lowers the pH within

the microenvironment of the hydrogel. There are two different macroscopic designs

in which this decrease in pH is used to actuate a change in swelling: matrix type

systems where the enzyme and insulin are contained within a bulk polymer, or

membrane type systems where the drug is contained in a reservoir within a

membrane. Kost and co-workers produced an example of the matrix system where

the insulin and enzyme were contained uniformly throughout a hydrogel. The

hydrogel was made from 2-hydroxyethyl methacrylate (HEMA), N,N-

dimethylaminoethyl methacrylate (DMAEMA) with tetraethylene glycol

dimethacrylate (TEGDMA) as a crosslinking agent. The presence of amines and the

low crosslinker concentration (between 0-0.95 %) meant that at low pH the amines

become ionised leading to an increase in swelling (schematic shown in Figure 2-11).

36


Figure 2-10. The enzymes and the reactions they catalyse used in glucose responsive hydrogels.

GOx, catalase and insulin were incorporated into the hydrogel during the

polymerisation step (free-radical initiation at room temperature). Figure 2-10 shows

the reactions driven by these enzymes, GOx catalyses the reaction of glucose to

gluconic acid forming hydrogen peroxide. A build up of hydrogen peroxide leads to

inhibition of the enzyme, and because oxygen is needed to form gluconic acid a

shortage of oxygen leads to slower swelling rates. For this reason catalase was also

incorporated into this system which serves to convert hydrogen peroxide to water

and oxygen. The effect of crosslinker concentration on the responsive swelling

behaviour was investigated and it was found that polymers prepared without

crosslinker led to the greatest increase in swelling. These polymers did not dissolve

in water even over prolonged periods, Kost and co-workers suggest that this stability

may be due to entanglements and non-covalent interactions between the polymer

chains. Due to the dynamic nature of conditions in vivo non-steady state experiments

investigating swelling changes in response to glucose were examined. The glucose

concentration was switched between a hyperglycaemic blood glucose concentration

and a normal blood glucose concentration, in these experiments they found that

deswelling occurred more rapidly than a further increase in swelling. Analysis of

glucose triggered release of insulin in matrices with varying crosslinker

concentration also showed that the shortest response time and the greatest amount of

insulin was released in matrices without crosslinking agent. The device was

implanted in rats and these in vivo experiments indicated that some of the entrapped

insulin retained its active form and was effective in reducing blood glucose levels.

Additionally, over the 2-3 weeks the matrices were implanted no fibrotic

encapsulation was observed demonstrating the biocompatibility of the devices. 36

37


Figure 2-11. Characterization of glucose-sensitive insulin release systems in simulated in vivo

conditions. Schematic presentation of a matrix system based on poly(HEMA-co-DMAEMA).

(top) unswollen matrix at time t=0, (bottom) swollen matrix.

Peppas and co-workers have previously developed a similar system consisting a co-

polymer of diethylaminoethyl methacrylate (DEAEMA) and poly(ethylene glycol)

monomethacylate (PEGMA) using tetra(ethylene glycol) dimethacryalte (TEGMA)

as the crosslinker. GOx and catalase were given vinyl functionality by reacting with

acryloyl chloride and mixed with the monomer solution prior to UV initiated

polymerisation to give hydrogel films. With this system they demonstrated pulsatile

pH-responsive swelling, but did not show glucose-responsive behaviour.54 Within

their following publication microparticles with the same chemistry were produced

by inverse suspension polymerisation with redox-initiators. These microparticles

demonstrated rapid swelling/deswelling dynamics in response to changes in pH and

it was determined that faster responses could be obtained from smaller particles.55

38


A membrane type system for insulin release was developed by Liang and co-

workers. Here, poly(acrylic acid) (PAAc) was grafted to a porous membrane of

polyvinylidene fluoride (PVDF), GOx was covalently bound to the PAAc by firstly

activating the carboxyl groups with a water soluble carbodiimide then immersing the

membrane in a aqueous solution of GOx. The PAAc with GOx covalently

immobilised effectively ‘gate’ the pores of the PVDF. At neutral pH and when there

is no glucose in the surrounding environment the pores within the membrane are

‘closed’. When the carboxyl groups present in the PAAc chains are dissociated and

negatively charged, these charges along the polymer chains electrostatically repel

one another forcing the chain to lengthen and extend. When glucose was present it

was oxidised into gluconic acid by the immobilised GOx, this led to a reduction of

pH in the microenvironment of the pores protonating the carboxylate groups on the

grafted PAAc chains. The gates then ‘opened’ because the PAAc chains were

collapsed on the removal of the electrostatic repulsion allowing the insulin to diffuse

out of the membrane. The grafting density of PAAc was varied to find the ideal

value for insulin release, at low values it was found that the PAAc chains were too

short/sparse to effectively close the pores. At higher densities the PAAc became too

long/dense and it was no longer possible for a conformation change to occur. Using

a grafting yield of 1.55% the insulin permeation coefficient after glucose addition

was 9.37 times greater than without glucose.56

Recently there have been fewer publications in which GOx has been utilised to

actuate insulin release (50 % decrease from 2003), it appears that this method has

reached it limits and recent approaches by researchers are based on effective oral

delivery of insulin rather than the development of glucose responsive polymers. An

obvious limitation within GOx hydrogel systems is that the treatment of diabetes is a

continuous long term process; any implanted devices must be able to contain large

quantities of insulin for release over this time frame in addition to maintaining their

dynamic response.

39


2.3.3.2. Electrostatic interactions between charges present in pendant actuators

The design of actuators in which the molecular actuation involves a change in the

net charge offers the potential to develop more intricate responsive systems. Current

approaches utilise enzyme cleavable peptides as the sensing element. Thornton et al

described the development of a functionalised hydrogel that alters its accessibility in

response to an enzyme with a selected specificity.57,58,62 This was achieved through

modification of poly(ethylene glycol)-co-acrylamide (PEGA) particles by solid

phase peptide synthesis with peptide actuators. Earlier work highlighted tri-peptides

consisting of an enzyme cleavable (di)peptide (ECP) and the charged amino acid

arginine. The presence of these charged groups within the polymer led to

electrostatically induced swelling. Upon cleavage of the ECP by an enzyme with

matching specificity these cationic groups were removed resulting in a decrease in

swelling. The highly hydrated and crosslinked structure of PEGA makes the interior

of the polymer network accessible to macromolecules. The maximum size

(molecular weight) of macromolecule that can diffuse into PEGA is well defined

and termed the molecular cut-off weight. Above this molecular weight molecules are

unable to diffuse into the interior of the polymer.63 An increase in swelling caused a

corresponding increase in this cut-off weight. Through the use of different molecular

weight Fluorescein isothiocyanate (FITC) labelled dextrans it was possible to

monitor the changes in polymer accessibility using two-photon microscopy (this

technique is described in section 8.1.2). If a dextran with a molecular weight greater

than the cut-off for the polymer could diffuse into the interior of the particles it

indicated there was an increase in accessibility (due to charge induced swelling). It

was found that enzymes with the correct specificity for the ECP (thus removing the

charged groups) led to a decrease in accessibility and diameter of the particles.62

Thornton et al went on to develop this system of hydrogel particles functionalised

with peptides for the controlled release of entrapped payloads. Here, the peptide

actuator consisted of two opposing charged amino acids separated by an uncharged

ECP, corresponding to the sequence Fmoc-D-(ECP)-R-PEGA (Figure 2-12 A). This

zwitterionic peptide had a net neutral charge but upon enzyme cleavage of the ECP

only the cationic amino acid arginyl and half the ECP remained covalently attached

to the hydrogel. With pKa of the amino group being 7.5, at pH values below this the

magnitude of response was greater due to the ionisation of amine group. This

40


enzyme responsive increase in swelling (a maximum increase in volume of 100 %)

corresponded to increase in accessibility which was used to release an entrapped

payload (Figure 2-12 B). Approximately 50 % of the payload being released in 30

minutes in response to an enzyme with the correct specificity.57 This type of release

system has a number of advantages over other release methods: the payload

molecule does not need to be covalently modified, enzymatic cleavage of the ECP

results in a tunable number of payload molecules being released and the payload

loading method is relatively mild (a pH switch). In a recent publication58 Thornton et

al modified the system to release proteins with different charges at physiological pH

(avidin pI= 10.0 and albumin pI= 4.7). This was achieved by tailoring the design of

the peptide actuator to give a net charge after ECP cleavage that was matched to the

charge on the protein. It was possible to release the positivity charged avidin with

the conventional peptide actuator (Fmoc-D-(ECP)-R-PEGA) due to the electrostatic

repulsion between the actuators and the protein as observed by two-photon

microscopy (Figure 2-13). In order to release albumin a new actuator was designed

(Fmoc-R-R-(ECP)-D-D-PEGA), again, this actuator had a net overall neutral charge

but upon enzyme specific hydrolysis of the ECP a net negative charge remained

coupled to the polymer (two negative carboxyl groups on the aspartic acid one

positive amine group). This allowed for release of the payload (albumin) due to

electrostatic repulsion. Analysis of the proteins released from the particles showed

that although some proteolysis did occur it was not a major concern. Another

limitation of this approach was the release of Fmoc-peptide fragments upon

enzymatic hydrolysis.

41


Figure 2-12. Enzyme-responsive hydrogel particles for the controlled release of proteins:

Designing peptide actuators to match payload. A: The peptide designed for the release of

positively charged proteins was comprised of Fmoc–D–AA–R, where the amide bond between

the two alanine residues is particularly liable to cleavage by our target enzyme. B: Generation

of positive charges by enzymatic cleavage of the bond between alanine residues allows protein

molecules to diffuse through the polymer pores for payload release.58

However, the main limitation of this concept was its inability to respond at

physiological ionic strength. When, counter-ions present in the solution screened

the electrostatic interactions between the peptide actuators, this led to an

insignificant release rate.58

42


Figure 2-13. Peptide actuator designed for the release of negatively charged protein molecules.

A: Two N-terminal arginine units are separated from two aspartic acid groups by two alanine

residues. A net negative charge remains on the particle following enzymatic hydrolysis. B:

Exclusion of albumin from the negatively charged swollen particle occurs following hydrolysis

of the bond between alanine residues.58

2.3.4. Actuation based on conformational changes

The third mechanism of actuation is based on changes in conformation of natural

proteins. These systems incorporate a natural protein into the hydrogel that

undergoes a conformation change and thus alters the characteristics of the material.

The use of proteins as actuators is a new development in bioresponsive hydrogels.64

An example of this system was developed by Mrksich and co-workers 65. In that

study the functional nature of the hydrogel was conferred to the system through the

introduction of the protein calmodulin (CaM). CaM is a 16.5-kDa protein with two

distinct conformational states. In the presence of calcium ions, CaM has an

extended, dumbbell-shaped conformation where the distance between the ends of the

protein is approximately 50 Å (extended CaM). This calcium-bound extended CaM

undergoes a transition from an extended dumbbell to a collapsed conformation

(collapsed CaM) upon the binding of ligands (with a distance between the protein

ends of approximately 15 Å). An engineered version of the CaM protein was

43


prepared in which the tyrosine residues at the ends of protein are replaced with

cysteine residues. This modification meant it was possible to selectively react the

acrylate end groups on the four-armed PEG molecules to the protein through a

Michael-type addition. This formed a water soluble conjugate. The success of this

reaction was shown by MALDI-TOF MS. A hydrogel was then formed by mixing

the conjugate with dithiothreitol (DTT) at room temperature crosslinking the

remaining acrylate groups to give a solid hydrogel. The hydrogel showed a

macroscopic decrease in volume when exposed to the trifluoperazine ligand (TFP).

TFP binds specifically to CaM, causing the CaM to undergo a conformation change

from extended to collapsed (Figure 2-14 A & B). This decrease in volume could be

reversed by chelating the calcium ions thus removing the calcium bound ligand.

Numerous cycles between and extended and collapsed material were possible

demonstrating the reversible nature of the hydrogel (Figure 2-14 C). More recently

this concept has been developed to incorporate a photochemical assembly allowing

spatial control of the location of the dynamic proteins.66

44


Figure 2-14. Ligand responsive hydrogel that relies on conformational changes. A: The two

conformational states of CaM, an extended conformation in the presence of calcium ions (left),

and a collapsed conformation upon binding to a ligand (right). B: A hydrogel with CaM in a

ligand-free state (left) and the same gel with CaM in a ligand-bound state (right) (scale bars: 1

mm). C: Hydrogels were exposed to TFP ligand, and the volume was measured at various

intervals for 2 h. The gel was then washed repeatedly and incubated in a calcium-containing

buffer to restore the extended CaM conformation. 65

45


2.3.5. Summary

Bioresponsive hydrogels are a relatively new area of research but there have already

been a range of different successful approaches to the design of these materials.

These systems present methods of: detecting biological compounds, controlling cell

modelling of scaffolds and the targeted/controlled release of active agents for

disease specific treatment. However, there are two main obstacles that need to be

overcome for many of these systems to provide actual medical usage: Few of these

systems offer true reversible responses, rather than one thermodynamically favoured

direction. Secondly, the high degrees of complexity within some of these materials

makes acquiring approval to use within the body difficult as it is essential to

understand what happens to all compounds once within the body.40 That said, the

developments within this field help outline the design rules for future researchers to

continue to progress and refine the concepts. In the future, research built upon this

groundwork should help to provide more effective treatments within the medical

field.

46


2.4. PEGA

2.4.1. The history of PEGA

In 1992, Meldal developed PEGA, a copolymer of poly(ethylene glycol) (PEG) and

acrylamide (Figure 2-15).67 This material was a highly polar solid support for solid

phase peptide synthesis particular continuous flow peptide synthesis. PEGA offered

a number advantageous properties: It was transparent with no absorbance in the

aromatic region allowing for easy spectrophotometric monitoring of the reaction

process. It had a highly branched polymer network with swelling in both organic and

polar solvents. Finally, the resin was highly polar assisting peptide solvation.67 This

section covers developments within the field of PEGA detailing methods and

techniques from the literature that have arisen since the first publication of the

material.

Figure 2-15. Chemical structure of PEGA.

PEGA provided an important new property to the field of solid phase peptide

synthesis (SPPS)68 in its compatibility with both organic and aqueous solvents. This

property allowed peptides to be incorporated onto the polymer which could then be

47


placed in an aqueous environment. The open structure of the polymer allowed

enzymes to diffuse into the interior of the polymer particles where they catalysed

reactions.69,70 This development inspired a number of publications in which

techniques used in SPPS such as split and mix synthesis were exploited to prepare

libraries of peptides. These peptide libraries were then exposed to selected enzymes

and their effect determined. An early example of this approach was demonstrated by

Meldal and co-workers. Here, they prepared a ‘one bead, two compounds’ (Figure 2-

16) library to screen for inhibitors for proteolytic enzymes that are essential for

parasite development. Two different peptides were incorporated into individual

particles (referred to as beads within SPPS literature) by firstly temporarily

protecting a fraction of the amine groups in the PEGA particles with

hydroxymethylbenzoic acid, while using the remaining amines to synthesise the

fluorescence quenched peptide substrate (FRET). These substrates allow for the

detection of enzyme action; upon hydrolysis of the substrate the fluorescence

quencher is cleaved resulting in a fluorescent molecule. The hydroxyl function on

the PEGA particles was then esterificated and a second peptide (to screen for

inhibition) prepared by split synthesis from the ester bond to the hydroxymethyl

benzamide. These particles were then exposed to the enzyme solution (cruzipain)

and hydrolysis of the fluorescence quenched peptide substrate resulted in highly

fluorescent particles. The fluorescence was only seen on the surface of the particles

indicating that the 57 kDa enzyme was not able to diffuse inside into the polymer

network. The darkest particles were manually collected and the peptides sequenced

by Edman degradation-gas phase sequencing. This method yielded a first generation

of effective cruzipain inhibitors.70

48


Figure 2-16. Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor

library assay. D-amino acid inhibitors for cruzipain, cathepsin B and cathepsin L. The strategy

used in the synthesis of the ‘one bead, two compounds’ libraries.70

The mesh size of the polymer matrix in PEGA is controlled by the length (molecular

weight) of the PEG chains cross-linking the polymer. Most earlier work on had been

carried out on PEGA1900 (subscript refers to the molecular weight of the PEG in

g/mol), it was found that enzymes up to 50 kDa could diffuse into the polymer

network.69,71 Meldal and co-workers then went on to address this limitation of the

earlier PEGA resin by preparing PEGA cross-linked with PEG with molecular

weights of 4000, 6000, 8000. Libraries of fluorescence quenched peptide substrates

(FRET) (described in more detail in section 8.1.3) were then prepared on these

resins and incubated with the enzyme, MMP-9 which has active forms of 67-83

kDa. Particles that appeared bright were manually isolated and sequenced to identify

substrates for MMP-9, effectively identifying MMP-9 substrates.72 This method of

fluorescence-quenched peptide substrates was later used to screen for substrates the

cysteine protease, Papain. Here, the limited loading of the PEGA4000 was doubled

through the incorporation of a K-K dipeptide as the first functionalisation step on the

resin.73

Interest in utilising PEGA in the preparation of peptide libraries for screening with

enzymes soon increased. This led to a drive to further understand the polymer’s

structure/property relationship with enzyme catalysed reactions. Bradley and co-

workers63 presented a paper in which confocal Raman microscopy was used to

49


investigate enzyme accessibility. Three different supports for SPPS; TentaGel,

PEGA1900 and controlled pore glasses (CPGs) were functionalised with a peptide

substrate terminated with 4-cyanobenzamide and incubated with five different

enzymes with a range of molecular weights. The library of particles were then

investigated by confocal Raman microscopy to ascertain if the peptide sequence was

hydrolysed by monitoring the stretching frequency of the cyano group. TentaGel

was not accessible to any of the enzymes, indeed, there was no detectable effect on

the peptide with even the smallest enzyme (MMP-12, 22 kDa). PEGA1900 displayed

peptide cleavage with all enzymes below 35 kDa but not enzymes above 42.5 kDa.

Finally, beaded CPG showed complete cleavage of the peptide for all enzymes

tested. This was because the pores in the CPG used in the study were 100 nm in

diameter which was much larger than the hydrodynamic radius of the enzymes. This

study provided a well defined accessibility for PEGA to enzymes from which the

research community to work.63

An alternative approach to that used by Meldal and co-workers to increase the

accessibility of PEGA was described by the groups of Flitsch and Gardossi in a

series of papers.74-77 Here, rather than increasing the length of the cross-linking PEG

chains they substituted some of the acrylamide in the polymerisation mixture with a

permanently charged acrylamide based monomer. These PEGA+ and PEGA-

particles demonstrated greater swelling than the neutral PEGA1900, although as the

ionic strength of aqueous solution was increased the swelling of the charged PEGAs

reduced to the same value as that of PEGA1900. The swelling behaviour of the

charged polymers was due to the electrostatic repulsion between adjacent polymer

chains, this resulted in a increase in pore size as determined by an increase in

enzyme accessibility. The substrate for the enzyme used (penicillin G amidase,

PGA, 88 kDa) was N-phenylacetylated L-Phe, and was only cleaved to low

conversions (10 %) when coupled onto PEGA1900 and PEGA- however, for PEGA+

much greater conversions of 50 % were observed.74 In their following paper these

electrostatic effects were further investigated with hydrolytic yields as high as 80 %

being obtained by increasing the amount of positive charges in the polymer network.

These electrostatically attracted the negatively charged enzyme (PGA, pI = 5.2-5.4)

thus favouring the accessibility of the bulky enzyme.75 Additionally, Gardossi and

50


co-workers showed that PEGA+ retained the most enzyme within the particles and

that PEGA- retained the least.77 In a final publication on this topic, they made use of

the improved yields of PGA catalysed reaction on PEGA+ to demonstrate a

hydrazide enzyme cleavable linker.76 Overall, the introduction of permanent charges

to the polymer network offers an interesting method for the adjustment of the

polymers properties and interaction with surrounding proteins.

Another analytical technique was introduced into the area of enzyme catalysed

reactions on PEGA in 2003 by Flitsch and co-workers in the form of two photon

microscopy (TPM) (described in section 8.1.2).78 This technique allows the

production of images detailing the spatial resolution of fluorophores within polymer

particles. PEGA1900 was functionalised with Fmoc-F-F and exposed to the protease

thermolysin for different lengths of time. Where hydrolysis had occurred free amine

groups were present, these were chemically acylated with dansyl chloride (Figure 2-

17 A). These areas then showed as fluorescent, revealing where enzyme hydrolysis

has occurred. Thermolysin treated particles initially displayed a bright ring on the

outside of particle, which then expanded into the particle until after approximately

45 minutes when the whole particle was fluorescent (Figure 2-17 B). This indicated

that the enzymatic action was limited by diffusion of the enzyme into in the polymer

particle. Indeed, it was determined that for the enzyme to diffuse the distance from

the outside of the particle to the centre (100 µm) in an aqueous environment would

take approximately one minute.79 The use of TPM presented an effective method to

determine spatial and temporal resolution of enzyme hydrolysis within polymer

particles.

Figure 2-17. Using two photon microscopy to quantify enzymatic reaction rates on polymer

beads. A: Thermolysin catalysed hydrolysis of solid supported Fmoc–Phe–Phe. B: Thermolysin

catalysed hydrolysis of PEGA1900 bound dipeptide 1 as examined by TPM. From left to right the

images represent 5, 10, 20, 45, 60, 90, 120 and 240 min.

51


In 2003 a new development in the use of combinatorial libraries on PEGA was

developed. With the establishment of large libraries of PEGA-bound peptide for

screening, a faster and effective method was needed to separate particles with ‘hits’

from particles that were ‘non-hits’. Previously, this process had been carried out

manually on PEGA. Meldal and co-workers prepared a ‘one bead, two compound’

combinatorial library; one compound consisted of a fluorescence quenched peptide

substrate (FRET) while the second compound was a randomly synthesised peptide

(by split and mix synthesis) to screen for inhibition of their test enzymes,

metalloproteinases (MMPs). These two compounds then competed for binding with

the same enzyme and if the FRET substrate was not cleaved this indicated inhibition

of the enzyme by the library compound. Once incubated with the enzyme the

particles were analysed with an instrument developed originally developed for high

throughput screening and sorting of different transgenic-fluorescent tagged

organisms. It was further adapted for the purpose of sorting labelled particles. This

device allowed ‘hits’, dark particles, to be separated from ‘non-hits’ in a very fast

and reliable manner. Using this approach ten dark particles were selected, sequenced

and resynthesised to provide very potent inhibitor activity towards a number of

MMPs.80

In 2006 Ulijn and co-workers described a different morphology of PEGA in the

form a micropatterned PEGA surface.81 Within this work three different techniques

were utilised to prepare a micropatterned surface: photolithography, capillary force

lithography (using a patterned stamp) and spotting with a manual microarray (Figure

2-18). These patterned surfaces maintained the same chemistry as PEGA particles

allowing for functionalisation with peptides by SPPS. Using this methodology

PEGA surfaces were prepared that could direct cell adhesion (using the peptide

RGD) or screen for protease specificity with FRET peptide substrates.81

52


Figure 2-18. A micropatterned hydrogel platform for chemical Synthesis and biological

analysis. Optical micrographs of fibroblasts cultured in serum-containing media for 48 h on

(functionalized) PEGA-patterned surfaces. (a) Unmodified PEGA surface; fibroblast cells

strictly adhere and spread only on the glass surroundings, and not to the PEGA surface. (b)

RGE-modified control surface that resists cell adhesion. (c) RGD-modified PEGA surface that

promotes cell adhesion.81

A recent development comes from the Halling and Flitsch groups.82 Within this

work they made use of TPM to demonstrate real-time spatially resolved

measurement of enzyme activity on polymer particles. Aminocoumarin-carboxylic

acid (a fluorescent derivative used extensively in biological assays) was coupled

onto PEGA1900 via a hexa-glycine linker. A peptide or amino acid was then coupled

to the aminocumarin resulting in quenching of its fluorescence (Figure 2-19 A).

When Bz-R-OH was coupled onto the aminocumarin and these particles were

treated with trypsin fluorescence appeared as an annular ring around the outside of

the particle which then gradually progressed inwards. This indicated that enzyme

hydrolysis was initially confined to the outside of the particles gradually diffusing

towards the centre of the particle (Figure 2-19 B). This was in agreement with the

non-real-time studies using TPM that were previously carried out by Flitsch and co-

53


workers.79 Indeed, it took over three hours for the fluorescence values of the centre

of the particle to match those of the outside, this delay was likely a result of the

electrostatic repulsion between the positively charged trypsin (pI = 8.69) and the

positively charged arginine covalently attached to the aminocumarin. When a

different enzyme (Subtilisin Carlsberg) was incubated with the functionalised

particles (Bz-R-OH was replaced with Z-G-G-L-OH) different behaviour was

observed; there were no defined rings noted and increase in fluorescence upon

enzyme action was relatively homogeneous across the particles (Figure 2-19 C).

This was presumably due to the smaller size of the Subtilisin Carlsberg (although

this is not detailed within the paper) resulting in fast enzyme diffusion that was not

rate limiting. In summary, TPM provides a useful tool for the real-time analyse of

enzyme hydrolysis although it not shown whether the aminocumarin itself effects

enzyme rate or hydrolysis yield.82

Figure 2-19. Real-time imaging of protease action on substrates covalently immobilised to

polymer supports. A: Schematic of coupling and enzymatic reaction processes on PEGA1900

particles. B & C: Two photon cross-section images of PEGA1900 particles treated with B; trypsin

and C; Subtilisin Carlsberg respectively.

2.4.2. Enzyme catalysed synthesis on PEGA

A different area of research on PEGA was the synthesis of peptides on solid support

by enzymatic means, the first example of this was published by Flitsch and co-

54


workers.83 In this research, PEGA particles functionalised with phenylalanine were

prepared using solid phase methodology, enzymatically catalysed amide bond

formation then occurred upon treatment of these particles with an excess of amino

acids (with their amines protected with Fmoc) in the presence of thermolysin. High

yields were found for more hydrophobic amino acids while more polar residues led

to lower yields. The authors suggested that amide formation was driven by three

main factors: the large excess of substrates, the removal of ionisation (amines)

within the solid support and the improved solvation of the hydrophobic (Fmoc

protected) acyl donors within the PEGA. This publication provided a method for

enzymatic synthesis of peptides allowing for high enantioselectivity and without the

need of side chain protection that is required in conventional chemical synthesis.83

Within a following publication Flitsch et al84 went on to develop this method to

obtain L,L and L,D diastereoisomers of dipeptides and L-amino acids in good yields

starting from enantiomeric mixtures of amino acids using the enantioselectivity of

the enzyme (thermolysin) catalysed reactions.84 Recently, Flitsch et al85 investigated

the main factors controlling the enzymatic synthesis of peptides. From the three

main factors described in their first publication it was determined that reduction in

the unfavourable hydrophobic hydration of the Fmoc group within the solid support

compared with the free amino acid in solution was the most important driving force

in the enzyme catalysed synthesis.85

A separate publication was also produced documenting enzymatic synthesis on

PEGA by another group of researchers. Burkart and co-workers published

describing a biomimetic approach to the synthesis of a cyclic peptide antibiotic,86 a

scheme is shown in Figure 2-20 A. A peptidic linkage was first synthesised on the

resin by traditional chemical means, this was structurally homologous to the tether

found naturally (phosphotpanteheine, PCP). A linear decapeptide, the substrate for

the enzymatic cyclisation was constructed from the linker (via an ester bond) via

conventional SPPS. Incubation of these functionalised PEGA particles with TycC

TE (the isolated C-terminal thioesterase domain excised from the larger synthetase

protein found naturally) resulted in the release of the cyclised product (tyrocidine A,

a cationic peptide antimicrobial). By employing SPPS to produce a library of

decapeptides in which the fourth amino acid (D-phenylalanine) was substituted for

55


either natural or non-natural amino acids an insight of the enzymology of TycC TE

was obtained (Figure 2-20 B). Furthermore, the effective substrates for the

enzymatic cyclisation were investigated as artificial analogies of the cyclic peptide

antibiotic.86 This paper demonstrated the enzymatic synthesis of cyclic peptides on

solid supports (PEGA) and how this methodology may be exploited to investigate

the specificity of the enzyme. This allowed for the production chemical analogies of

the peptide offering the possibility of finding peptides with greater therapeutic

effect.

Figure 2-20. Biomimetic synthesis and optimization of cyclic peptide antibiotics. A: Natural

versus biomimetic macrocycle synthesis. The enzymatic assembly line involved in biosynthesis

of the cyclic cationic antimicrobial peptide, tyrocidine A. A carrier protein (PCP) in each

module is loaded with a phosphopantetheine prosthetic group (red). The individual modules

contain domains that load amino-acid building blocks onto the thioester tether and condense

via successive peptide bond giving the terminal PCP domain loaded with a linear decapeptide.

The terminal thioesterase domain (TE) catalyses head-to-tail cyclisation. In the biomimetic

synthetic strategy, a linker (red) that mimics phosphopantetheine was chemically synthesized

onto a solid-phase resin. Solid-phase peptide synthesis is used to construct a tethered linear

peptide, which can then serve as a substrate for cyclisation by the TE domain excised from the

synthetase proteins. B: Addition of the TE catalyses formation of the cyclisation product or the

hydrolysis product.

56


2.4.3. Summary

PEGA, a material initially developed as a new resin for use in continuous SPPS,

offers a number of unique properties that make it highly suitable for the preparation

of peptides. These peptides covalently attached to solid supports can then be

exposed to enzymes in aqueous solutions. This concept has been used to screen for

protease inhibitors and determine protease specificity. Effective methods have been

shown that have allowed enzyme activity within the hydrated polymer to be better

understood, as well as providing useful tools for future researchers. Additionally,

PEGA polymers have been created that contain permanent charges that give

increased yields for reactions catalysed by enzymes with the opposing charge.

Protease responsive PEGA particles have been demonstrate through the

incorporation of charged peptide actuators (described in detail in section 2.3.3.2)

Finally, PEGA has been used to demonstrate enzymatic synthesis of peptides on

solid supports and employed to use a biomimetic approach for the partially

enzymatic synthesis of cyclic peptides allowing for the screening of effective

substrates and production of analogies.

57


2.5. Microfluidic polymerisation of particles

Polymerisation processes in which a monomer solution is dispersed within another

immiscible solvent may overcome many problems that occur with other

polymerisation techniques such as autoacceleration and heat transfer limitations.87

There are numerous techniques (such as suspension polymerisation88 and emulsion

polymerisation89) used to form discrete polymer particles through the formation of a

monomer emulsion however, these are “top-down” approaches where the mixing of

the two liquids occurs as a bulk process. This leads to little control over the

formation of individual droplet dimensions and therefore typically results in

particles with a relatively broad size distribution. Ideally a “bottom-up” approach to

emulsification in which the formation of each individual droplet is controlled and

defined would be ideal. One strategy to achieve these objectives is based on

microfluidic devices. A early example of the controlled formation of liquid

dispersions using a microfluidic flow-focusing device (MFFD) was shown by Stone

and co-workers.90 Here, a pressure gradient along the long axis of the device forced

the two immiscible liquids through the orifice of the MFFD. The continuous phase

(supplied by the two outside channels) surrounded the dispersed phase (flowing

through the central channel) (Figure 2-21 A) causing the inner flow to become

unstable. This flow then broke in the orifice to give discrete droplets which then

entered the outlet channel (Figure 2-21 B & C). The planar microchannel design was

made by soft lithography in poly(dimethylsiloxane) (PDMS) and the smallest

droplets produced were much smaller than the orifice radius. Ultimately the flow

ratios of the water and oil through the device determined the size of droplets sizes

formed.

58


Figure 2-21. Formation of dispersions using "flow focusing" in microchannels. A: Flow-

focusing geometry implemented in a microfluidic device. An orifice is placed downstream of

three coaxial inlet streams. Water flows in the central channel and oil flows in the two outer

channels. B & C: Droplets are formed at the orifice and move into the outlet channel 90

In 2005 the idea of combining microfluidic droplet formation with a polymerisation

process was utilised to produce polymer particles.91 By dissolving the monomer in

the dispersed phase uniform monomer droplets were produced, a method of

initiation was then included to give polymer particles. These systems typically use

UV radiation to initiate polymerisation although interfacial polymerisation and

thermally initiation have also been used.92

Whitesides and co-workers produced a MFFD in either PDMS or polyurethane, this

allowed both water-in-oil and oil-in-water dispersion to be produced.91 They found

that it was possible to also produce uniform non-spherical particles by restricting the

dimensions of the outlet channel. If one of the dimensions is less than that of the

diameter of a regular droplet then disks were formed, by restricting both the height

and the width of the channel polymer ellipsoids or rods were produced. A number of

different monomer were used and with a single MFFD device they produced up to

59


250 polymer particles per second with a polydispersity (defined as the standard

deviation in the particle diameter divided by the mean particle diameter) of 1.5 %.91

At the same time Whitesides and co-workers also published a variation of the flow-

focusing microfluidic device in the form of an axisymmetric flow-focusing

microfluidic device (AFFD) (Figure 2-22 A).93 This device was fabricated from

PDMS but rather than using photolithography to create microchannels, glass

microfibres and polyethylene tubing were used to template the design (Figure 2-22

B). The main advantage of an axisymmetric design over the conventional setup was

that AFFD confines droplets to the central axis of the channel, protecting droplets

from shear or damage resulting from adhesion or wetting of the walls of the outlet

channel. Polymer membranes of Nylon-6,6 enclosing aqueous solutions of either

ions or superparamagnetic particles were produced by interfacial polymerisation.

The polydispersity or coefficient of variation (CV) of these microcapsules was

approximately 5 %. The orientation of the device was found to be very important

due to the relative densities of the two liquids, the dispersed phase (water) had a

higher density than the continuous phase (hexadecane). This would lead to the

droplets settling against the floor of the channel where they formed a high volume

fraction emulsion, this altered the flow of the continuous phase providing a higher

resistance to flow in the outlet channel, increasing the pressure in the orifice region.

Through the vertical orientation of the device these problems were avoided (as the

flow and gravity were in the same orientation) (Figure 2-22 C). The interfacial

polymerisation could be quenched by flowing the nylon coated droplets into a

beaker of dodecan-1-ol in hexadecane (Figure 2-22 D).93

60


Figure 2-22. An axisymmetric flow-focusing microfluidic device (AFFD) A: An axisymmetrical

flow focussing channel, composed of a cylindrical tube with a narrow cross-section halfway

along its length. The narrow region serves as the orifice where fluid is focused and breaks into

aqueous droplets. B: A scheme depicting the fabrication process of an AFFD. C: a) The

diameter of droplets at various flow rates of continuous phase. (b & c) images showing droplets

created in the AFFD oriented (b) horizontally and (c) vertically. D: a) A collection of nylon-6,6-

coated aqueous droplets. b) A coated-droplet containing 50 nm diameter magnetic particles in

an applied magnetic field.93

McQuade and co-workers described a simplified microfluidic device that produced

capsules by interfacial polymerisation without the need for any form of

microfabrication.92 This setup required only needles and tubing (Figure 2-23 A); the

continuous phase (30 % w/v aqueous solution of glycerol) flowed through the tubing

and the dispersed phase (3:1 cyclohexane/chloroform with 2 % Tween 80

(surfactant)) was introduced via a 30 gauge needle inserted through the wall of the

tubing into the middle of the channel (Figure 2-23 B). Analogous to Whiteside and

co-workers’ axisymmetric device, droplets produced with the needle and tubing

were entirely surrounded by continuous phase (coaxial flow) with the additional

advantage that any blocking of the device was very simple to overcome by replacing

the tubing and/or needle. Oil filled polyamide capsules were produced by

introducing polyethyeneimine (PEI) to the continuous phase and sebacoyl and

61


trimesoyl chloride to the dispersed phase. By varying the continuous flow rate

capsules between 313 - 865 µm in diameter with a coefficient of variation (CV) of

3.3 - 8.6 % were obtained (Figure 2-23 C).92

Figure 2-23. Interfacial Polymerization within a Simplified Microfluidic Device: Capturing

Capsules. A: Photograph of fluidic device including needle and dye-filled organic droplets

dispersed in the continuous aqueous phase. B: Schematic of fluidic device. C: Light microscope

images of capsules in water formed with constant organic flow rate (0.141 mL min-1) and

increasing aqueous flow rate (clockwise). 92

Kumacheva and co-workers published two papers on the use of microfluidic reactors

for the production of polymer particles.94,95 The first paper demonstrated a novel

approach to the continuous production of core-shell droplets and polymer capsules.

In this work, the microfluidic reactor was fabricated in polyurethane by soft-

lithography to give a MFFD with five separate channels approaching the orifice.

This design allowed three immiscible liquids to be supplied to the flow-focussing

region; the outer channels contained an aqueous solution containing surfactant

62


(sodium dodecyl sulphate) while the middle channels contained the oil phase

(silicone oil) and the monomer phase (tripropyleneglycol diacrylate (TPGDA) or

ethyleneglycol dimethacrylate (EGDMA)) flowed through the centre channel.

Photoinitiator was contained in the monomer phase and SPAN 80 in the oil phase.

Upon a pressure gradient along the long axis of the axis of the MFFD the three

liquids were forced through the orifice and the continuous water phase surrounded

the monomer-oil thread which adopted a circular cross-section (Figure 2-24 A). To

minimise interfacial tension this coaxial jet broke into segments of with a spherical

shape. These monomer droplets were then photopolymerised in the wavy channel

(this shape maximises the curing time with minimum size) of the microfluidic

reactor. By controlling the flow rates of the three phases the diameter of the cores,

the size of the core-shell droplets and the thickness of the shells were controlled.

Generally if the ratio of flow rates of outer to inner phases was increased the size of

droplets was reduced due to the increased shear stress imposed on the undulated jet

of the dispersant liquid. If the flow rate of the aqueous phase was increased smaller

droplets with thinner cores were formed (with thicker shells). While at higher oil

phase flow rates the diameter of the cores increased and the shell thickness

decreased. It was also demonstrated that it was possible to control the number of

cores within the droplets. This was achieved by varying the value of interfacial

capillary wavelength and shifting the length and phases of capillary waves

(undulations). The oil cores could be removed after the photopolymerisation of the

monomer by using acetone to obtain particles with different shapes (Figure 2-24 B).

Overall productivity of the microfluidic reactor was 200 to 1000 s -1 with particles

polydispersity not exceeding 2.5%.94

63


Figure 2-24. Polymer particles with various shapes and morphologies produced in continuous

microfluidic reactors. A: (a) Schematic of production of droplets in MFFD by laminar co-flow

of silicone oil (A), monomer (B), and aqueous (C) phases. (b) Schematic of the wavy channel

used for photopolymerization of monomer in core-shell droplets. (c) Optical microscopy image

of core-shell droplets. The scale bar is 200 µm. (d) Photograph of a PU microfluidic system. The

arrow is pointing to the orifice. B: (a-e) Scanning electron microscopy images of polymer

microparticles obtained by polymerizing monomer in droplets, after removing a silicon oil core.

(Inset) Cross section of the core-shell particle. (f) Cross section of a polymer particle with three

cores obtained by polymerizing core-shell droplets with three cores. Scale bar is 40 µm.94

A second paper by the Kumacheva group95 that was published at the same time was

a comprehensive paper detailing the use of MFFD to produce polymer particles

(Figure 2-25 A). As in the Whitesides and co-workers MFFD publication, these

devices were produced using soft lithography in either poly(dimethylsiloxane)

(PDMS) or polyurethane (PU). Here, four different multifunctional acrylate

monomers were emulsified. Three major regimes in the formation of monomer

64


droplets were found; at low flow rates for the aqueous continuous phase (Qc) and

low flow ratios of continuous to dispersed phases (Qc/Qd) the device operated in a

dripping regime with the monomeric thread breaking into monomer droplets behind

the orifice with droplet size significantly larger than the orifice size. The second

flow regime observed was at moderate values of Qc and Qc/Qd in which the

monomer droplets were generated by the breaking up of the monomer thread in or

behind the orifice, upon releasing a droplet the monomeric thread retracted break

upstream. The final flow regime was jetting mode and this was observed at high

flow rates and high Qc/Qd, the monomeric thread remained behind the orifice

breaking into droplets (Figure 2-25 B). Under certain conditions the formation of

smaller satellite droplets were observed along with the main droplet population. The

size of the droplets produced was shown to be governed by the properties of the

monomer liquid (viscosity and interfacial tension) and the flow rates of the

continuous and dispersed phases. Therefore, the conditions with which monomer

droplets with a very narrow size distribution varied for each monomer and by

varying these conditions along with the design of the MFFD particularly the size of

the orifice droplets (Figure 2-25 C) as small as 18 µm were formed. By adding

photoinitiator to the monomer liquid and exposing the monomer droplets to UV light

after the orifice polymer particles were formed, additionally, it was demonstrated

that by changing the dimensions of the channel in which polymerisation took place

different shape particles could be obtained. These included discs of various

morphologies if the height of the channel was less that the initial diameter of the

monomer droplet or rods if both the height and width of the channel were less than

the diameter (Figure 2-25 D). This paper provided an in-depth study of the use of

MFFDs to produce monodisperse polymer particles.95

65


Figure 2-25. Continuous microfluidic reactors for polymer particles. A:(a) Schematic of droplet

formation in the microfluidic flow-focusing device. (b) Wavy channel for the

photopolymerization of monomer droplets. B: Breakup of the monomer thread in 2 wt %

aqueous SDS solution. (a) Regime 1, (b) Regime 2, (c) Regime 3. C: SEM image of polymer

particles. Scale bar is 100 µm. D: Schematic (a-c) and optical microscopy (a’-c’) images of

polymer particles with different shapes: microspheres (a, a’), disks (b, b’), (c, c’) and rods of

obtained via photopolymerization of droplets. Scale bar is 50 µm.95

An alternative microfluidic reactor has been described by Goddard et al,96 this

system utilises a 35 µm diameter hole through which the dispersed phase was

introduced into spiral channel where photopolymerisation took place (Figure 2-26 A

& B). This device was fashioned from a polycarbonate sheet using a precision

milling machine. Using this device it was possible to produce poly(methacrylate)

(pMA) particles with a low CV (typically around 2%) in a range of diameters

between 10-115 µm (Figure 2-26 C). Additionally, it was possible to produce

molecularly imprinted particles through the incorporation of propranolol into the

monomer solution prior to polymerisation, these particles showed the same uptake

of propranolol as particles produced by conventional suspension polymerisations.96

66


Figure 2-26. A micro-reactor for preparing uniform molecularly imprinted polymer beads A:

Schematic of the spiral micro-flow-reactor showing the overall layout and the sample and oil

inlet points. B: Detail of the tapered region of the reactor where the monomer is introduced into

the flowing continuous phase. Monomer is extruded into the flowing oil, where it breaks off into

droplets. These are swept into the spiral polymerisation reactor where they are polymerised by

UV light. C: SEM of typical particles produced in oil using the micro-reactor.96

A different route to prepare near monodisperse particles was demonstrated by

Hadziioannou and co-workers,97 this was an axisymmetrical needle/tubing device

that could be prepared without the need for any microfabrication techniques (Figure

2-27 A). Within conventional planar microfluidic devices surface modification is

usually required to prevent an inverse emulsion. The axisymmetrical setup overcame

this requirement by avoiding direct contact of the dispersed phase with the channel

walls.93 A simple T-junction and needle were used to introduce the dispersed phase

into the continuous phase, both removing the need for microfabrication and avoiding

the clogging problems that microchannels often suffer from. By varying the flow

rates and solvent viscosities they were able to produce poly(methylmethacylate)

(poly(MMA)) particles with a narrow distribution between 150-550 μm in diameter

(Figure 2-27 B & C).97

67


Figure 2-27. A predictive approach of the influence of the operating parameters on the size of

polymer particles synthesized in a simplified microfluidic system. A: Schematic of the

microfluidic system for the synthesis of controlled-size polymer particles. The dispersed phase

is injected via a thin needle positioned along the main axis. B: Effect of flow rate ratio of

continuous and dispersed phases for two different continuous phase flow rates on average

diameter, Dp, of polymer particles. C: Effect of flow ratio and continuous phase viscosity on the

average diameter of polymer particles.97

2.5.1. Summary

Microfluidic polymerisation techniques offer a highly controlled approach to the

preparation of polymer particles. There are a number of different configurations

available that allow particles to be produced with a very narrow size distribution

(generally with polydispersities less than 2.5 %). Additionally, by introducing

further co-flowing solvents it is possible to make particles with very well defined

morphologies. However, there are a number of limitations relating to the preparation

of polymer particles based on microfluidics. These include: the overall production

68


rate of particles is low. Microfluidic devices cannot be ‘scaled up’ (although this

might be addressed though the combination of many microfluidic reactors in

parallel). Additionally, microfabrication techniques are required to construct many

microfluidic devices (those fabricated by soft lithography) making these approaches

inaccessible for many researchers.

Overall, microfluidics presents a versatile method for the small scale production of

polymer particles with very narrow size distribution and desired morphology.

69


2.6. Aims of thesis

The overall objective of this thesis was to synthesise and characterise enzyme

responsive hydrogel particles functionalisation with peptide actuators. These

functionalised particles were then to be applied to the triggered release of a

macromolecule payload.

Firstly, PEGA microparticles (µPEGA) were to be produced and characterised with

the aim of establishing the optimum support of the enzyme responsive system.

Previous approaches to modifying the properties of PEGA such as the introduction

of charged monomers74,75 were to be assessed for µPEGA. Particles are then to be

modified with peptide actuators57,58 in order to demonstrate enzyme responsive

release of an entrapped payload.

Peptide actuators with enhanced functionality were to be designed and incorporated

into µPEGA, the enzyme responsive behaviour of this system could then be

characterised and utilised for the triggered release of an entrapped macromolecule.

Finally, based on the literature, a simplified microfluidic device97 was to be used

produced and used to prepare PEGA particles with the smallest diameter possible

and a very narrow size distribution.

70


3 PEGA polymerisation, characterisation and enzyme

responsive swelling through functionalisation with

peptide actuators†

3.1. Abstract

Within this chapter the polymerisation of PEGA hydrogel microparticles (µPEGA)

with either neutral, positive or negative charge were demonstrated. The swelling of

these different particles was characterised and neutral µPEGA were selected for

further investigation. These particles had a mean diameter of 16 µm (similar to that

of biological cells) and were compatible with different enzymes. Furthermore, it was

demonstrated that enzyme catalysed reactions occur faster with these microparticles

than with commercially available macrobeads which are typically 200-400 µm in

diameter. μPEGA was then functionalised with peptide actuators. These particles

demonstrated an enzyme specific increase in accessibility allowing fluorescently

labelled dextran to diffuse into the particles. The pH responsive nature of the linear

peptide actuators was shown and utilised for the physical entrapment of a

macromolecule payload. Release of this payload was only possible when the

functionalised particles were exposed to the target enzyme. However, at

physiological ionic strength no response of the particles was observed due to

electrostatic screening.

3.2. Introduction

PEGA is a polymer hydrogel that was developed as a resin for solid phase peptide

synthesis (SPPS). A particularly attractive property of this material is its

compatibility with both aqueous and organic solvents. This allows for easy chemical

modification through SPPS67 as well as compatibility with aqueous solutions

containing biomolecules. There has been thorough research into using enzymes, † Published in part as: McDonald, T. O., Christensen, S., and Ulijn, R. V., Making peg-based

microparticles for applications in biology and medicine. Mater. Res. Soc. Symp. Proc 1008E (T05),

18 (2007).

71


specifically proteases to catalyse reactions on peptides synthesised on PEGA.63,72,74

Several workers have shown that PEGA is accessible to small enzymes and that

enzyme activity can occur inside the resin.63,69,73,98 PEGA polymers possess a highly

crosslinked network which gives the material a molecular cut-off weight, above

which molecules are not able to diffuse in or out of the polymer.63 Further details on

the development and further investigation of enzyme reactions on PEGA polymers

are outlined in section 2.4 within the literature review.

Currently, PEGA particles are commercially available between 200 - 400 µm in

diameter, for convenience in solid phase chemistry applications.72 Depending on the

biomedical applications smaller size ranges are desirable (such as injected into tissue

(<200 μm), inhaled (<100 μm) or released into circulation (<10 μm).99 Smaller

particles also offer an increase the rate of response due to their greater surface area

to volume ratio.55 Additionally, microparticles allow for possible use in automated

analysis of ‘on-bead’ libraries using a cell sorter. Therefore, PEGA microparticles

were prepared that are smaller in size, thereby enhancing responses, but still

conveniently handled for solid phase synthesis and analysis by fluorescence

microscopy.100 Work carried out in the author’s MSc project investigated the effect

of stirring speed and surfactant concentration on mean particle size.101

Numerous stimuli have been exploited in the development of responsive materials in

a biomedical context including pH,102 temperature,9 ionic strength35 and small

molecules (e.g. glucose).36 Bioresponsive materials21 are stimuli-responsive surfaces,

self-assembled structures or chemically crosslinked polymers that change their

properties in response to biochemical recognition events, offering potential

applications in biosensing,103 tissue regeneration28 and controlled release.54 The

responsiveness of these materials is determined by a biorecognition moiety that,

upon a recognition event, actuates structural changes in the material. In systems

established using hydrogels there are three main categories of molecular actuation

based on changes in: (i) crosslinking density, 11,27,28,41 (ii) electrostatic

interactions36,56,57 or (iii) molecular conformation changes.65,66 These systems have

been discussed in detail in section 2.3.

72


There has been significant interest in exploiting enzyme catalysed reactions as the

stimulus to trigger molecular actuation in polymer hydrogels.8,51,104,105 Enzymes

generally function under mild conditions and possess a high degree of selectivity.

Also, they are vital to the function of all living systems, catalysing and controlling

healthy and diseased biological processes. In particular proteases (enzymes that

hydrolyse peptide bonds) have been shown to be specific markers in many disease

states including cancers13 and chronic wounds.106

Most enzyme responsive systems that have been described make use of enzyme

cleavable linkers which covalently attached the drug to a polymer whereby enzyme

action detaches the drug.107-109 An alternative approach through the development of

peptide actuators has been shown by Ulijn and co-workers. 57,58 These actuators

constructed by SPPS on PEGA, have the ability to release entrapped payloads in

response to a specific enzyme by exploiting changes in electrostatic interactions

resulting in an increase in the swelling of the polymer (shown schematically in

Figure 3-28). This method of release offers advantages over pro-drug approaches:

There is no need to covalently modify the drug molecules and drug release is not

directly governed by enzyme kinetics, i.e. instead of the enzymatic hydrolysis of one

bond corresponding to the release of one drug molecule. This system allows for a

variable number of payload molecules to be released upon enzymatically induced

swelling. Using designed peptides allows the possibility to develop a range of

different actuators using the varied functionalities that are offered by nature’s

building blocks, for example the benefit of designing the actuator to match payload

charge has previously been shown (described within section 2.3.3.2).58

73


Figure 3-28. Schematic of the enzyme responsive swelling of PEGA particles functionalised with

linear peptide actuators. The linear peptide actuator is made up of 2 parts, a sensor and an

actuator. The actuator consists of the 2 oppositely charged amino acids (a zwitterion) and the

sensor is the amino acids between the charged residues. The sensor is hydrolysed by an enzyme

with the correct specificity; therefore it is termed the enzyme cleavable peptide or ECP.

Therefore, the aims of this chapter were to: (i) Produce and characterise PEGA

microparticles (µPEGA) and to investigate the effect of changing the chemical

structure of PEGA on its swelling behaviour. (ii) Study the compatibility of µPEGA

with enzyme activity using TPM. (iii) To establish the ideal experimental conditions

to produce PEGA particles that offer the suitable properties (loading and particle

size distribution) for use in the enzyme responsive release systems. (iv) Incorporate

peptide actuators on µPEGA, (v) characterise the enzyme specific changes in

accessibility of the functionalised particles, (vi) demonstrate the possibility of

loading these particles with a payload and (vii) use enzymes to trigger specific

release of the payload.

74


3.3. Experimental

3.3.1. Materials

All chemicals were used as supplied from Sigma with the exception of amino acids

(Bachem) and O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate (HBTU) (AGTC Bioproducts Ltd). The enzymes used were

thermolysin (EC 3.4.24.27), 36.5 U/mg and chymotrypsin (EC 3.4.21.1), 60 U/mg

(both were supplied by Sigma).

3.3.2. Inverse suspension polymerisation

μPEGA was prepared by inverse suspension polymerisation. A stainless steel baffle-

less reactor (250 ml) stirred with an anchor-style agitator was used for the

polymerisation reactions. 3.14 g (3.5 mmol) of the PEGA800 macromonomers (a 2:1

(molar) mixture of acrylamide-PEG-acrylamide and amino-PEG-acrylamide) was

dissolved in 10 ml of distilled water along with 0.156 g (2.2 mmol) of acrylamide

(the chemical structures of the monomers are shown in Figure 3-29). This solution

was then purged for 30 minutes with N2 gas. 50 ml of Isopar M (isoparaffin) was

added to the reactor and was also purged for 30 minutes. The reactor was heated to

80°C. After 20 minutes of purging 0.16 ml (1.0 mmol) of N,N,N ′,N′-

tetramethylethylenediamine (TEMED) was added to the oil phase, and 0.156 g (2.2

mmol) of acrylamide to the dissolved macromonomer solution. 0.164 g (0.47 mmol)

of Span 20 (sorbitan monolaurate) was dissolved in the oil, which was stirred at 500

rpm for 30 seconds to ensure the surfactant was fully dispersed in the oil phase.

0.070 g (0.30 mmol) of ammonium persulfate (APS) was dissolved in the

macromonomer solution, which was added to the oil phase in the reactor which was

stirred at 2000 rpm for a further 30 minutes. The particles were washed with (3 x 50

ml) dichloromethane (DCM), (3 x 50 ml) Tetrahydrofuran (THF), (3 x 50 ml)

methanol and (4 x 50 ml) distilled water (retained by centrifugation at each step).

3.3.3. Charged PEGA polymerisation

Particles that incorporate permanent charges were prepared by substituting the

acrylamide with either (3-trimethylammonium chloride) propyl acrylamide to

produce positive particles or 1,1-dimethyl-2-(sulphonate) ethyl acrylamide to give

75


negative particles (the chemical structures of these monomers are shown in Figure 3-

29). All remaining aspects of the polymerisation were the same as with the inverse

suspension of neutral particles

Figure 3-29. Monomers used in the synthesis of PEGA and its charged variants. From the top

down: amino-PEG-acrylamide, acrylamide-PEG-acrylamide, acrylamide, (3-

trimethylammonium chloride) propyl acrylamide and 1,1-dimethyl-2-(sulphonate) ethyl

acrylamide.

3.3.4. Microscopy and particle size analysis

An optical microscope was used to acquire images of the particles and

environmental scanning electron microscopy (ESEM) images were taken on a FEI

Quanta 200 ESEM, using ESEM low vac mode at 10.0 KV. Size distribution

analysis of the microparticles was carried out using a Malvern Mastersizer particle

size analyser. Deionised water was used to fill the small volume dispersion unit and

the dispersion control unit set to 1500 rpm, the polymer particles were added until

an obscuration value of above 10% was obtained. Mastersizer Microplus software

version 2.18 was used to analyse the results and plot distribution graphs.

76


3.3.5. Solid phase peptide synthesis

Peptides can be prepared synthetically in good yields, solid phase peptide synthesis

(SPPS)68 is a popular synthetic method used to achieve this. Here, amine

functionalised crosslinked polymers (resins) are reacted with amino acids (with

protected their amines protected) resulting in amide bond formation (shown

schematically in Figure 3-30, while the chemical reactions used in this work can be

seen in Figure 3-31). The removal of the protecting group allows for this process to

be repeated in a step-wise process to build up the desired peptide.

Figure 3-30. Solid phase peptide synthesis scheme. A: A scheme of the step by step process in

the synthesis of a peptide from protected amino acids. B: The key, (left) amines are present in

PEGA particles, (right) Fmoc protected amino acid.

77


Figure 3-31. Chemical reactions involved in peptide synthesis. A: Formation of an amide bond.

Firstly, the carboxyl of the amino acid/peptide reacts with O-(Benzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium hexafluorophosphate (HBTU) in the presence of N,N-

diisopropylethylamine resulting in an activated ester. The amine of the free amino acid then

reacts with the ester resulting in amide bond formation. B: The deprotection of the Fmoc

protected amine group with piperidine.

3.3.5.1. Solid phase synthesis of dipeptides and enzyme treatment

To quantify the loading of the microparticles Fmoc Amino acids (3 equiv.) were

coupled to PEGA particles using DIC (di-isopropyl carbodiimide) (6 equiv.) and

HOBt (hydroxybenzotriazole) (6 equiv.) in DMF. The first coupling was performed

for 3 hours and the second overnight. A roller mixer at room temperature was used

to agitate the solutions. Between steps the resin was washed extensively using 5 ml

volumes of 5 x MeOH, 5 x 50 : 50 (v/v) DMF : MeOH and 5 x DMF. A 20 %

78


solution of piperidine in DMF was used for Fmoc deprotection for 2 hours. Enzyme

reactions: 1.5 mg of thermolysin per 1 ml 0.01 M phosphate buffer solution of pH

7.5, reactions were at room temperature on a roller mixer. To determine how quickly

the enzyme catalysed hydrolysis has occurred, a thermolysin solution was added to

the Fmoc-A-A modified particles the reaction was then stopped with 0.1%

trifluoroacetic acid (TFA) in deionised water at seven different times and analysed

with high performance liquid chromatography (HPLC) to quantify any cleaved

residues.

3.3.5.2. Solid phase peptide synthesis of peptide actuators

Peptide actuators (Fmoc-DAAR-PEGA) were prepared by solid phase peptide

synthesis using Fmoc protected amino acids. 8 equivalents of the amino acid and 7.8

equivalents of HBTU were dissolved in 2 ml of N,N-Dimethylformamide (DMF). 16

equivalents of N,N-Diisopropylethylamine (DIPEA) was added to this solution prior

to its addition to the PEGA particles. The coupling reaction was left for 16 hours,

and the Kaiser test26 was used to ensure complete coupling. Deprotection was

achieved using 20 % piperidine in DMF for 2 hours. This cycle was repeated to

build up the required peptide sequence with thorough washing between steps (5 x 5

ml methanol, 5 x 5 ml 1:1 methanol:DMF, 5 x 5 ml DMF). A solution 95 %

Trifluoroacetic acid (TFA) 5 % water was used to remove the side chain protecting

groups.

3.3.6. HPLC

HPLC experiments were undertaken on a Dionex HPLC (P680 pump, ASI-100

Automated sample injector, Nucleosil 100-5-C18 column with a UVD170U

detector), using a solvent ramp of 20 % ACN and 80 % water to 80 % ACN 20 %

water over 30 minutes (0.1 % TFA was present in both phases) shown in Figure 8-

66. Chromeleon 6.60 software was used for analysis.

3.3.7. Two-photon microscopy

The distribution of amine groups and homogeneity of enzyme hydrolysis were

assessed with TPM. Firstly, three differently modified batches of particles were

prepared; unmodified, Fmoc-A-A (prepared with the previously described SPPS

method) and Fmoc-A-A treated with thermolysin solution. 0.1 g of these particles

79


were then placed in a filter column and rinsed with DMF (3 x 5 ml). 0.036 g (0.133

mmol) of dansyl chloride was dissolved in 2 ml of DMF and 25 μl (0.143 mmol)

DIPEA was then added to this solution. This coupling solution was then added to the

PEGA particles and put on the blood rotator in the dark for 2 hours. The polymer

was then rinsed with DMF (3 x 5 ml), ethanol (3 x 5 ml) and water (3 x 5 ml). TPM

was used to take visual cross-sections of the particles to determine the distribution of

free amine groups. A Ti:Sapphire laser was tuned to 770 nm and fluorescence from

the sample was filtered using a 525/550 nm filter.

3.3.8. Determining particle swelling

The neutral, positive and negative PEGA microparticles were swollen in water or

buffer 0.1 M (pH 7.5) and centrifuged at 3000 rpm. 0.50 g of the hydrated polymers

were weighed out and placed in a Gallenkamp vacuum oven at room temperature at

100 mbar. Every three hours the polymers were weighed, and when no further

weight loss was recorded this mass was taken to be the dry mass.

3.3.9. Assessing accessibility

To analyse the change in accessibility of the particles a 2 mg/ml solution of 40 kDa

fluorescently labelled dextran was prepared in water. Approximately 50 mg of

PEGA particles were added to these solutions. 10 minutes was left to allow diffusion

into the hydrogel prior to imaging. The confocal microscope used was a Lecia sp2

AOBS. The laser was an Argon laser tuned to 488 nm, the fluorescence from the

sample was filtered to leave 490-550 nm.

3.3.10. Entrapping payload

The particles (0.1 g) were loaded with a 40 kDa FITC Dextran using the following

method; 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg/ml) was added to

the particles for 30 minutes (pH 2.5). After this time 0.4 ml of 0.05 M NaOH was

added dropwise (pH 7). The particles were then filtered and washed (2 x 5 ml water,

2 x 5 ml 0.18 M buffer, 2 x 5 ml methanol).

3.3.11. Fluorimetry

The release of entrapped dextran was determined using the following method;

loaded particles were treated in solution for 80 minutes, these samples were

80


centrifuged and the supernatant taken off. A Jasco FP – 6500 Spectrofluorometer

using an excitation wavelength of 490 mn was used to obtain the fluorescent

intensity at 519 nm. Treated particles were filtered and imaged using fluorescence

microscopy.

3.3.12. Microscopy and determination of swelling

Swelling measurements were obtained using a Zeiss Imager A1 microscope, a

Leistungselektronik mbq 52 AC power source (Jena, Germany) equipped with a

Canon Powershot G6 camera. For pH responsive swelling the diameter of a

minimum of 300 particles was measure and the mean volume calculated. For

enzyme responsive swelling individual particles were observed throughout the time

course and ImageJ 1.38x analysis software was used to determine the change in

particle diameter. An average V/Vo was determined from at least six representative

particles, where V is the volume of the particle at time t and Vo is the volume of the

particle immediately after exposure to enzyme solution. Enzyme solutions were

made up at a concentration of 1 mg/ml and buffers were prepared by using the

appropriate amounts of sodium phosphate dibasic heptahydrate and sodium

phosphate monobasic.

81


3.4. Results and Discussion

3.4.1. Production of µPEGA800

PEGA microparticles (µPEGA) were prepared by inverse suspension polymerisation

using thermal initiators.67 The water soluble monomers were dispersed along with

the initiator in the oil phase, which contained the co-initiator and the surfactant

(Figure 3-32). Upon stirring monomer droplets were formed and undergo continuous

break-up and coalescence, this leads to a dynamic equilibrium being established

giving a stationary mean droplet size.110 The size and the distribution of the particles

formed were controlled by the imposed shear field within the reactor and the

stabilisation effect of the surfactant. Currently PEGA particles are commercially

available in size ranges above 150 μm for ease of handling in SPPS. Depending on

the biomedical applications smaller size ranges are desirable (such as injected into

tissue (<200 μm), inhaled (<100 μm) or released into circulation (<10 μm).99 Smaller

particles also offer the potential to give faster response times due to their greater

surface area to volume ratio (as shown by Peppas and co-workers).55 In previous

work, the stirring speed and the surfactant concentration were varied in order to find

the optimum conditions to produce microparticles.101 There are three monomers used

in the preparation of PEGA; aminoacrylamido PEG, bisacrylamido PEG and

acrylamide. The amino functionalised monomer imparts the amine functional group

or ‘handle’ on a flexible chain allowing easy modification by solid phase peptide

synthesis (SPPS). The bisacrylamide PEG was the crosslinking agent in the

polymerisation while the acrylamide acts as a spacer to separate the PEG based

monomers. The PEG macro-monomers used had an average molecular weight of

800 g/mole, the molar mass of the PEG chain in the resulting cross-linked

polyacrylamide-graft-PEG co-polymer. This structure leads to PEGA’s well defined

molecular weight cut-off, 63 with molecules above this unable to diffuse in or out of

the polymer matrix.

82


Figure 3-32. Polymerisation of PEGA particles by inverse suspension polymerisation. The

monomers; acrylamide, aminoacrylamido PEG and bisacrylamido PEG along with the initiator

APS were dissolved in water and dispersed in an oil phase containing surfactant and co-

initiator. Upon stirring, monomer droplets are formed and undergo continuous break-up and

coalescence and a dynamic equilibrium is established giving a stationary mean particle size.

3.4.1.1. Particle morphology and size characterisation of µPEGA

In order to produce μPEGA with the desired size distribution a stirring speed 2000

rpm was employed with 3.28 mg/ml of surfactant in the oil phase. These particles

had a mean diameter of 15 µm. Further investigation was carried out on these

particles, including analysis of the size distribution, determining the loading capacity

and its homogeneity throughout the particles as well as their compatibility with

enzymes. Study of the particles by optical microscopy (Figure 3-33 A) showed that

the microparticles are separate spherical entities, with no obvious surface detail or

texture on the sub-micron scale between the range of about 5-25 µm. The data

provided by particle size analysis (Figure 3-33 B) gave a mean particle diameter of

15 µm with a polydispersity ((standard deviation/mean particle diameter) x 100) of

43 %. Images of the particles obtained by environmental scanning electron

microscopy (ESEM) (Figure 3-33 C & B) concurred with the optical microscopy.

The ESEM images showed that the majority of particles have diameters between 1-

83


10 µm. This difference in size measurement is due to the low pressure within the

ESEM chamber resulting in removal of water from the particles (hence reducing the

particle diameters). Individual spherical PEGA microparticles with a mean diameter

of 15 µm and polydispersity of 43 % were produced as a result of the combination of

shear forces and stabilisation effect of surfactant on the droplets within the reactor.

Figure 3-33. A: Optical micrograph of microparticles is water. B: Size distribution of

microparticles in water obtained by particle size analyser. C and D: ESEM micrographs of

PEGA microparticles. (Scale bar in all figures is 20 µm).

3.4.2. Production of µPEGA+ and µPEGA-

Other researchers74-77 have shown that by modifying the chemical structure of PEGA

to include permanent charges within the acrylamide backbone improved swelling,

accessibility and yields in enzyme catalysed reactions. This change in properties is

due primarily to the electrostatic repulsion between neighbouring charges in the

polymer structure but also offers the possibility to use electrostatic interactions

between the enzyme and the polymer. By substituting the acrylamide in PEGA with

an acrylamide-based monomer carrying a permanent charge it maybe possible to

introduce a charge into the backbone of the polymer. Two different monomers were

84


used; (3-trimethylammonium chloride) propyl to produce positively charged PEGA

(PEGA+) and 1,1-dimethyl-2-(sulphonate) ethyl to give negatively charged PEGA

(PEGA-) (Figure 3-34). To determine whether the conditions used to polymerise the

neutral PEGA800 microparticles would successfully produce charged particles,

positive and negative PEGA polymers were prepared and analysed for mean

diameter and size distribution.

Figure 3-34. Chemical structure of PEGA and its charged variants.75

3.4.2.1.1 Particle morphology and size characterisation of µPEGA+

The inverse suspension polymerisation of the PEGA monomers with the acrylamide

substituted with (3-trimethylammonium chloride) propyl acrylamide produced

PEGA+ particles.74 Optical and ESEM microscopy images (Figure 3-35) show the

positively charged particles produced are highly aggregated with a narrow size

distribution. Based on optical micrograph analysis the particle diameter were

approximately 10 μm, whilst analysis of the ESEM images indicates the particles

had a diameter of approximately 8 μm. Much like the neutral PEGA800 particles this

was because the polymer is not fully hydrated within the ESEM chamber. When the

85


pressure within the ESEM chamber was further lowered there was an appearance of

a textured surface, this was water being drawn out of the hydrogel onto the surface

prior to evaporating (Figure 3-35 D). The positively charged particles have no

apparent surface features or texture on the sub-micron level. The aggregation of the

particles meant that determining a size distribution using the particle size analyser

did not yield any quantative information. The PEGA+ particles had an aggregated

structure which may have be due to incomplete polymerisation during stirring

leading to coalescence of the partially polymerised monomer droplets upon settling.

It is possible that the negatively charged initiator (ammonium persulfate) cancelled

the electrostatic repulsion between the positively charged polymerising monomer

droplets. This may have removed the electrostatic stabilisation within the system

allowing the partially polymerised particles to aggregate during the sticky period of

the reaction.111

Figure 3-35. PEGA+ microparticles. A & B: ESEM micrograph of positively charged PEGA

particles (scale bar in A is 50 μm and in B is 10 μm). C: Optical micrograph of positively

charged PEGA particles (scale bar is 15 μm). D: ESEM micrograph of positively charge PEGA

particles, arrows indicate examples of water droplet forming on the surface as the pressure is

reduced (scale bar is 5 μm).

86


3.4.2.1.2 Particle morphology and size characterisation of µPEGA-

By carrying out the inverse suspension polymerisation reaction with 1,1-dimethyl-2-

(sulphonate) ethyl acrylamide replacing the acrylamide (with all other conditions

kept the same) PEGA- was prepared.74 Optical microscopy of these negative

microparticles (Figure 3-36) produced images similar to μPEGA although with a

slightly larger and more polydisperse. Unlike the PEGA+ there was no aggregation

visible. Data produced from the particle size analyser agrees with this comparison (a

mean diameter of 20 μm with a polydispersity of 59 % compared to 15 μm and 43 %

respectively for μPEGA). It is likely that the different between PEGA800 and PEGA-

is due to batch-to-batch variation. PEGA- particles were produced with morphology

similar to that of neutral PEGA.

Figure 3-36. Right: Optical micrographs PEGA-, scale bar is 20 μm. Left: Size distribution of

PEGA-.

3.4.2.1.3 Comparison of swelling the different types of PEGA

The swelling properties of a hydrogel are dependent on the ability of the material to

absorb water. This ability is determined of a number of structural factors such as:

crosslinking density, hydrophilicity of the polymer and the number of

polar/ionisable groups. Greater swelling may be advantageous as in certain cases,

greater swelling of the network results in increased mesh size of the polymer and

therefore accessibility. The uptake of water and 0.1 M buffer (pH 7.4) into the

particles was determined for dry particles from each of the three different PEGA

polymers. This was achieved by measuring the dry and hydrated masses of the

87


particles, providing information about polymer’s interactions with water

(hydrophilicity) and neighbouring polymer chains (Figure 3-37).

Figure 3-37. The uptake of water by the three different types of dry PEGA microparticles and

the effect of ionic strength on the swelling of the three different types of PEGA microparticles.

Blue; uptake of water, red; uptake of 0.1 M buffer.

The positively charged particles gave the highest values for uptake of water because

of the electrostatic repulsion between chains resulted in an osmotic driving force

drawing more water into the polymer. The swelling of negative PEGA was much

less than seen for PEGA+, this is likely due to interactions between the amines

present (positively charged at pH below 7.5 in a test solution (water) which has a pH

of 6.5) and the negatively charged acrylamide. This finding is in agreement with

published work in which it was shown that PEGA- swelled less than PEGA+.75

Increasing the ionic concentration of the uptake solution resulted in a decrease in

swelling in all three polymers. This observation was a result of the dissolved ions

associating with the oppositely charged permanent groups with the effect of

screening the charges reducing the effect of electrostatic repulsion. This reduction

was least pronounced with PEGA800 this is because this polymer has the lowest

density of charges (only the protonated pendant amines). Indeed, the charge

densities of within the three different polymers were calculated to be 0.46, 0.37 and

88


1.2 mmol/g for PEGA-, µPEGA and PEGA+ respectively. A similar trend was found

by other researchers on PEGA1900, and charged PEGA macroparticles.75 Although

they found that increasing the ionic strength with neutral PEGA system showed ‘no

measureable effect’ on swelling. This difference may be the result of the

experiments being carried out on PEGA1900 whereas this work was using PEGA800

which has double the loading and therefore charged amines. Overall, the charged

PEGAs particularly PEGA+ had greater swelling in water than µPEGA. Increasing

the ionic strength of the solution absorbed by the particles reduced the swelling of

all three polymers.

3.4.3. Further characterisation of µPEGA

µPEGA was selected as the most promising polymer for the incorporation of the

enzyme responsive functionalisation. PEGA+ and PEGA- offered greater swelling

and have been demonstrated in the literature to have higher accessibilities than

neutral μPEGA. An important criterion for the material selection is accessibility; it

must high enough to allow the target enzyme to diffuse into the particles while being

low enough to entrap the payload molecules. Neutral µPEGA offered this, as well a

simpler base to understand the effect of peptide actuator design of particle swelling.

Therefore, further characterisation was carried out on µPEGA.

3.4.3.1. Amine characterisation by two-photon microscopy and enzyme

compatibility

The ease with which PEGA could be chemically modified with amino acids or

peptides by SPPS and compatibility with aqueous environments makes it a

particularly interesting material. This capacity for functionalisation is determined by

the loading; the amount of amines per unit mass of polymer, usually quoted in

mmol/g. In order to access this property, µPEGA was functionalised with an Fmoc

protected amino acid using standard DIC/HOBT chemistry was undertaken. Within

SPPS, piperidine is used to remove the Fmoc protecting group. These Fmoc groups

were quantified by HPLC against Fmoc standards and the loading was found to be

0.37 mmol/g of (dry) µPEGA. This is in close agreement with the declared loading

of commercially available PEGA800 macrobeads (0.4 mmol/g). Furthermore, the

89


distribution of the amine groups was analysed by TPM.79 Here, the amine groups

within the particles were reacted dansyl and the spacial distribution of the

fluorophore determined. In TPM the sample is irradiated with a laser with a

wavelength approximately twice that of the excitation length wavelength of the

fluorophore. This means that excitation can only occur when two photons are

absorbed simultaneously. This is extremely unlikely and therefore only occurs at

very high photon density, the focal point of the laser beam. This method of imaging

is well established for the characterisation of PEGA particles as the long wavelength

of the infrared excitation laser gives good penetration into solid objects allowing for

optical cross-sections within the z plane of the particles to be obtained.79,82 The TPM

analysis of the µPEGA are shown in Figure 3-38. Here, an optical cross-section

obtained was at the equatorial plane (the widest point) and fluorescence indicates

where amines were present. Firstly, unmodified µPEGA were treated with dansyl

chloride and imaged. From Figure 3-38 A it is clear that the free amine groups are

homogeneously distributed throughout the particles. Other microparticles were

functionalised with the protected dipeptide; Fmoc-AA and some then treated with

dansyl chloride and imaged (Figure 3-38 B), it can be observed that 90 % of the

amine groups have been functionalised with the protected dipeptide.

The enzyme compatibility of µPEGA was then assessed. Here, Fmoc-AA

functionalised particles were also exposed to a solution of the enzyme thermolysin

for 12 hours before being reacted with dansyl chloride and imaged. Thermolysin, a

protease which has been used previously on PEGA particles was used as the model

enzyme. 62,63,75,85 Thermolysin is a proteolytic enzyme with a well-known specificity

for hydrophobic amino acids in the P’1 and little specificity for the P1 position.112

Additionally, thermolysin is known to be active on peptides linked to PEGA

particles.62,79. Figure 3-38 C shows that ~70 % of the original number of amine

groups available (due to the cleavage of the peptide bond) and that enzyme action

occurs evenly throughout the interior of the particles. This result agrees with HPLC

results, in that >100 minutes the enzyme cleaves a maximum of around 70 % of the

peptide bonds.

90


Figure 3-38. Assessment of the distribution of the amines within μPEGA by TPM. A: Schematic

of the process used to determine the homogeneity of free amine groups and enzyme activity

using dansyl labelling. Three different batches of particles were prepared: a) unmodified and

labelled with dansyl-chloride, (b) functionalised with Fmoc-AA then labelled with dansyl-

chloride and (c) functionalised with Fmoc-AA then treated with thermolysin and labelled with

dansyl-chloride (scale bar is 15 μm). B: Two photon micrographs (TPM) of representative

microparticles for each of the different treatments. C: Cross-section of the intensity of

fluorescence in each of the 3 microparticles

3.4.3.2. Comparison of enzymatic hydrolysis within µPEGA800 and macroparticles

Enzyme hydrolysis is the process that initiates a responsive change in particle

swelling (when peptide actuator functionality has been incorporated). Therefore, the

rate at which enzyme hydrolysis occurs within the PEGA particles was the main

factor determining the response time of these functionalised particles. In order to

assess how quickly the enzyme catalysed hydrolysis occurs, a thermolysin solution

91


was added to Fmoc-AA modified particles. The reaction was then stopped with

0.1% TFA at different times and analysed with HPLC to quantify any cleaved

residues. The Figure 3-39 shows that a maximum hydrolysis of the dipeptide was

attained after approximately 110 minutes for µPEGA. This experiment was repeated

on larger (200-400 µm diameter) commercially available PEGA800 macroparticles,

where maximum hydrolysis was found after 150 minutes with an initial rate three

times slower than the enzyme reaction on the microparticles. Other workers79 have

also shown that on larger commercially available particles maximum hydrolysis was

obtained after a much longer time, typically ~ 4 hours.

Figure 3-39. Comparison of time dependence of enzyme reactions on ♦ (blue): µPEGA and ■

(red): commercially available macroparticles (curves are guides to the eye).

3.4.4. Functionalisation with peptide actuators

Enzyme responsiveness was incorporated into PEGA microparticles through SPPS.

The peptide actuator functionality is shown schematically in Figure 3-28. These

peptide actuators are made up of four amino acids and which can be divided into two

sections based on their role; in the centre of the peptide actuator is an uncharged

dipeptide (shown in green) this is the sensing element the enzyme cleavable peptide

(ECP), while at each end of the peptide actuator is an oppositely charged amino acid

92


(making the peptide actuator a zwitterion). If a protease with specificity for the ECP

is present it hydrolyses the amide bond in the ECP and only the positively charged

half of the peptide actuator remains covalently attached to the polymer.

Additionally, dependant on the pH of the surrounding solution (if below pH 7.5) the

amine on the remaining half of the ECP may become protonated and therefore

positively charged. Within the polymer network these neighbouring cationic

fragments of the peptide actuators electrostatically repel one another resulting in an

increase in the swelling of the polymer particles. These peptide actuators have

already been shown on macroparticles (this is covered in more detail in the

preceding literature review, within section 2.3.3.2). By making use of the faster rates

of complete hydrolysis observed on microparticles it should be possible to

demonstrate a faster enzyme responsive system.

3.4.4.1. Enzyme responsive increase in accessibility

Enzymes are highly specific and in order to determine whether this specificity could

be used to for selective release, the change in accessibility of the microparticles was

measured after exposure to three different enzymes. µPEGA was firstly

functionalised with peptide actuator with three different ECPs, these were AA, GG

and FF. The change in accessibility upon enzyme treatment was then determined by

immersing the particles in a solution containing FITC labelled dextrans of known

molecular weight, then using confocal microscopy to visualise the location of

fluorescence. Confocal microscopy allows fluorescent cross-sectional images of the

particles to be obtained. This technique can have problems with quenching occurring

in the centre of hydrogel particles79 however, because of the small size of the

microparticles quenching was not observed and the technique is suitable for this

purpose.

The three proteolytic enzymes tested were Thermolysin, Chymotrypsin and Elastase.

Each of these enzymes has a different specificity (i.e. the composition of the

peptides that they will hydrolyse), details of these specificities and the molecular

weight of the enzymes is shown in Table 3-4. Thermolysin, a thermostable

metalloproteinase produced in the culture broth of Bacillus thermoproteolyticus113

has a well-known specificity for hydrophobic amino acids in the P’1 and little

specificity for the P1 position.112 Chymotrypsin, an enzyme found in the digestive

93


systems of mammals was chosen due to its complementary specificity towards

substrates with aromatic groups or to a lesser extent, hydrophobic amino acids with

bulky side chains in the P1 position with little specificity for the amino acid in

P’1.114 Finally, Elastase was included due to its clinically relevance in its

involvement in breaking down the extracellular matrix (ECM); it preferentially

cleaves peptide bonds between amino acids with small uncharged side chains

(alanine or valine in the P1).114

Table 3-4. Specificity of the enzymes used towards amino acids (AA) in the substrate and their

molecular weight.

Enzyme Thermolysin Chymotrypsin Elastase

Specificity Hydrophobic AA in

P’1

Aromatic/Hydrophobic

AA in P1

Small uncharged

AA in P1 and P’1

Molecular

Weight

(kDa)

35 25 25

Table 3-5 shows the relative cleavages of each ECP for each enzyme, in which

100% cleavage was taken as the maximum peak area detected on HPLC for that

ECP (therefore discounting the dipeptides at sites inaccessible to the enzymes). As

expected, thermolysin was found to give the highest cleavages for both the AA and

FF enzyme cleavable peptides due to its specificity for hydrophobic residues. Some

hydrolysis also occurred for ECP the composed of GG indicating that the enzyme is

somewhat unspecific. Chymotrypsin would be expected to cleave the FF ECP more

than the other ECPs, however, the comparatively high cleavage of the peptide

actuator with GG as the ECP was not expected (due to the hydrophilic nature of

glycine). Standard chymotrypsin is known to contain trypsin impurities, as it is

derived from the proteolytic cleavage of chymotrypsinogen by trypsin.115 Trypsin

has been shown to cleave peptides when hydrophobic residues or lysine or arginine

are in the P’1 position (except when praline is in the P1),116 therefore, the higher

observed value of hydrolysis for the GG ECP may be due to some cleavage

occurring between the ECP and arginine. Elastase was observed to give high

94


percentage cleavages for the AA ECP due to its preference for cleaving peptide

bonds between amino acids with small uncharged side chains.

Table 3-5. Values for HPLC relative enzyme cleavage for each ECP.

ECP Thermolysin Chymotrypsin Elastase

Ala-Ala (AA) 100% 4% 99%

Gly-Gly (GG) 9% 20% 6%

Phe-Phe (FF) 100% 23% 14%

When the peptide actuator was intact (the peptide actuators were zwitterions) the

accessibility was determined to be less than 40 kDa. The 40 kDa fluorescently

labelled dextran was not able enter the particles. This was because the net charge of

the peptide actuators was neutral. However, if the ECP was cleaved then there was a

switch from uncharged to positively charged resulting in an increase in swelling of

the particles and therefore mesh size of the network. This made the polymer network

more accessible allowing the 40 kDa dextran to diffuse into the microparticles. This

process is shown schematically in Figure 3-40. The confocal micrographs of

representative microparticles observed in the enzyme specificity investigation are

provided in Figure 3-41. It can noted that a sufficient increase in accessibility to

allow the FITC dextran to enter the particles is only seen for thermolysin treatment

of AA and FF, while elastase only initiated a response with AA as the ECP. HPLC

was used to provide quantitative information about the hydrolysis of the peptide

actuators (Table 3-5) showing that there was still some hydrolysis of the peptide

actuators for all ECPs. However, the hydrolysis (up to 23 %) was insufficient to

trigger an increase in accessibility.

95


Figure 3-40. A schematic of enzyme responsive microparticles illustrating both successful (top)

and unsuccessful (bottom) cleavage of the ECP resulting in a change of accessibility to a 40 kDa

FITC labelled dextran.

Figure 3-41. Confocal microscopy images of representative microparticles in an aqueous

solution of 40 kDa fluorescently labelled dextran. Scale bar is 20 µm.

3.4.4.2. Demonstration of the encapsulation of a payload

In order to be able to release a payload it must first be encapsulated. To achieve this

the pH responsive nature of the peptide actuators was utilised. The charges within

the peptide actuator are due to the acidic and basic amino acid side groups. By

96


lowering the pH below the pKa of the carboxylate side group it becomes protonated,

changing the net charge of the peptide actuator from neutral to positive. This results

in electrostatically induced swelling leading to an increase in accessibility. Through

the use of confocal microscopy to observe the spatial confinement of a FITC

labelled 40 kDa dextran the loading and encapsulation process was demonstrated

(Figure 3-42). At pH 7 fluorescence was not observed within the particles indicating

that the dextran was not able to diffuse into the polymer. Upon lowering the pH to 3

the 40 kDa FITC dextran was able to diffuse into the particles. The apparent

decrease in the fluorescence at pH 3 is due to the pH dependence of the fluorophore

efficiency. By returning the pH to around pH 7 by the addition of base the dextran

remained in the particles. After repeated washing with water, fluorescence remained

inside the particles.

Figure 3-42. The pH responsive loading of the 40 kDa FITC labelled dextran (1 mg/ml) into the

PEGA particles, gain of images varied. (A) Particles in a solution of the dextran in water. (B)

Particles in a solution of dextran in dilute HCL at pH 3, purple colour represents detector

saturation. (C) Particles after being lowered to pH 3, neutralised to pH 7 and washed several

times with water. Scale bar is 15 µm.

3.4.4.3. Enzyme specific release

Utilising the method described in section 3.4.4.2 the peptide actuator functionalised

particles were loaded with 40 kDa FITC dextran and washed. The particles were

then treated with either thermolysin or chymotrypsin in water. Thermolysin has

specificity for the ECP (AA) in the peptide sequence therefore it should cleave the

sequence triggering an increase in swelling and thus release. The fluorescent

intensity of the solution surrounding the particles was then measured (after 85

minutes), determining whether the FITC labelled dextran has been released. The

result showed there was a dramatic difference between the amount of release for

thermolysin and chymotrypsin with approximately 15 times more release occurs

97


when treated with thermolysin compared to chymotrypsin. This is due to the specific

enzymatically hydrolysis of the ECP that only occurs with thermolysin.

3.4.4.4. Enzyme responsive increase in swelling; effect of ionic strength

The responsive nature of the functionalised particles could also be assessed by

monitoring the change in size of individual particles when treated with different

enzymes. Figure 3-43 shows that when the particles were treated with thermolysin

dissolved in water there is an increase in volume of ≈ 60 % after 15 minutes.

Additionally, we observe the specificity of the response as chymotrypsin did not

trigger an increase in swelling. However, if the linear peptide actuator functionalised

µPEGA was treated with thermolysin in a solution at ionic strength of 0.18 M no

change in swelling was observed. This was a result of the electrostatic screening of

the charges on the peptide actuators by the ions within the solution. This finding has

also been observed on peptide actuator-functionalised PEGA macroparticles by

other researchers.58

Figure 3-43. Change in volume of individual particles over time with different treatments: ♦

(blue): Thermolysin in water. ■ (red): Chymotrypsin in water. ▲ (green): thermolysin in buffer

at an ionic strength of 0.18 M.

98


3.5. Conclusions

Three different types of PEGA microparticles have been prepared. PEGA hydrogels

incorporating charges within the polymeric backbone displayed increased swelling

in water due to the electrostatic repulsion resulting in a greater uptake of water.

Within a buffer solution 0.1 M all PEGA hydrogels had similar swelling ratios due

to the screening of some of the longer range electrostatic interactions within the

polymers. Neutral µPEGA was chosen for further investigation these cell-sized

particles had a mean diameter of 15 µm and a polydispersity of 43 %. The

distribution of amines was homogeneous throughout the particles. It was shown that

enzyme action on coupled peptides was also homogeneous. These microparticles

gave rise to faster enzymatic hydrolysis than commercially available large PEGA

particles (macroparticles) due to the greater surface area to volume ratio of smaller

particles. When µPEGA was functionalised with peptides actuators an enzyme

specific response through the increase in the accessibility was observed. The pH

responsiveness of these particles has been demonstrated by the loading of the

particles with a fluorescently labelled macromolecule. Enzyme specific release of

this payload was possible. However, at physiological ionic strength no enzyme

triggered swelling of the functionalised particles was observed. This limitation will

be addressed in the following chapter with the design of branched peptide actuators.

99


4 Designing new peptide actuators for improved enzyme

responsive behaviour‡

4.1. Abstract

This chapter describes the functionalised μPEGA with branched peptide actuators.

These peptide actuators provided enhanced charge density and responded through an

increase in swelling to the target enzyme at physiological ionic strength. Analysis of

enzymatic activity revealed that the target enzyme (thermolysin) could access the

core of particles when linear peptides are used, while access was restricted to the

surface when using branched actuators due to electrostatic interactions. These

responsive μPEGA particles were then loaded with a fluorescent labelled dextran by

application of a sequential pH change. The macromolecule payload could be

selectively released at physiological ionic strength when exposed to the target

enzyme.

4.2. Introduction

4.2.1. Branched peptide actuators

As demonstrated at the end of Chapter 3 and earlier systems from the Ulijn and co-

workers,58 responsive systems based on electrostatic actuation using peptide

actuators are inherently sensitive to the ionic strength of the solution. This limitation

meant that they were unable to function in solutions with physiological ionic

strength due to electrostatic screening.58 In this chapter this problem is addressed

through the design of branched peptide actuators with enhanced charge density.

The inability of the linear peptide actuator to respond at physiological ionic strength

was related to the electrostatic screening of neighbouring charges.58 For charge

induced swelling to occur the distance between the charges must be less than the

‡ This chapter has been published as: Mcdonald, T.O., Qu, H., Saunders, B.R. and Ulijn, R.V., Branched Peptide Actuators for Enzyme Responsive Hydrogel Particles. Soft

Matter. (2009).

100


Debye length. At physiological ionic strength (0.15 M) the Debye length is 0.8 nm.

By increasing the space occupied by the actuator we aimed to reduce the distance

between neighbouring charges to less than 0.8 nm. The approach used to was to

incorporate the di-amino functionalised amino acid lysine (K) to act as a branch

point thus causing the peptide actuator to occupy more space (Figure 4-44). Each

arm consists of three parts: i) a di-glycine (G) spacer to enable enzyme access, ii)

oppositely charged actuation amino acids and which are iii) separated by an enzyme

cleavable peptide (ECP) sequence which serves as the sensing element. By matching

the ECP to the specificity of a target protease, the material may be programmed to

respond exclusively to a target enzyme. Enzymatic hydrolysis of the ECP leads to

release of anionic fragments and conversion of the branched zwitterionic peptide

actuator to cationic groups remaining covalently bonded to the polymer (Figure 4-

44). These neighbouring cationic groups induce swelling and an increase in the mesh

size within the polymer which can be exploited in triggered release of pre-entrapped

payload molecules.

The objectives within this chapter were to: (i) synthesise and characterise branched

peptide actuators on µPEGA, (ii) investigate the enzyme response behaviour of the

functionalised particles and (iii) utilise these particles for triggered enzyme of a

payload at physiological ionic strength.

101


Figure 4-44. Schematic of enzyme responsive branched peptide actuator. Left; peptide

actuator, Right; upon cleavage of peptide by an enzyme only the cationic groups remain

attached. These charged peptide fragments then electrostatically repel one another leading to

an increase in swelling of the polymer.

102


4.3. Materials and methods

4.3.1. Materials

All chemicals were used as supplied from Sigma with the exception of amino acids

(Bachem), PEGA macromonomers (Versamatrix), Isopar M (Multisol) and N,N,N′,N

′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)

(AGTC Bioproducts Ltd). The enzymes used were thermolysin (EC 3.4.24.27), 36.5

U/mg and chymotrypsin (EC 3.4.21.1), 60 U/mg.

4.3.2. Inverse suspension polymerisation and polymer characterisation

A stainless steel baffleless reactor (250 ml) stirred with an anchor-style agitator was

used for the polymerisation reaction. 3.14 g (3.4 mmol) of the PEGA800

macromonomers (2:1 ratio of acrylamide-PEG-acrylamide to amino-PEG-

acrylamide) and 0.156 g (2.2 mmol) of acrylamide were dissolved in 10 ml of

distilled water and purged for 30 minutes with N2 gas. 50 ml of Isopar M

(isoparaffin) was added to the reactor and was also purged for 30 minutes. The

reactor was heated to 70°C. After 20 minutes of purging 0.16 ml (1.0 mmol) of

N,N,N′,N′- tetramethylethylenediamine (TEMED) was added to the oil phase. 0.164

g (0.47 mmol) of Span 20 (sorbitan monolaurate) was dissolved in the oil, which

was stirred at 500 rpm for 30 seconds to ensure the surfactant was fully dispersed in

the oil phase. 0.070 g (0.30 mmol) of ammonium persulfate (APS) was dissolved in

the macromonomer solution, which was added to the oil phase in the reactor and

stirred at 2000 rpm for a further 30 minutes. The particles were washed with (3 x 50

ml) dichloromethane (DCM), (3 x 50 ml) Tetrahydrofuran (THF), (3 x 50 ml)

methanol and (4 x 50 ml) distilled water. Particle size distribution and mean particle

diameter were determined using a Malvern Mastersizer particle size analyser,

Mastersizer Microplus software version 2.18 was used to analyse the results.

Environmental scanning electron microscopy (ESEM) images were taken on a FEI

Quanta 200 ESEM, using low vac mode at 10.0 KV.

103


4.3.3. Solid phase peptide synthesis

Peptide actuators (linear: Fmoc-DAAR-PEGA) and branched: (Fmoc-DAARGG)2-

K-PEGA) were prepared by solid phase peptide synthesis using Fmoc protected

amino acids. 8 equivalents of the amino acid and 7.8 equivalents of HBTU were

dissolved in 2 ml of N,N-Dimethylformamide (DMF). 16 equivalents of N,N-

Diisopropylethylamine (DIPEA) was added to this solution prior to its addition to

0.1 g of µPEGA. Coupling was left for 16 hours, and Kaiser test117 was used to

check for complete coupling. Deprotection was achieved using 20 % piperidine in

DMF for 2 hours. This procedure was repeated to build up the peptide sequence with

thorough washing between steps (5 x 5 ml methanol, 5 x 5 ml 1:1 methanol:DMF, 5

x 5 ml DMF). A solution of 95 % Trifluoroacetic acid (TFA) and 5 % water was

used to remove the amino acid side chain protecting groups.

4.3.4. Microscopy and determination of swelling

Swelling measurements and fluorescent images were obtained using a Zeiss Imager

A1 microscope, a Leistungselektronik mbq 52 AC power source (Jena, Germany)

equipped with an HBO 50 mercury lamp and Canon Powershot G6 camera. A Zeiss

filter set 09 (excitation 450 - 490 nm, emission 515 + nm) was used to visualise

FITC. For pH responsive swelling peptide actuator functionalised PEGA particles

were immersed in either water (CHROMASOLV plus for HPLC) or 0.01 M HCl

(pH 2.5) and images obtained using the microscope. ImageJ 1.38x analysis software

was used to determine the change in particle diameter. For pH responsive swelling

the diameter of a minimum of 300 particles was measure and the mean volume

calculated. For enzyme responsive swelling individual particles were observed

throughout the timecourse and an average V/Vo was determined from at least six

representative particles, where V is the volume of the particle at time t and Vo is the

volume of the particle immediately after exposure to enzyme solution. Enzyme

solutions were made up at a concentration of 1 mg/ml and buffers were prepared by

using the appropriate amounts of sodium phosphate dibasic heptahydrate and

sodium phosphate monobasic.


µPEGA functionalised with either linear or branched peptide actuators was exposed

to an aqueous thermolysin solution (1 mg/ml) for 40 minutes. The particles were

104


then washed with (5 x 1 ml) Acetonitrile (AcN):H2O (50:50) containing 0.1 % TFA

and then (5 x 1 ml) DMF. 6 equivalents of dansyl-Cl were dissolved in 2 ml DMF

along with 10 equivalents DIPEA this solution was added to the particles and

incubated in the dark at room temperature for 2 hours. The dansyl-labelled particles

were washed with (3 x 1 ml) DMF and (3 x 1 ml) water. Two-photon microscopy

images of the particles in water were obtained on a Leica TCS SP2 inverted

microscope. ImageJ software was used to produce the surface plots of intensity. The

intensities at the centre, the outside (1 µm from the edge of the particle) and the

average intensity across the particle were determined for 15 particles functionalised

with either the linear or branched peptide actuators.

4.3.6. Entrapping payload

The particles (0.1 g) were loaded with a 40 kDa FITC Dextran using the following

method; 2 ml of solution of FITC dextran in 0.01 M HCl (10 mg/ml) was added to

the particles for 30 minutes (pH 2.5). After this time 0.4 ml of 0.05 M NaOH was

added dropwise (pH 7). The particles were then filtered and washed (2 x 5 ml water,

2 x 5 ml 0.18 M buffer, 2 x 5 ml methanol).

4.3.7. Release measurements

The release of entrapped dextran was determined using the following method;

loaded particles were treated in solution for 80 minutes, these samples were

centrifuged and the supernatant was removed. A Jasco FP – 6500

Spectrofluorometer using an excitation wavelength of 490 nm was used to obtain the

fluorescent intensity at 519 nm. Treated particles were filtered and imaged using

fluorescence microscopy.

4.3.8. HPLC and LCMS

HPLC experiments were undertaken on a Dionex HPLC (P680 pump, ASI-100

Automated sample injector, Nucleosil 100-5-C18 column with a UVD170U

detector), using a solvent ramp of 20 % ACN and 80 % water to 80 % ACN 20 %

water over 30 minutes (0.1 % TFA was present in both phases). Chromeleon 6.60

software was used for analysis. All LCMS analyses were carried out on a reverse-

phase Luna C18(2), 250 x 2mm, 5 micron column (Phenomenex). The LC-MS

instrument was an Agilent 1100 Series HPLC, coupled to an Agilent 1956B Mass

105


Detector. The solvent ramp of 90 % water 10 % ACN to 15 % water 85 % AcN over

14 minutes was used in all analyses; the flow rate was set at 1.0 mL min -1. Mass

detection was set to analyse in SCAN mode with electrospray ionisation.

106


4.4. Results and discussion

4.4.1. Microparticle characterisation and peptide functionalisation

µPEGA was produced (described in detail in Chapter 3) and peptide

functionalisation was achieved through solid phase peptide synthesis using Fmoc

protected amino acids. Use of the Kaiser test and the quantification of Fmoc

removed at each deprotection step indicated high yield peptide formation with over

90 % of the initial loading achieved for the final step (Figure 4-45).

Figure 4-45. HPLC quantification of Fmoc removed after each coupling step for linear and

branched peptide actuators.

The charged structure of the peptide actuators can be indirectly determined by

examining the pH responsive swelling of the peptide actuators. The charged state of

these peptide actuators is pH dependant, as the amino acid side chains have pKa

values of 4.4 and 12.0 for aspartic acid (D) and arginine (R) respectively. Therefore,

if the pH was lowered below 4.4, the previously anionic carboxylate groups became

protonated giving the peptide actuators a net positive charge. Electrostatic repulsion

between neighbouring positively charged actuators led to an increase in swelling of

the particles. This can be seen in Figure 4-46 where at pH 2.5 there was a 30 %

increase in volume for particles functionalised with linear peptide actuator while a

57 % increase in volume was noted with branched peptide actuators. This difference

can be attributed to the higher density present in the branched actuator.

107


Figure 4-46. pH responsive swelling behaviour of peptide actuators on PEGA microparticles

(average of at a minimum of 300 particles). Student’s t-test shows a difference is significant at

98 %.

4.4.2. Actuator design and responsiveness of peptide functionalised particles

The responsive increase in particle swelling is dictated by the extent of electrostatic

repulsion between neighbouring charged groups. Figure 4-47 A shows the enzyme

responsive swelling of PEGA microparticles functionalised with linear peptide

actuators in water. The distance between charged groups in linear actuators (Fmoc-

DAAR-PEGA) is greater than the Debye length at 0.15 M (0.8 nm) and therefore did

not give rise to sufficient electrostatic repulsion to cause swelling. The branched

design of the new actuator occupies more space than the linear actuator resulting in

the cationic groups being closer together. Figure 4-47 B shows that when the

particles functionalised with the branched actuator are treated with thermolysin

(from Bacillus thermoproteolyticus rokko E.C. 3.4.24.27) at an ionic strength of

0.18 M an increase in volume is observed reaching a maximum of ~1.3 V/Vo after

20 minutes. This response is exclusively observed upon treatment with thermolysin.

This enzyme has a relatively broad specificity preferring hydrophobic residues in the

P1’ position.112 While treatment with α-chymotrypsin from bovine pancreas (E.C.

232.671.2), which has a preference for large hydrophobic residues in the P1

108


position,114 did not give rise to a change in swelling (visualised in Figure 4-47 C).

Indeed, HPLC and LCMS analysis of the solutions obtained after enzyme treatment

shows that ECP cleavage occurs exclusively with thermolysin (Figure 4-48 A).

Furthermore, LCMS data after enzyme treatment shows two main peaks consisting

of fragments of the desired peptide sequence (Figure 4-48 B & C). Therefore,

functionalisation of µPEGA with the branched peptide actuator has achieved

enzyme specific response at physiological ionic strength.

Next, the ionic strength dependence of the branched actuators (Figure 4-49) was

investigated by determining the final increase in swelling. It was found that, due to

the enzyme triggered increased charge density maximum swelling (~1.3 V/Vo) was

observed at and above physiological ionic strength (0.15 M)118,119 up to 0.3 M. This

corresponds to a Debye length of 0.55 nm. Presumably, at higher ionic strengths the

mobile ions in solution begin to screen the static peptide charges effectively

resulting in a decrease in the amplitude of the response. This is most pronounced at

an ionic strength of about 0.45 M where V/Vo ≈ 1.15. As expected, chymotrypsin

did not initiate a response at any ionic strength tested. To conclude this section, the

branched peptide actuator functionalised particles demonstrate specific enzyme

responsive at physiological ionic strength and above.

109


Figure 4-47. Enzyme responsive swelling behaviour of peptide actuator functionalised µPEGA

at pH 7. A: Swelling of particles functionalised with the linear peptide actuator; ●:

Thermolysin in water, ¯ : Thermolysin at ionic strength 0.18 M. B: Swelling of particles

functionalised with the branched peptide actuator; ¯: Thermolysin at ionic strength 0.18 M, r:

Thermolysin at ionic strength 0.76 M, ¢: Chymotrypsin in water. C: Swelling of individual

particles treated with either Thermolysin or Chymotrypsin at 0.18 M ionic strength (circle

shows original size, scale bars are 15µm).

110


Figure 4-48. HPLC and MS analysis of enzyme hydrolysis of branched peptide actuators. A:

HPLC traces for thermolysin (blue) and chymotrypsin (pink) treated particles modified with

branched peptide actuators. Absorbance at 254 nm. The ratio of peak areas is 3:1 (1st peak : 2nd

peak). B: Mass spectra for thermolysin treated particles modified with branched peptide

actuators peak at 22.1 minutes. C: Mass spectra for thermolysin treated particles modified with

branched peptide actuators peak at 24.8 minutes. D: Key of peptide fragments found.

111


Figure 4-49. Effect of ionic strength on the maximal enzyme responsive swelling of branched

peptide actuator functionalised µPEGA at pH 7 (after 40 minutes) ¯ :Thermolysin , r: No

enzyme, ¢: Chymotrypsin. Dashed line indicates physiological ionic strength.

4.4.3. Characterisation of enzyme action on peptide actuators

Enzymatic hydrolysis of the peptide actuators produces polymer-bond peptide

fragments with free amines (see Figure 4-44). The distribution of amines can be

examined through labelling with dansyl which allows the location of enzyme

hydrolysis within the particles to be investigated (as also previously described in

section 3.4.3.1). TPM was used to assess the distribution of fluorophores (dansyl)

within PEGA particles functionalised with either linear or branched peptide

actuators after 40 minutes thermolysin treatment. As seen in Figure 4-50 A & B

enzyme cleavage of the linear peptide actuators on PEGA particles was

homogeneous on the micrometer scale, while branched peptide actuator

functionalised particles had greater cleavage in the outer regions of the particles. The

pI of thermolysin is 4.97120 and the protein was therefore negatively charged at

neutral pH. It is likely that electrostatic attraction between the cationic fragments on

the particle formed as a result the enzymatic hydrolysis of the ECP and the anionic

enzyme lead to enzyme retention. It is expected that this interaction would to be

stronger for branched actuators (double the positive charge). Therefore, enzyme

diffusion may be slower in particles containing branched actuators (i.e. enzymes are

112


held in place after enzymatic hydrolysis). Indeed, similar electrostatic retention has

been observed previously.58 Linear peptide actuators have been shown in water to

produce a maximal V/Vo of 1.6 (Figure 4-47 A) while the maximal swelling for the

branched peptide actuators observed was 1.35 (Figure 4-47 B). The heterogeneous

enzymatic cleavage apparent for the branched peptide actuators explains the reduced

maximum swelling observed when compared to the linear peptide actuator.

Figure 4-50. Comparison of thermolysin action on both linear and branched peptide actuators

on µPEGA. A: Two-photon micrographs of representative particles labelled with dansyl (scale

bar is 15 μm). B: Surface plot of two-photon images indicating qualitatively the fluorescent

intensities of the particles. C: Quantification of fluorescent intensities of dansyl labelled,

enzyme treated particles functionalised with either linear or branched peptide actuators.

113


4.4.4. Enzyme triggered release

The applicability of branched peptide actuator functionalised particles to

enzymatically triggered release a payload was assessed. Firstly, a payload was

entrapped within the particles. This was achieved by utilising the pH responsiveness

of the peptide actuators.58 Here, fluorescein isothiocyanate (FITC) dextran (40 kDa)

was the payload macromolecule. By lowering the pH of the payload solution to pH

2.5 the aspartic acid residue side chain is protonated (while the R side chain remains

protonated) making the net charge of the peptide actuator positive, resulting in an

increase in the swelling (Figure 4-46 B) and thus mesh size of the polymer

microparticles. The pH switch therefore allowed the 40 kDa dextran to diffuse into

the particle. By returning the system to pH 7 the D side chain carboxyl group again

becomes ionised. In this case the net charge of the peptide actuator becomes zero

and the repulsive forces are removed. The polymer particle deswells and entraps the

dextran. Figure 4-51 A shows a fluorescent micrograph of these particles after

washing demonstrating that the FITC labelled dextran was entrapped within the

particles. In Figure 4-51 B & C the particles are treated with buffer and

chymotrypsin respectively (both at 0.18 M ionic strength), because no actuation had

occurred the majority of the FITC dextran remained entrapped with some leakage

apparent. It is likely that some of the dextran was not completely entrapped within

the particles resulting in leakage.

114


Figure 4-51. µPEGA particles functionalised with the branched peptide actuator at pH 7 (scale

bars are 100 μm). A: After loading with FITC labelled 40 kDa dextran and washing. B: After

loading and 80 min treatment with Thermolysin. C: After loading and 80 min treatment with

Chymotrypsin. D : After loading and 80 min treatment with buffer E: mmoles of FITC labelled

dextran released per g of particles after 80 min treatment. (all experiments were carried out at

an ionic strength of 0.18 M)

When the particles were treated with thermolysin (Figure 4-51 D) the increase in

swelling (and corresponding increased pore size) allowed the dextran to diffuse out

of the particles, resulting in a reduction of fluorescence of the particles. By

measuring the fluoroscence of the solution surrounding the particles it was possible

to measure the amount of FITC dextran released (Figure 4-51 E). When the particles

were exposed to either buffer alone or chymotrypsin a small amount of leakage was

observed (~ 0.02 mg dextran/mg particles). While treatment with thermolysin

resulted in a 350 % increase in dextran release (Figure 4-51 E). Hence the enzyme

triggered increase in swelling and accessibility was responsible for the greater

release of the entrapped macromolecules.

115


To summarise, by exploiting the enzyme responsive swelling it was possible to

release an entrapped macromolecule in the presence of the target enzyme.

Electrostatically induced swelling occurs upon cleavage of the ECP within the

peptide actuator at physiological ionic strength.

4.5. Conclusions

Branched peptide actuators have been synthesised and incorporated into μPEGA.

These functionalised particles were capable of specifically responding to selected

enzymes at a variety of ionic strengths. Through the physical entrapment of a

macromolecule payload within the functionalised particles it was possible to obtain

payload release at physiological ionic strength in response to target enzyme. This

system may have applications in a number of areas including drug delivery and

automated bio-sensing of ‘on-bead’ libraries.

116


5 Microfluidic preparation of low polydispersity PEGA

particles

5.1. Abstract

This chapter details the preparation of PEGA particles by a simplified microfluidic

setup assembled using a needle and tubing. The size of the particles produced was

determined by the surfactant concentration and the relative flow rates of the

dispersed and continuous phases. The minimum particle size obtained was limited

by the diameter of the needle (335 μm). The development of a flow-focussing setup

allowed for the production of smaller particles down to 100 μm in diameter.

Optimum conditions were determined allowing for the production of 4000 particles

a minute with a mean diameter of 160 μm.

5.2. Introduction

This chapter describes the development of microfluidic devices for the preparation

of near monodisperse§ polymer particles. Monodisperse particles present a number

of advantages over polydisperse samples: the sample of particles is well defined, any

reaction occurring on or in the particles occurs at the same rate. Additionally, any

change in swelling or size is easy to determine without the need for a large sample

size.

Microfluidic systems offer the potential to produce particles with a very narrow

distribution because the size each monomer droplet formed is determined by the

conditions at the mixing point. As each droplet is formed under constant conditions

the size of the droplets are the same. The introduction of a polymerisation method,

normally UV initiation results in the production of polymer particles. In these

systems there are a number of variables that control the mixing process and therefore

§ According to the standards of the National Institute of Standards and Technology (NIST): “a

particle distribution may be considered monodisperse if at least 90% of the distribution lies within

5% of the median size” (Particle Size Characterization, Special Publication 960–961, January 2001).

117


ultimately determined particle size. The following work aims to find the optimum

conditions to produce near monodisperse µPEGA. These particles would be better

defined and more homogenous than the samples prepared by inverse suspension

polymerisation in Chapter 3. The very narrow size distribution would offer the

potential for rapid screening with an automated cell sorter. Using this technique it

could be possible to rapidly screen for enzyme action on µPEGA using peptide

libraries.72,73,80

The polymerisation of monodisperse particles using microfluidic devices was first

demonstrated in 2005 by Whitesides and co-workers,91 since then there has been

great interest and developments in the use for microfluidics for the preparation of

polymer particles.92,93,95-97,121 These systems conveniently allow for the size of the

droplets (and therefore particles) to be controlled by the flow rates of the continuous

and dispersed phases. However, in order to prepare most microfluidic devices

microfabrication techniques are needed; typically, soft-lithography is used to form

planar microchannels.91,95,122 There are a number of limitations associated with these

devices: Usually a clean room is required for device production. Surface treatment

of the channel walls can be needed to prevent an inverse emulsion forming.

Additionally it is often easy for the microchannels to become blocked by solid

polymer. Simplified microfluidics setups have been shown that do not require no

specialist fabrications techniques. These typically make use of needles and/or tubing

to create near monodisperse droplets.92,97 These publications have been covered in

more detail in the preceding literature review (Section 2.5). The research in this

section is based on a paper by Hadziioannou and co-workers in which the dispersed

phase is introduced to the continuous phase via a needle positioned along the main

axis of the tubing.97 The axisymmetric setup of the device prevents the need of

surface modification because the dispersed phase droplets do not come into contact

with the channel walls.93 While any blockages in the tubing due to polymer build up

can simply be rectified through a fast and low cost replacement of that section of

tubing. In this chapter a microfluidic device as described by Hadziioannou (shown

schematically in Figure 5-52) was used to produce PEGA particles.

118


Figure 5-52. Schematic of microfluidic setup for the synthesis of controlled-size polymer

particles. The dispersed phase (containing the monomer) and the continuous phase are

delivered by syringe pumps. The dispersed phase is introduced to the co-flowing continuous

phase the centre of the PTFE tubing by means of a 26 gauge needle.

The objectives of this chapter were to: (i) Optimise the conditions to manufacture

particles with the smallest diameter possible with a very narrow size distribution. (ii)

Develop strategies to produce smaller particles, through the use of a simplified flow-

focusing setup.

119


5.3. Materials and methods

5.3.1. Microfluidic system

Figure 5-52 shows a schematic of the microfluidic system. A stainless steel needle

blunt tip needle (26 gauge) internal diameter (ID) 240 μm was inserted into a T-

junction (Swaglok T-junction SS-100-3). The needle tips exits the T-junction in the

centre of the polytetrafluoroethylene (PTFE) tubing. Two syringe pumps (New Era

Pump Systems Inc. NE-1000) were used to deliver the continuous and dispersed

phases at a specific flow rate. The two phases were carried in PTFE tubing with an

internal diameter (ID) of 1.6 mm. The dispersed phase was injected through the

needle while the continuous phase was injected perpendicular to the main axis of the

T-junction. The outlet tubing was also PTFE with ID 1.6 mm tubing with a length of

120 cm. The exit of the needle was located in the centre of the outlet tubing. The

outlet tubing entered a UV box where it was exposed to UV light (320-390 nm

wavelength) from a Dymax model 5000 Flood using a Dymax 400 W power source.

Residence time within the UV box was 30-240 seconds depending on the flow rates.

Particles were collected at the end of the outlet in a water bath.

5.3.2. Flow focussing setup

A modified T-junction was used to produce smaller droplets. Here, a 26 G needle

was inserted though a Fisher Tubing connector T connector Nylon 1/16in Masterflex

entering a 21 G needle. The internal needle ends approximately 2/3 of the way down

the external needle (Figure 5-58). Two syringe pumps were used to deliver the

continuous and dispersed phases at a specific flow rate. The two phases were carried

in PTFE tubing with an ID of 1.6 mm. The dispersed phase was injected through the

central needle while the continuous phase was injected perpendicular to the main

axis of the T-junction and flowed through the outer needle. The outlet tubing was

also PTFE ID 1.6 mm tubing with a length of 30 cm. The exit of the outer needle

was located in the centre of the outlet tubing. The outlet tubing entered a UV box

where it was exposed to UV light (320-390 nm wavelength) from a Dymax model

5000 Flood using a Dymax 400 W power source. Residence time within the UV box

was 30-240 seconds depending on the flow rates. Particles were collected at the end

of the outlet in a water bath.

120


5.3.3. Monomer and continuous phase preparation

All materials were used as supplied from Sigma-Aldrich with the exception of

PEGA macromonomers (Versamatrix), photoinitiator (Ciba) and Isopar M

(Multisol)

The dispersed phase consisted of an aqueous solution (10 ml ultrapure water)

containing 3 g (3.34 mmol) PEGA macromonomers, 0.15 g (2.11 mmol) acrylamide

and 0.10 ml darocure 1173 photoinitiator. 0.05 ml of red food colouring (Supercook)

was included in early experiments to give greater contrast to the monomer droplets.

The density of the solution was 1.008 g/ml. The viscosity solution as measured

using a Hydramotion Viscolite 700HP model VL700-T15HP was 4.8 cP at 20 ˚C.

The continuous phase consisted of Isopar M (viscosity of 2.1 cP) in which the

surfactant Span 20 (Sorbitan monolaurate) was dissolved, the amount of surfactant

was varied. The free radical polymerisation of the dispersed phase droplets led to the

formation of insoluble particles, these particles were collected in a water bath at

room temperature. Silicone oil (20 cSt) was used without surfactant (viscosity of 20

cP).

5.3.4. Particle size measurement

The polymerised particles were collected at the exit of the outlet tube and washed (2

x 10 ml DCM, 2 x 10 ml THF, 2 x 10 ml methanol and 4 x 10 ml water). The

diameter of the particles (fully swollen in water) was measured using an optical

microscope (a Zeiss Imager A1 microscope and Canon Powershot G6 camera).

Particles above 1500 µm in diameter were photographed without use of the

microscope over grid marker paper. ImageJ was used to determine the mean

diameter of the particles, due to the sometimes asymmetrical shape of the particles

the cross sectional area of each individual particle was measured and a mean

diameter then determined from it. The mean diameter and standard deviation were

obtained by measuring the diameter of at least 30 particles.

121


5.4. Results and discussion

5.4.1. Microfluidic system

Droplet formation in microfluidic systems is determined by the balance between the

shear forces imparted on the forming droplet by the co-flowing continuous phase

and the interfacial energy between the two phases. These hydrodynamic conditions

within the microfluidic device are usually described by two dimensionless numbers;

the Reynolds number and the capillary number.95 The Reynolds number is defined in

Equation 1 and the capillary number in Equation 2. Where ρ and µ are the density

and viscosity of the liquid, respectively, D is the diameter of the outlet tubing, V is

the velocity of the liquid and γ is the interfacial tension.95,97 It has been shown that

increasing the value of the capillary number for the dispersed phase (Cad) produces

smaller droplets.95 Therefore, by increasing the velocity or viscosity of the

continuous phase or decreasing the interfacial tension smaller droplets should be

produced.

Re ≡ ρDV/µ

Equation 1. Reynolds number

Ca ≡ µV/γ

Equation 2. Capillary number.

5.4.1.1. Variation of particle size with flow rate ratios and surfactant

concentration

Initial experiments produced droplets with diameters equal to that of the tubing

(Figure 5-53 A & B). Increasing Qc/Qd (where Qc and Qd are the flow rates of the

continuous and dispersed phases respectively), did not lead to a significant decrease

in droplet size and the particles formed (once washed and fully swollen in water)

had diameters greater than 1800 μm (Figure 5-53). This result suggested that the

interfacial energy between the aqueous monomer solution and organic continuous

phase (isoparaffin) was relatively high. Indeed, the interfacial tension of linear

alkanes with boiling points in the range of Isopar M (190 - 260 ˚C) have been shown

to be 53.1 - 54.5 mN/m.123 Higher interfacial tensions result in higher capillary

122


pressures, (the pressure difference between inside and outside of a droplet) as given

by the Young-Laplace (Equation 3).

Δp = γ(1/R1 + 1/R2)

Equation 3. Young-Laplace equation

Where Δp is the pressure difference across the liquid-liquid interface, γ is the surface

tension and R1 and R2 and the principal radius of curvature.124 This means that larger

droplets have a lower capillary pressure and are therefore more thermodynamically

favourable than smaller droplets. The highest calculated Cad for the experimental

results shown in Figure 5-53 was ~ 3 x 10-5.

This setup did not lead to control of particle size, as presumably it was not possible

to overcome the interfacial forces with shear forces.

Figure 5-53. A: Effect of Qc/Qd on mean particle diameter with no surfactant in the continuous

phase, Qc= 2 ml/min. B: Photograph of PEGA particles produced using a Qc/Qd of 10 and a Qc

of 2 ml/min (scale bar is 3500 µm).

The addition of surfactant to the continuous phase results in reduced interfacial

energy due to the surfactant molecules sitting at the interface. The use of surfactants

is well established within conventional dispersion type polymerisations.88,125

Additionally, surfactants have often been used within other publications describing

the controlled formation of droplets by microfluidics.95 The surfactant used within

this work was Span 20 (sorbitan monolaurate), which has been shown to reduce the

interfacial tension of linear alkanes with water to between 20 – 26 mN/m (dependant

123


on concentration).126 The effect of including surfactant in the continuous phase was

dramatic (Figure 5-54 A & B), even at the lowest Qc/Qd (10) and lowest surfactant

concentration (0.01 g/ml (three times the concentration used in the inverse

suspension polymerisation)) there was a large reduction in mean particle diameter

(625 µm compared to 2105 µm when no surfactant is present). The lowered

interfacial energy allowed the shear forces to influenced droplet size. As Q c/Qd was

increased particle size decreased reaching a plateau in mean particle diameter of

about 450 µm at flow ratio of 200. Slightly smaller particles (422 µm) were obtained

at very high values of Qc/Qd. This relationship between mean particle diameter and

Qc/Qd was seen as surfactant concentration was increased (further lowering the

interfacial forces increasing the dominance of shear forces), albeit with smaller

particles produced at higher surfactant concentrations. Upon the addition of

surfactant the value for Cad was greatly increased to ~ 9 x 10-5. Surfactant was

removed from the outside of the polymerisation particles by repeated washing in

solvents with a range of polar/non-polar characters.

124


Figure 5-54. Effect of surfactant concentration on particle size. A: Effect of Qc/Qd and

surfactant concentration (♦ (blue): 0.01 g/ml, ■ (red): 0.04 g/ml, ▲ (green): 0.08 g/ml) on mean

particle diameter, Qc= 2 ml/min. B: Effect of increasing surfactant concentration on mean

particle diameter at varying values of Qc/Qd. C: Effect of increasing surfactant concentration on

mean particle diameter at varying values of Qc/Qd excluding 0 g/ml surfactant concentration.

125


At a Qc/Qd of 10 the mean diameter of particles produced is very similar independent

of whether 0.04 g/ml or 0.08 g/ml of surfactant was used, indicating that the

minimal particle size at that flow rate had been obtained. However, as the shear

forces are increased particle diameter is also decreased. A surfactant concentration

of 0.08 g/ml and Qc/Qd of 1000 offered the smallest particles for this setup (335 µm).

Typically upon the polymerisation of a monomer droplet the resulting polymer

particle has a smaller volume. This corresponds to the increase density of the

polymer verses the monomer.91,97 With this system the dispersed phase was an

aqueous solution of the monomers. This was done to maintain a comparable method

to the inverse suspension polymerisation. It was found that monomer droplets

formed at the diameter of the tubing (≈1600 µm) initially give PEGA particles of

similar diameter (if collected in oil) which swelled after washing in water to give

much larger particles (≈2100 µm). The size of particular importance was that of the

hydrated particles as further applications of the polymer would be in aqueous

environments. Further investigation of swelling this behaviour was undertaken.

Particles were initially collected in oil then measured then washed and measured

again in water. Using this method it was determined that on average fully swollen

particles had diameter 35 % (± 3 %) larger than initial monomer droplets.

Considering this information a diameter of 335 µm which was found to be the lower

limit for particle size corresponds to droplet diameter of 248 µm, very close to the

internal diameter of the 26 gauge needle (240 µm). Earlier work using a similar

setup determined that the smallest droplets that could be produced had diameters just

above that of the needle.97 Surfactant concentrations above (0.08 g/ml) were not

assessed because above this concentration the surfactant was not completely soluble.

In summary, the introduction of surfactant to the continuous phase lowered the

interfacial tension resulting in the production of smaller particles. While increasing

Qc/Qd led to greater shear forces during droplet formation resulting in smaller

droplets and therefore particles, the lower limit in particle size was due to the

droplets formed at the needle diameter.

126


5.4.1.2. Effect of increasing total flow rate on particle size

Figure 5-55. A: Effect of total flow rate (at constant Qc/Qd) on mean particle diameter. B: Effect

of Qc/Qd and total flow rate (♦ (blue): 2 ml/min, ■ (red): 4 ml/min) on mean particle diameter,

0.08 g/ml surfactant concentration.

Very high Qc/Qd ratios did not offer efficient particle production; the rate of particles

was slow and the process was wasteful with a large amount of oil is used per particle

produced. Higher surfactant concentrations might have shown slightly reduced

diameters at lower flow ratios however, at concentrations higher than 0.08 g/ml the

surfactant did not fully dissolve in the oil. Another approach was to increase the

127


shear forces by increasing the total flow rate, a preliminary investigation into the

effect of total flow rate (at a constant Qc/Qd) on mean particle diameter showed as

flow rate was increased there was slight size decrease ( A). However, flow rates

higher than 4 ml/min did not lead to completely cured particles. Using a Qc of 4

ml/min over a range of Qc/Qd produced particles of only slightly reduced diameter

compared to 2 ml/min (Figure 5-55 B). Hadziioannou and co-workers produced

similar findings showing that similar values of Qc/Qd led to similar diameters

independently of the total flow, Qc + Qd.97

5.4.1.3. Effect of flow ratios and surfactant concentration on polydispersity

The polydispersity** of the particles increases when there is variation between

droplet to droplet formation or when coalescence occurs prior to polymerisation. In

order to determine whether the variables that were changed within the microfluidic

setup produced measureable trends in the polydispersity of the particles the data

shown in Figure 5-56 was obtained. At the highest and lowest surfactant

concentrations (0.01 g/ml and 0.08 g/ml) used there was a tendency for the

polydispersity to increase at higher values of Qc/Qd. While using 0.04 g/ml produced

particles with polydispersities in the range of 2-3 % over the range of Q c/Qd values

tested. The overall variation of polydispersity was found to be small (between 1.4 -

4.3 %).

5.4.1.4. Variation of particle production rates with the conditions assessed

In determining the optimum conditions with which to produce PEGA particles the

production rate was assessed (Figure 5-57). Lower values of Qc/Qd led to higher

production rates (due to higher flow rate of the continuous phase) while higher

surfactant concentrations offered higher production rates as a greater number of

smaller particles were produced from the same volume of monomer solution

(dispersed phase).

** Defined as the standard deviation in the particle diameter divided by the mean particle diameter.91

128


Figure 5-56. Effect of Qc/Qd and surfactant concentration (♦: 0.01 g/ml ■: 0.04 g/ml ▲: 0.08

g/ml) on polydispersity (Qc=2 ml/min).

Figure 5-57. Effect of Qc/Qd and surfactant concentration on particle production rate (♦ (blue):

0.01 g/ml ■ (red): 0.04 g/ml ▲ (green): 0.08 g/ml) (Qc=2 ml/min).

129


5.4.2. Using a simplified microfluidic flow-focussing device to produce

polymer particles

The minimum size of particles of particles obtained using the microfluidic setup

described up to this point was limited to the diameter of the needle from which the

dispersed phase was introduced into the continuous phase. To overcome this

minimum size limitation an adjustment to the microfluidic setup was made (Figure

5-58). Flow-focussing (FF) is a technique that allows for the formation of droplets

smaller than the orifice of the dispersed phase of the device by focussing the two

phases through a second orifice, this leads to greater shear forces. This technique is

well described within the literature for planar type microfluidic devices and also for

glass capillaries93,94,122,127, but to our knowledge this is the first time that this process

has been shown simply using needles. Therefore a FF type design was developed for

use at the mixing point of our microfluidic system as shown schematically in Figure

5-58.

Figure 5-58. Schematic representation of the ‘flow-focussing’ device. The internal needle fits

within the external needle.

5.4.2.1. Orientation of device

The shear forces at the point of mixing between the two phases is controlled by the

relative flow rates of the two phases. By reducing the diameter of the outlet at the

end of the needle (from which the continuous phase flows) the shear forces are

increased due to the greater velocity of the liquids. This led to small droplets being

produced at a high rate however, the high production rate of droplets often lead to

130


the coalescence of the droplets. The density of oil phase was 0.79 g/cm3 while the

density of the monomer solution was 1.00 g/cm3. This led to the monomer droplets

settling on the bottom of the PTFE tubing. Additionally, this difference in density

meant that the orientation of setup up was especially important because the speed of

the monomer droplets was determined by both the flow rate of the continuous phase

and gravity. If the flow direction opposed gravity the monomer droplets would

cluster upon leaving the external needle, this resulted in a polydisperse sample with

a larger mean diameter as some of the droplets coalesced before being polymerised.

If the flow direction and gravity were in the same direction the distance between the

monomer droplets was greater than when the device was horizontal (i.e. flow

direction was at 90˚ to gravity) (Figure 5-59 A & B), however, because the PTFE

tubing had to pass through the UV box in a relatively horizontal position

coalescence of droplets also occurred where the tubing was bent from vertical to

horizontal. This bend led to a reduction in the speed of the monomer droplets,

greatly reducing their separation from neighbouring droplets causing coalescence.

The optimum setup was found to be a constant slightly ‘downhill’ gradient (20˚

from horizontal) from the needle to the end of the outlet tubing. The slight slope led

to a slight increase in the separation between monomer droplets at the tip of the

needle preventing coalescence (except at very high monomer flow rates). The

constant gradient removed the problem of coalescence due to the changing of

droplet speed (and therefore separation) caused by a change in the angle of the

tubing. The FF design lead to the production of smaller droplets that the earlier

microfluidic setup, however the high production rate of droplets and the difference

in density meant that the orientation of the device had to be adjusted to minimise

coalescence of droplets.

131


Figure 5-59. Effect device orientation on droplet and resulting particle formation. A: Schematic

representation of effect of gravity and flow direction on droplet formation (left; the flow of

gravity opposes gravity, right; flow direction and gravity are in the same direction. B: Optical

micrographs of particles produced (left; when flow opposes gravity, right; when flow is with

gravity) scale bar is 110 µm.

5.4.2.2. Optimising conditions with the FF device

Using the optimised device orientation, a range of flow rates were tested to

determine the conditions for the production of monodisperse particles (Figure 5-60

A). At high values of Qd polydisperse particles were polymerised as a result of

insufficient droplet separation leading to coalescence of droplets. While values of Qc

above 1.5 ml/min produced particles that were not fully polymerised. A narrow

132

A

B

Gravity


range of conditions were found to give near monodisperse particles, these conditions

were investigated to determine the variation of particle size with Qc/Qd (Figure 5-60

B) using 0.08 g/ml of surfactant in the continuous phase. As Qc/Qd was increased

particle size decreased until an apparent minimum diameter of approximately 100

µm at Qc/Qd values of 375 and above. The effect of surfactant concentration was not

tested for the FF setup as the highest concentration (0.08 g/ml) had already been

established to give the smallest particle diameters with the earlier microfluidic setup.

The optimised flow focussing setup produced particles below the limit of the earlier

microfluidic setup down to a minimum of 100 µm in diameter. However, it offered a

less robust production with near monodisperse particles only found under a narrow

range of conditions.

Figure 5-60. Development of flow-focussing setup. A: Particle morphology as a function of the

flow rate of the dispersed phase (Qd) and continuous phase (Qc) where ■; polydisperse particles

(polydispersity > 8 %), ♦; near monodisperse particles (polydispersity < 8 %) and ▲; particles

did not fully polymerise. B: Effect of Qc/Qd on mean particle diameter using flow-focussing.

133


5.4.2.3. Effect of changing oil phase

The shear forces exerted on the disperse phase are determined by the velocities and

the viscosities of the two phases. Up to this point only the velocities have been

varied, therefore, in order to achieve smaller particle sizes the continuous phase was

changed to oil with higher viscosity. Silicone oil was chosen as it was obtainable in

a range of different viscosities additionally it had a density of 0.95 g/ml which

would reduce the problems associated with the difference in density found for Isopar

M. Initial polymerisations using silicone oil (with a viscosity of 20 cP) as the

continuous phase produced either polydisperse or highly aggregated particles

(Figure 5-61).

Figure 5-61. Optical micrographs of PEGA particles produced using FF device with silicone oil

(50 cSt viscosity) at a variety of conditions (clockwise from top right: Qc/Qd = 100 with Qc = 0.5,

Qc/Qd = 100 with Qc = 0.1, Qc/Qd = 450 with Qc = 0.25, and top left Qc/Qd = 650 with Qc = 0.1.

Scale bar is 200 µm.

Particles with a narrow size distribution were not formed at any of the conditions

investigated. At high total flow (Qc + Qd) rates polydisperse particles were formed,

while at lower flow rates aggregated particles were produced (Figure 5-61). The

aggregated particles appear to be of a relatively narrow size distribution with a mean

134


diameter of approximately 60 µm. Presumably, droplets underwent coalescence

prior to polymerisation at higher flow rates while at lower flow rate droplets began

to polymerise before coming into contact with one another resulting a mass of

aggregated particles. An increase in surfactant concentration within the continuous

phase was investigated however it was still not possible to produce individual

particles. Coalescence occurred as a result of slight variations in the speed of

movement of the dispersed droplets causing the separation between droplets to

become insufficient. It is likely that this is an intrinsic limitation of microfluidic

devices fabricated from flexible tubing. This problem is not reported in planar ‘chip’

type designs. As a result of these findings silicone oil was not further investigated as

the continuous phase.

5.4.3. Optimum conditions for particle production

The aim of this work was to produce particles with the smallest possible diameter

with a very narrow size distribution. However, the rate of particle production was

slower at higher values Qc/Qd. With these factors in mind the optimum conditions

used in the FF design were: 0.08 g/ml of surfactant in the oil phase, a Qc/Qd of 175

and a Qc of 1.5 ml/min.

5.4.3.1. Particle size distribution

Optimum conditions (described above) were used to produce approximately one

gram of polymer. These particles were further analysed by optical microscopy

(Figure 5-62 A) and a size distribution obtained (Figure 5-62 B). The majority of the

polymer volume was in the size range (150-170 μm) a low volume of smaller

satellite were also observed. The mean particle diameter was 161 μm with a

polydispersity of 3.3 %. The rate of particle production was 4000 particles min-1.

135


Figure 5-62. PEGA Particles produced under the optimum conditions. A: Optical microscopy

of particle (scale bar is 160 μm). B: Particle size distribution.

5.4.4. Morphology of particles

The particles produced by this technique often had an elliptical morphology with

aspect ratios128 ranging from 1.006-1.098. While Hadziioannou and co-workers97 did

not report this; no images of the particles that were produced were shown in the

paper. Within this work the formation of these asymmetrical particles seems to be a

result of the difference in density of the water (dispersed phase) and the oil

(continuous phase). This led to the monomer droplets descending and flowing along

the bottom of PTFE tubing. This contact with the tubing wall would have lead to

asymmetrical flow around the droplets.

136


Figure 5-63. The typically slightly asymmetric shape of particles produced by microfluidics

each image is of particles produced under different conditions. Scale bar is 200 µm.

5.5. Conclusions

This work provides an overview of using simplified microfluidic devices to produce

PEGA particles. The creation of the device required no specialist fabrication

methods and could be assembled using readily available lab supplies. Surfactant was

required to obtain control of the particle size by varying Qc/Qd, upon increasing

Qc/Qd smaller particles were obtained. Additionally, increasing the surfactant

concentration led to a reduction to the particle size. A minimum particle diameter of

335 µm was determined. A flow-focussing setup was devised to overcome this

limitation and allowed for the production of smaller particles with a diameter of 99

µm and a polydispersity of 3.2 %. This low value for polydispersity was a dramatic

improvement compared to the particles prepared by inverse suspension

polymerisation (43 %).

137


6 Conclusions

This thesis describes the design, synthesis and application of enzyme responsive

hydrogel particles through the use and development of peptide actuators.

Initially, PEGA hydrogel microparticles (μPEGA) were produced as the support for

the enzyme responsive system based on peptide actuators. These particles had a

homogeneous distribution of amines and were used as the chemical ‘handle’ from

which peptides could be synthesised. Enzymatic hydrolysis on the coupled peptides

was shown to be homogeneous throughout the particles. Additionally, μPEGA was

found to give rise to faster enzyme hydrolysis than commercially available large

PEGA particles (macroparticles) due to reduced diffusion distance. μPEGA was then

functionalised with linear peptide actuators. These zwitterionic molecules were

shown to actuate changes in the accessibility of the crosslinked PEGA particles. This

occurred through a switch in the overall charge balance of the polymer network

(from neutral to cationic) upon enzymatic hydrolysis of the enzyme cleavage peptide

(ECP) within the peptide actuator. The resulting electrostatic repulsion between

neighbouring peptide actuator fragments led to an increase in the mesh size of the

polymer network. By selecting the composition of the ECP to match an enzyme’s

specificity it was possible respond to different enzymes. The pH responsiveness of

the linear actuator functionalised particles was demonstrated. This behaviour was

utilised for the loading of the particles with a fluorescently labelled macromolecule.

Enzyme specific release of this payload was then possible. However, at

physiological ionic strength no enzyme triggered swelling of the functionalised

particles was observed. This was a result of the mobile ions in solution screening out

the interactions between the peptide actuators. This limitation was addressed with

the design of branched peptide actuators.

138


Branched peptide actuators consisted of two linear actuators using the amino acid

lysine as the branch point. This structure was built up from each of the amines

within μPEGA. Upon enzyme hydrolysis there was double the charge density within

particles (compared to when using linear peptide actuators) and the distance between

charged groups was likely reduced. Therefore, particles functionalised with

branched peptide actuators capable of specifically responding to enzymes at

physiological ionic strength (by overcoming electrostatic screening). Much like the

linear peptide actuator functionalised particles this system capable for physically

entrapping a macromolecular payload, was could be released only in response to a

defined enzyme at physiological ionic strength.

The final experimental chapter provided an overview of using simplified

microfluidic devices to produce PEGA particles. Rather than requiring specific

microfabrication techniques it was possible to produce the device using a needle and

tubing. It was found that by varying the surfactant concentration and the flow rates

of the continuous and dispersed phase particles of different sizes were obtained.

Increasing the surfactant concentration or increasing the ratio of continuous to

dispersed phase flow rates led to smaller particles. The minimum particle diameter

possible was found to be determined by the diameter of the needle. Therefore, a

flow-focussing setup was devised to overcome this limitation and allowed for the

production of smaller particles.

139


7 Future work

The most obvious future work at this stage would be using PEGA particles produced

by microfluidics as the basis of the peptide actuator system. These particles with

their narrow size distribution would allow for the kinetics of both enzyme and pH

responsive swelling behaviour to be characterised in greater depth. As narrow size

distribution of these PEGA particles would allow for a relatively small number of

particles to be measured in order to obtain the change in swelling.

It would also be of interest to use microfluidics to prepare polymer particles

crosslinked by enzyme cleavable peptides. This would be possible by employing a

strategy similar to Moore and co-workers.11 Monomers capable disulphide transfer

reactions to thiols would be synthesised (this has been carried out details are

contained in appendix section 8.2.2. These could then be conjugated to peptides

containing two cysteine residues resulting in a peptide crosslinker. A mixture of

monomer and peptide crosslinker could then be polymerised into particles using the

microfluidic device. Much like the enzyme responsive particles using peptide

actuators, these particles would also allow for enzyme responsive release of a

macromolecule (by tuning the crosslinking density). However, if required these

peptide crosslinked particles could be formulated so that they undergo complete

dissolution upon enzyme action (along with release of the payload). Additionally, it

may be possible to combine an enzyme cleavable peptide crosslinker and peptide

actuators into the same polymer. These systems could potentially have greater

amplitudes of response or by using different peptide substrates in the crosslinker and

ECP only response to a solution containing two different target enzymes.

Rapid and high throughput analysis of the response of the functionalised particles

may be possible if monodisperse PEGA particles of around 20 μm in diameter could

be produced (using a microfluidic chip or possibly by membrane

140


emulsification129,130). These particles could then be passed through an automated cell

sorter (flow cytometry)131. Using a split and mix method it would be possible to

prepare PEGA particles functionalised with a variety of different ECP compositions.

If these particles were then exposed to an enzyme and then sorted by size it would

be possible to screen for effective substrates for the enzyme. By designing the

peptide actuator and tuning the molecular weight cut-off of the functionalised

particles it may be possible to use enzyme hydrolysis of the ECP to trigger a

deswelling of the particles. This offers the potential for the specific entrapment of

the enzyme responsible for hydrolysing the ECP and additionally, might allow for

removal of a desired protease from a complex solution.

Ultimately, there are a number of steps that need to be overcome in order to use

particles functionalised with peptide actuators as drug delivery vehicles: Depending

on target site and method of introduction to the body (i.e. into circulation or injected

directly into the tissue) particles of smaller size may need to be produced.

Emulsion132 or miniemulsion133,134 may offer this possibility. Additionally, the

particles must respond only to the target enzyme (i.e. the marker for the disease to be

treated), to achieve this goal the ECP must be a substrate of that enzyme. This would

be assessed by synthesising the ECP as the substrate of a known disease specific

protease, then exposing these particles to that enzyme in an appropriate complex

mixture. Finally, the ultimate destiny of the particles within the body must be

determined. If the particles are not able to be cleared from the body then some form

possible degradation must be introduced into the polymer.

141


8 Appendices

8.1. Background

8.1.1. Polymers

Polymers have been used as structural materials within nature since life began with

substances such as DNA, polysaccharides and peptides playing crucial roles in

animal and plant life. The term polymer is defined from the Greek words poly and

meros, meaning many parts. In the strictest sense a polymer is a substance composed

of molecules consisting of one or more types of atoms linked to each other by

primarily, usually covalent bonds.87,135 Polymers are created through the linking

together of the small monomer molecules through chemical reactions known as

polymerisation reactions. There are two main types of polymerisation reaction;

addition or chain-growth polymerisation in which, typically a vinyl monomer

(CH2=CHX) is attacked by an initiator to yield an active centre that can then attack

another vinyl monomer linking them together by covalent bonds. Condensation or

step-growth is the second type of polymerisation, here, monomers have different

functional groups (e.g. A-A + B-B → A-a-b-B or A-B + A-B → A-b-a-B) that react

together in a condensation type reaction to form larger molecules consisting of

covalent bonds.

Individual polymer chains can be linked together through the introduction of a di-

functional (or multi-functional) monomer or crosslinker and high levels of

crosslinking result in the formation of a three-dimensional network that is insoluble.

8.1.1.1. Peptides and proteins

Peptides are polymers of amino acids. These substances exhibit a wide range of

differing biological properties allowing them to act as; antibiotics, hormones, food

additives, poisons or pain-killers. Peptides are formed by a condensation reaction

142


between a carboxylic acid and an (primary) amine forming a peptide bond.

Essentially peptide and proteins are the same at the molecular level. The term

protein is used to refer to large molecules typically containing at least fifty amino

acids with a well defined three-dimensional structure. There are twenty amino acids

that are encoded by DNA, each amino acid has the same generalised structure but

with a different side group. These twenty amino acids offer a wide range of

functionalities that can be incorporated into peptides (Figure 8-64). This gives as the

possibly of preparing a huge number of different peptides, for example, for a

pentapeptide there are 3,200,000 possible combinations.136 Within this thesis amino

acids will be referred to either by their full name or the 1-letter abbreviation shown

in Figure 8-64.

143


Figure 8-64. The structures of the twenty DNA encoded amino acids. The single letter

abbreviation is indicated with the brackets.


Two-photon microscopy (TPM)78 is a fluorescent microscopy technique traditionally

used in the fluorescent imaging of biological cells. In this technique the sample is

irradiated with a laser with a wavelength approximately twice that of the excitation

144


length wavelength of the fluorophore. This means that excitation can only occur

when two photons are absorbed simultaneously. This is extremely unlikely and

therefore only occurs at very high photon density; the focal point of the laser beam.

TPM has a number of advantages over the similar technique, confocal microscopy

(in which the sample is irradiated with ultraviolet light with a wavelength equal to

that of the excitation wavelength of the fluorophore) seen in Figure 8-65. The use of

a longer wavelength from the laser for excitation allows for deeper penetration into

the sample. Additionally, because excitation of the fluorophore only occurs at the

focal point the problem of photobleaching is greatly reduced.131

Figure 8-65. Jablonski energy diagram showing a comparison of the excitation of a fluorophore

with a single photon (confocal microscopy) and two photons (two-photon microscopy).

8.1.3. Fluorescence resonance energy transfer

Fluorescence (or Förster) resonance energy transfer (FRET) microscopy is a

technique used for quantifying the distance between two different fluorophores. 137,138

FRET involves the transfer of energy from a fluorescent donor in its excited state to

another excitable moiety, the acceptor, by a non-radiative dipole-dipole interaction

In order for FRET to occur the distance between the donor and the acceptor must be

small (1-10 nm) and results in a decrease in donor emission and an increase in

acceptor emission.

145


FRET allows the determination of whether there is a close association between the

donor and the acceptor. This technique has been used to assess (amongst others)

calcium ion concentration,139 protein-protein colocalisation140 and enzyme

hydrolysis.141

8.2. Supplementary data

8.2.1. HPLC solvent gradient

Figure 8-66. HPLC solvent gradient used in analytical runs. Concentration of buffer B over the

length of a HPLC run.

8.2.2. Synthesis of activated disulfide-methacrylamide monomer

An activated disulfide-methacrylamide monomer was synthesised with the aim of

preparing enzyme responsive particles using on peptide crosslinkers based on the

work of Moore and co-workers.11 This monomer, N-[2-(2-pyridyldithio)]ethyl

methacrylamide (PDTEMA) (Figure 8-67 was initially described by Ruffner and co-

workers for conjugation to oligonucleotides and oligopeptides.142 The procedure

described in this paper was followed, resulting in the successful synthesis of the

monomer (as determined by NMR, Figure 8-68).

146


Figure 8-67. Synthesis of PDTEMA.142

Figure 8-68. Characterisation of PDTEMA by NMR. 1H NMR (CDCl3, 200 MHz): δ (ppm) 7.0-8.6

(m, 5H, Ar-H and -NH), 5.809 (s, 1H, one of d, CH2), 5.361 (s, 1H, one of dCH2), 3.603 (m, 2H, -CH2-

NR), 2.960 (t, 2H, -S-CH2-), 2.002 (d, 3H, -CH3, the split of this peak is due to the tautomerization of

the adjacent double bond).

147


9 References

1 Ratner, B. D. and Bryant, S. J., Biomaterials: Where we have been and where we are going. Annu. Rev. Biomed. Eng. 6, 41-75 (2004).

2 Peppas, N. A. and Langer, R., New challenges in biomaterials. Science 263 (5154), 1715-1720 (1994).

3 Stauffer, R. N., 10-year follow-up-study of total hip-replacement - with particular reference to roentgenographic loosening of the components. J. Bone Joint Surg.-Am. Vol. 64 (7), 983-990 (1982).

4 Conrad, H. J., Seong, W. J., and Pesun, G. J., Current ceramic materials and systems with clinical recommendations: A systematic review. J. Prosthet. Dent. 98 (5), 389-404 (2007).

5 Langer, R., Drug delivery and targeting. Nature 392 (6679), 5-10 (1998).

6 Refojo, M. F., Current status of biomaterials in ophthalmology. Surv. Ophthalmol. 26 (5), 257-265 (1982).

7 Ratner, B.D., in Biomaterials science: An introduction to materials in medicine, edited by AS Hoffman BD Ratner, FJ Schoen, JE Lemons (Academic Press, San Diego, California, 1996), pp. 1-10.

8 Langer, R. and Tirrell, D. A., Designing materials for biology and medicine. Nature 428 (6982), 487-492 (2004).

9 Zhang, X. Z. and Zhuo, R. X., Preparation of fast responsive, temperature-sensitive poly(n-isopropylacrylamide) hydrogel. Macromol. Chem. Phys. 200 (12), 2602-2605 (1999).

10 Tauro, J. R. and Gemeinhart, R. A., Matrix metalloprotease triggered delivery of cancer chemotherapeutics from hydrogel matrixes. Bioconjugate Chem. 16 (5), 1133-1139 (2005).

11 Plunkett, K. N., Berkowski, K. L., and Moore, J. S., Chymotrypsin responsive hydrogel: Application of a disulfide exchange protocol for the preparation of methacrylamide containing peptides. Biomacromolecules 6 (2), 632-637 (2005).

148


12 Nelson, D. L. and Cox, M. M., Principles of biochemistry, Fourth edition ed. (W. H. Freeman and Company, New York, 2005).

13 Duffy, M. J., Proteases as prognostic markers in cancer. Clinical Cancer Research 2 (4), 613-618 (1996).

14 Wysocki, A. B., Staianocoico, L., and Grinnell, F., Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases mmp-2 and mmp-9. J. Invest. Dermatol. 101 (1), 64-68 (1993).

15 Visse, R. and Nagase, H., Matrix metalloproteinases and tissue inhibitors of metalloproteinases - structure, function, and biochemistry. Circ.Res. 92 (8), 827-839 (2003).

16 Hoffman, A. S., Hydrogels for biomedical applications. Advanced Drug Delivery Reviews 54 (1), 3-12 (2002).

17 Stupp, S. I., LeBonheur, V., Walker, K., Li, L. S., Huggins, K. E., Keser, M., and Amstutz, A., Supramolecular materials: Self-organized nanostructures. Science 276 (5311), 384-389 (1997).

18 Petka, W. A., Harden, J. L., McGrath, K. P., Wirtz, D., and Tirrell, D. A., Reversible hydrogels from self-assembling artificial proteins. Science 281 (5375), 389-392 (1998).

19 Zhang, S. G., Marini, D. M., Hwang, W., and Santoso, S., Design of nanostructured biological materials through self-assembly of peptides and proteins. Curr. Opin. Chem. Biol. 6 (6), 865-871 (2002).

20 Osada, Y. and Gong, J. P., Soft and wet materials: Polymer gels. Adv. Mater. 10 (11), 827-837 (1998).

21 Miyata, T., Uragami, T., and Nakamae, K., Biomolecule-sensitive hydrogels. Advanced Drug Delivery Reviews 54 (1), 79-98 (2002).

22 Jeong, B. and Gutowska, A., Lessons from nature: Stimuli-responsive polymers and their biomedical applications. Trends Biotechnol. 20 (7), 305-311 (2002).

23 Mano, J. F., Stimuli-responsive polymeric systems for biomedical applications. Adv. Eng. Mater. 10 (6), 515-527 (2008).

24 Qiu, Y. and Park, K., Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews 53 (3), 321-339 (2001).

25 Gil, E. S. and Hudson, S. A., Stimuli-reponsive polymers and their bioconjugates. Progress in Polymer Science 29 (12), 1173-1222 (2004).

149


26 Kim, J. S., Singh, N., and Lyon, L. A., Displacement-induced switching rates of bioresponsive hydrogel microlenses. Chem. Mat. 19 (10), 2527-2532 (2007).

27 Miyata, T., Asami, N., and Uragami, T., A reversibly antigen-responsive hydrogel. Nature 399 (6738), 766-769 (1999).

28 Ehrbar, M., Rizzi, S. C., Hlushchuk, R., Djonov, V., Zisch, A. H., Hubbell, J. A., Weber, F. E., and Lutolf, M. P., Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering. Biomaterials 28 (26), 3856-3866 (2007).

29 Ahn, S. K., Kasi, R. M., Kim, S. C., Sharma, N., and Zhou, Y. X., Stimuli-responsive polymer gels. Soft Matter 4 (6), 1151-1157 (2008).

30 Wichterle, O. and Lim, D., Hydrophilic gels for biological use. Nature 185 (4706), 117-118 (1960).

31 Barr, J. T., 2004 annual report, Available at http://www.clspectrum.com/article.aspx?article=12733, (2005).

32 Gupta, P., Vermani, K., and Garg, S., Hydrogels: From controlled release to ph-responsive drug delivery. Drug Discov. Today 7 (10), 569-579 (2002).

33 Rzaev, Z. M. O., Dincer, S., and Piskin, E., Functional copolymers of n-isopropylacrylamide for bioengineering applications. Progress in Polymer Science 32 (5), 534-595 (2007).

34 Sershen, S. and West, J., Implantable, polymeric systems for modulated drug delivery. Advanced Drug Delivery Reviews 54 (9), 1225-1235 (2002).

35 Ozbas, B., Kretsinger, J., Rajagopal, K., Schneider, J. P., and Pochan, D. J., Salt-triggered peptide folding and consequent self-assembly into hydrogels with tunable modulus. Macromolecules 37 (19), 7331-7337 (2004).

36 Traitel, T., Cohen, Y., and Kost, J., Characterization of glucose-sensitive insulin release systems in simulated in vivo conditions. Biomaterials 21 (16), 1679-1687 (2000).

37 Goessl, A., Tirelli, N., and Hubbell, J. A., presented at the Symposium on Gels, Genes, Grafts and Giants held in Celebration of the 70th Birthday of Allan Hoffman, Maui, HI, 2002 (unpublished).

38 Kikuchi, A. and Okano, T., Pulsatile drug release control using hydrogels. Advanced Drug Delivery Reviews 54 (1), 53-77 (2002).

39 Roy, I. and Gupta, M. N., Smart polymeric materials: Emerging biochemical applications. Chem. Biol. 10 (12), 1161-1171 (2003).

150


40 Ulijn, R. V., Bibi, N., Jayawarna, V., Thornton, P. D., Todd, S. J., Mart, R. J., Smith, A. M., and Gough, J. E., Bioresponsive hydrogels. Materials Today 10 (4), 40-48 (2007).

41 Lutolf, M. P., Raeber, G. P., Zisch, A. H., Tirelli, N., and Hubbell, J. A., Cell-responsive synthetic hydrogels. Adv. Mater. 15 (11), 888-+ (2003).

42 Kim, S. and Healy, K. E., Synthesis and characterization of injectable poly(n-isopropylacrylamide-co-acrylic acid) hydrogels with proteolytically degradable cross-links. Biomacromolecules 4 (5), 1214-1223 (2003).

43 Hu, B. H. and Messersmith, P. B., Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels. J. Am. Chem. Soc. 125 (47), 14298-14299 (2003).

44 Ehrbar, M., Rizzi, S. C., Schoenmakers, R. G., San Miguel, B., Hubbell, J. A., Weber, F. E., and Lutolf, M. P., Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions. Biomacromolecules 8 (10), 3000-3007 (2007).

45 Goldbart, R., Traitel, T., Lapidot, S. A., and Kost, J., Enzymatically controlled responsive drug delivery systems. Polym. Adv. Technol. 13 (10-12), 1006-1018 (2002).

46 Ruoslahti, E. and Pierschbacher, M. D., New perspectives in cell-adhesion - rgd and integrins. Science 238 (4826), 491-497 (1987).

47 Patel, M., Viscoelastic properties of polystyrene using dynamic rheometry. Polym. Test 23 (1), 107-112 (2004).

48 Jones, M. E. R. and Messersmith, P. B., Facile coupling of synthetic peptides and peptide-polymer conjugates to cartilage via transglutaminase enzyme. Biomaterials 28 (35), 5215-5224 (2007).

49 Goldbart, Riki and Kost, Joseph, Calcium responsive bioerodible drug delivery system. Pharmaceutical Research 16 (9), 1483-1486 (1999).

50 Wu, K., Yang, J. Y., Konak, C., Kopeckova, P., and Kopecek, J., Novel synthesis of hpma copolymers containing peptide grafts and their self-assembly into hybrid hydrogels. Macromol. Chem. Phys. 209 (5), 467-475 (2008).

51 Zhang, R., Bowyer, A., Eisenthal, R., and Hubble, J., A smart membrane based on an antigen-responsive hyrogel. Biotechnology and Bioengineering 97 (4), 976-984 (2007).

52 Li, K. and Stover, H. D. H., Synthesis of monodisperse poly(divinylbenzene) microspheres. Journal of Polymer Science Part a-Polymer Chemistry 31 (13), 3257-3263 (1993).

151


53 Lupas, A., Coiled coils: New structures and new functions. Trends Biochem.Sci. 21 (10), 375-382 (1996).

54 Podual, K., Doyle, F. J., and Peppas, N. A., Preparation and dynamic response of cationic copolymer hydrogels containing glucose oxidase. Polymer 41 (11), 3975-3983 (2000).

55 Podual, K., Doyle, F. J., and Peppas, N. A., Dynamic behavior of glucose oxidase-containing microparticles of poly(ethylene glycol)-grafted cationic hydrogels in an environment of changing ph. Biomaterials 21 (14), 1439-1450 (2000).

56 Chu, L. Y., Li, Y., Zhu, J. H., Wang, H. D., and Liang, Y. J., Control of pore size and permeability of a glucose-responsive gating membrane for insulin delivery. J. Control. Release 97 (1), 43-53 (2004).

57 Thornton, P. D., Mart, R. J., and Ulijn, R. V., Enzyme-responsive polymer hydrogel particles for controlled release. Adv. Mater. 19 (9), 1252-+ (2007).

58 Thornton, P. D., Mart, R. J., Webb, S. J., and Ulijn, R. V., Enzyme-responsive hydrogel particles for the controlled release of proteins: Designing peptide actuators to match payload. Soft Matter 4 (4), 821-827 (2008).

59 Hassan, C. M., Doyle, F. J., and Peppas, N. A., Dynamic behavior of glucose-responsive poly(methacrylic acid-g-ethylene glycol) hydrogels. Macromolecules 30 (20), 6166-6173 (1997).

60 Zhang, K. and Wu, X. Y., Modulated insulin permeation across a glucose-sensitive polymeric composite membrane. J. Control. Release 80 (1-3), 169-178 (2002).

61 Goldraich, M. and Kost, J., presented at the Conf on Biomedical Polymers, Jerusalem, Israel, 1991 (unpublished).

62 Thornton, P. D., McConnell, G., and Ulijn, R. V., Enzyme responsive polymer hydrogel beads. Chem. Commun. (47), 5913-5915 (2005).

63 Kress, J., Zanaletti, R., Amour, A., Ladlow, M., Frey, J. G., and Bradley, M., Enzyme accessibility and solid supports: Which molecular weight enzymes can be used on solid supports? An investigation using confocal raman microscopy. Chem.-Eur. J. 8 (16), 3769-3772 (2002).

64 Ehrick, J. D., Deo, S. K., Browning, T. W., Bachas, L. G., Madou, M. J., and Daunert, S., Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics. Nature Materials 4 (4), 298-302 (2005).

65 Murphy, W. L. Dillmore, S. Modica, J. Mrksich, M., Dynamic hydrogels: Translating a protein conformational change into macroscopic motion. Angewandte Chemie International Edition 46 (17), 3066-3069 (2007).

152


66 Sui, Z. J., King, W. J., and Murphy, W. L., Dynamic materials based on a protein conformational change. Adv. Mater. 19 (20), 3377-+ (2007).

67 Meldal, M., Pega - a flow stable polyethylene-glycol dimethyl acrylamide copolymer for solid-phase synthesis. Tetrahedron Lett. 33 (21), 3077-3080 (1992).

68 Merrifield, R. B., Solid phase peptide synthesis .1. Synthesis of a tetrapeptide. J. Am. Chem. Soc. 85 (14), 2149-& (1963).

69 Meldal, M., Auzanneau, F. I., Hindsgaul, O., and Palcic, M. M., A pega resin for use in the solid-phase chemical-enzymatic synthesis of glycopeptides. J. Chem. Soc.-Chem. Commun. (16), 1849-1850 (1994).

70 Meldal, M., Svendsen, I., Juliano, L., Juliano, M. A., Del Nery, E., and Scharfstein, J., Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin b and cathepsin l. J. Pept. Sci. 4 (2), 83-91 (1998).

71 Renil, M. and Meldal, M., Synthesis and application of a pega polymeric support for high-capacity continuous-flow solid-phase peptide-synthesis. Tetrahedron Lett. 36 (26), 4647-4650 (1995).

72 Renil, M., Ferreras, M., Delaisse, J. M., Foged, N. T., and Meldal, M., Pega supports for combinatorial peptide synthesis and solid-phase enzymatic library assays. Journal of Peptide Science 4 (3), 195-210 (1998).

73 St Hilaire, P. M., Willert, M., Juliano, M. A., Juliano, L., and Meldal, M., Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity. J. Comb. Chem. 1 (6), 509-523 (1999).

74 Basso, A., De Martin, L., Gardossi, L., Margetts, G., Brazendale, I., Bosma, A. Y., Ulijn, R. V., and Flitsch, S. L., Improved biotransformations on charged pega supports. Chem. Commun. (11), 1296-1297 (2003).

75 Basso, A., Ulijn, R. V., Flitsch, S. L., Margetts, G., Brazendale, I., Ebert, C., De Martin, L., Linda, P., Verdelli, S., and Gardossi, L., Introduction of permanently charged groups into pega resins leads to improved biotransformations on solid support. Tetrahedron 60 (3), 589-594 (2004).

76 Basso, A., Ebert, C., Gardossi, L., Linda, P., Phuong, T. T., Zhu, M., and Wessjohann, L., Penicillin g amidase-catalysed hydrolysis of phenylacetic hydrazides on a solid phase: A new route to enzyme-cleavable linkers. Adv. Synth. Catal. 347 (7-8), 963-966 (2005).

77 Basso, A., Maltman, B. A., Flitsch, S. L., Margetts, G., Brazendale, I., Ebert, C., Linda, P., Verdelli, S., and Gardossi, L., Optimized polymer-enzyme

153


electrostatic interactions significantly improve penicillin g amidase efficiency in charged pega polymers. Tetrahedron 61 (4), 971-976 (2005).

78 Denk, W., Strickler, J. H., and Webb, W. W., 2-photon laser scanning fluorescence microscopy. Science 248 (4951), 73-76 (1990).

79 Bosma, A. Y., Ulijn, R. V., McConnell, G., Girkin, J., Halling, P. J., and Flitsch, S. L., Using two photon microscopy to quantify enzymatic reaction rates on polymer beads. Chem. Commun. (22), 2790-2791 (2003).

80 Christensen, C., Groth, T., Schiodt, C. B., Foged, N. T., and Meldal, M., Automated sorting of beads from a "One-bead-two-compounds" Combinatorial library of metalloproteinase inhibitors. QSAR Comb. Sci. 22 (7), 737-744 (2003).

81 Zourob, M., Gough, J. E., and Ulijn, R. V., A micropatterned hydrogel platform for chemical synthesis and biological analysis. Adv. Mater. 18 (5), 655-+ (2006).

82 Deere, J., McConnell, G., Lalaouni, A., Maltman, B. A., Flitsch, S. L., and Halling, P. J., Real-time imaging of protease action on substrates covalently immobilised to polymer supports. Adv. Synth. Catal. 349 (8-9), 1321-1326 (2007).

83 Ulijn, R. V., Baragana, B., Halling, P. J., and Flitsch, S. L., Protease-catalyzed peptide synthesis on solid support. J. Am. Chem. Soc. 124 (37), 10988-10989 (2002).

84 Ulijn, R. V., Bisek, N., and Flitsch, S. L., Enzymatic optical resolution via acylation-hydrolysis on a solid support. Org. Biomol. Chem. 1 (4), 621-622 (2003).

85 Ulijn, R. V., Bisek, N., Halling, P. J., and Flitsch, S. L., Understanding protease catalysed solid phase peptide synthesis. Org. Biomol. Chem. 1 (8), 1277-1281 (2003).

86 Kohli, R. M., Walsh, C. T., and Burkart, M. D., Biomimetic synthesis and optimization of cyclic peptide antibiotics. Nature 418 (6898), 658-661 (2002).

87 Young, R.J. Lovell, P.A., Introduction to polymers, Second Edition ed. (Chapman & Hall, London, 1991).

88 Dowding, P. J. and Vincent, B., Suspension polymerisation to form polymer beads. Colloid Surf. A-Physicochem. Eng. Asp. 161 (2), 259-269 (2000).

89 Dimitratos, J., Elicabe, G., and Georgakis, C., Control of emulsion polymerization reactors. Aiche J. 40 (12), 1993-2021 (1994).

154


90 Anna, S. L., Bontoux, N., and Stone, H. A., Formation of dispersions using "Flow focusing" In microchannels. Appl. Phys. Lett. 82 (3), 364-366 (2003).

91 Xu, S., Nie, Z., Seo, M., Lewis, P., Kumacheva, E., Stone, H. A., Garstecki, P., Weibel, D. B., Gitlin, I., and Whitesides, G. M., Generation of monodisperse particles by using microfluidics: Control over size, shape, and composition (vol 44, pg 724, 2005). Angew. Chem.-Int. Edit. 44 (25), 3799-3799 (2005).

92 Quevedo, E., Steinbacher, J., and McQuade, D. T., Interfacial polymerization within a simplified microfluidic device: Capturing capsules. J. Am. Chem. Soc. 127 (30), 10498-10499 (2005).

93 Takeuchi, S., Garstecki, P., Weibel, D. B., and Whitesides, G. M., An axisymmetric flow-focusing microfluidic device. Adv. Mater. 17 (8), 1067-+ (2005).

94 Nie, Z. H., Xu, S. Q., Seo, M., Lewis, P. C., and Kumacheva, E., Polymer particles with various shapes and morphologies produced in continuous microfluidic reactors. J. Am. Chem. Soc. 127 (22), 8058-8063 (2005).

95 Seo, M., Nie, Z. H., Xu, S. Q., Mok, M., Lewis, P. C., Graham, R., and Kumacheva, E., Continuous microfluidic reactors for polymer particles. Langmuir 21 (25), 11614-11622 (2005).

96 Zourob, M., Mohr, S., Mayes, A. G., Macaskill, A., Perez-Moral, N., Fielden, P. R., and Goddard, N. J., A micro-reactor for preparing uniform molecularly imprinted polymer beads. Lab Chip 6 (2), 296-301 (2006).

97 Serra, C., Berton, N., Bouquey, M., Prat, L., and Hadziioannou, G., A predictive approach of the influence of the operating parameters on the size of polymer particles synthesized in a simplified microfluidic system. Langmuir 23 (14), 7745-7750 (2007).

98 Leon, S., Quarrell, R., and Lowe, G., Evaluation of resins for on-bead screening: A study of papain and chymotrypsin specificity using pega-bound combinatorial peptide libraries. Bioorg. Med. Chem. Lett. 8 (21), 2997-3002 (1998).

99 LaVan, D. A., McGuire, T., and Langer, R., Small-scale systems for in vivo drug delivery. Nature Biotechnology 21 (10), 1184-1191 (2003).

100 McDonald, T. O. , Christensen, S. , and Ulijn, R. V., Making peg-based microparticles for applications in biology and medicine. Mater. Res. Soc. Symp. Proc 1008E (T05), 18 (2007).

101 McDonald, T. O., Making size and distribution controlled pega hydrogels for use in biology and medicine, University of Manchester, (2005)

155


102 Freemont, T. J. and Saunders, B. R., Ph-responsive microgel dispersions for repairing damaged load-bearing soft tissue. Soft Matter 4 (5), 919-924 (2008).

103 Kim, J., Nayak, S., and Lyon, L. A., Bioresponsive hydrogel microlenses. J. Am. Chem. Soc. 127 (26), 9588-9592 (2005).

104 Patel, K., Angelos, S., Dichtel, W. R., Coskun, A., Yang, Y. W., Zink, J. I., and Stoddart, J. F., Enzyme-responsive snap-top covered silica nanocontainers. J. Am. Chem. Soc. 130 (8), 2382-2383 (2008).

105 Duxbury, C. J., Hilker, I., de Wildeman, S. M. A., and Heise, A., Enzyme-responsive materials: Chirality to program polymer reactivity. Angew. Chem.-Int. Edit. 46 (44), 8452-8454 (2007).

106 Trengove, N. J., Stacey, M. C., Macauley, S., Bennett, N., Gibson, J., Burslem, F., Murphy, G., and Schultz, G., Analysis of the acute and chronic wound environments: The role of proteases and their inhibitors. Wound Repair and Regeneration 7 (6), 442-452 (1999).

107 Duncan, R., Gac-Breton, S., Keane, R., Musila, R., Sat, Y. N., Satchi, R., and Searle, F., Polymer-drug conjugates, pdept and pelt: Basic principles for design and transfer from the laboratory to clinic. J. Control. Release 74 (1-3), 135-146 (2001).

108 Duncan, R., Vicent, M. J., Greco, F., and Nicholson, R. I., Polymer-drug conjugates: Towards a novel approach for the treatment of endrocine-related cancer. Endocrine-Related Cancer 12, S189-S199 (2005).

109 Shabat, D., Self-immolative dendrimers as novel drug delivery platforms. Journal of Polymer Science Part a-Polymer Chemistry 44 (5), 1569-1578 (2006).

110 Hamielec, A.E. Tobita, H., Polymerization processes, ullmann's encyclopedia of industrial chemistry. (VCH publishers, lnc., New York, 1992).

111 Arshady, R., Beaded polymer supports and gels .1. Manufacturing techniques. Journal of Chromatography 586 (2), 181-197 (1991).

112 Morihara, K., Tsuzuki, H., and Oka, T., Comparison of specificities of various neutral proteinases from microorganisms. Archives of Biochemistry and Biophysics 123 (3), 572-& (1968).

113 Yasukawa, K., Kusano, M., and Inouye, K., A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in escherichia coli. Protein Eng. Des. Sel. 20 (8), 375-383 (2007).

156


114 Harris, J. L., Backes, B. J., Leonetti, F., Mahrus, S., Ellman, J. A., and Craik, C. S., Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proceedings of the National Academy of Sciences of the United States of America 97 (14), 7754-7759 (2000).

115 Creighton, Thomas C, Encyclopedia of molecular biology. (John Wiley & Sons, 1999).

116 Norioka, . S. and Sakiyama, . F., in Handbook of proteolytic enzymes, edited by. A. J. Barrett, . N. D. Rawlings, and . J. R. Woessner (Elsevier, 2004), Vol. 2, pp. 1483-1487.

117 Kaiser, E., Colescot.Rl, Bossinge.Cd, and Cook, P. I., Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34 (2), 595-& (1970).

118 Medlock, K., Harmer, H., Worsley, G., Horgan, A., and Pritchard, J., Ph-sensitive holograms for continuous monitoring in plasma. Analytical and Bioanalytical Chemistry 389, 1533-1539 (2007).

119 Cortese, J. D., Voglino, A. L., and Hackenbrock, C. R., Ionic-strength of the intermembrane space of intact mitochondria as estimated with fluorescein-bsa delivered by low ph fusion. Journal of Cell Biology 113 (6), 1331-1340 (1991).

120 Miki, Y., Kidokoro, S., Endo, K., Wada, A., Yoneya, T., Aoyama, A., Kai, K., Miyake, T., and Nagao, H., Effect of a charged residue at the 213th site of thermolysin on the enzymatic activity. J. Mol. Catal. B-Enzym. 1 (3-6), 191-199 (1996).

121 Whitesides, G. M., The origins and the future of microfluidics. Nature 442 (7101), 368-373 (2006).

122 Garstecki, P., Gitlin, I., DiLuzio, W., Whitesides, G. M., Kumacheva, E., and Stone, H. A., Formation of monodisperse bubbles in a microfluidic flow-focusing device. Appl. Phys. Lett. 85 (13), 2649-2651 (2004).

123 Goebel, A. and Lunkenheimer, K., Interfacial tension of the water/n-alkane interface. Langmuir 13 (2), 369-372 (1997).

124 Lu, N. and Likos, W. J., Unsaturated soil mechanics. . (John Wiley & Sons., 2004).

125 Capek, I., Sterically and electrosterically stabilized emulsion polymerization. Kinetics and preparation. Adv. Colloid Interface Sci. 99 (2), 77-162 (2002).

126 Peltonen, L., Hirvonen, J., and Yliruusi, J., The behavior of sorbitan surfactants at the water-oil interface: Straight-chained hydrocarbons from

157


pentane to dodecane as an oil phase. J. Colloid Interface Sci. 240 (1), 272-276 (2001).

127 Shah, R. K., Shum, H. C., Rowat, A. C., Lee, D., Agresti, J. J., Utada, A. S., Chu, L. Y., Kim, J. W., Fernandez-Nieves, A., Martinez, C. J., and Weitz, D. A., Designer emulsions using microfluidics. Materials Today 11 (4), 18-27 (2008).

128 Barreiros, F. M., Ferreira, P. J., and Figueiredo, M. M., Calculating shape factors from particle sizing data. Part. Part. Syst. Charact. 13 (6), 368-373 (1996).

129 Cheng, C. J., Chu, L. Y., Zhang, J., Zhou, M. Y., and Xie, R., Preparation of monodisperse poly(n-isopropylacrylamide) microspheres and microcapsules via shirasu-porous-glass membrane emulsification. Desalination 234 (1-3), 184-194 (2008).

130 Qu, H. H., Gong, F. L., Ma, G. H., and Su, Z. G., Preparation and characterization of large porous poly(hema-co-edma) microspheres with narrow size distribution by modified membrane emulsification method. Journal of Applied Polymer Science 105 (3), 1632-1641 (2007).

131 Robinson, R. K., Encyclopedia of food microbiology. (Elsevier., 2000).

132 Kriwet, B., Walter, E., and Kissel, T., Synthesis of bioadhesive poly(acrylic acid) nano- and microparticles using an inverse emulsion polymerization method for the entrapment of hydrophilic drug candidates. J. Control. Release 56 (1-3), 149-158 (1998).

133 Asua, J. M., Miniemulsion polymerization. Progress in Polymer Science 27 (7), 1283-1346 (2002).

134 Antonietti, M. and Landfester, K., Polyreactions in miniemulsions. Progress in Polymer Science 27 (4), 689-757 (2002).

135 Hiemenz, P.C., Polymer chemistry. (Marcel Dekker, New York and Basel, 1984).

136 Bailey, P.D., An introduction to peptide chemistry. (John Wiley & Sons, Chichester, 1990).

137 Gordon, G. W., Berry, G., Liang, X. H., Levine, B., and Herman, B., Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74 (5), 2702-2713 (1998).

138 Jares-Erijman, E. A. and Jovin, T. M., Fret imaging. Nature Biotechnology 21 (11), 1387-1395 (2003).

158


139 Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y., Fluorescent indicators for ca2+ based on green fluorescent proteins and calmodulin. Nature 388 (6645), 882-887 (1997).

140 Kenworthy, A. K., Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24 (3), 289-296 (2001).

141 Meldal, M., The one-bead two-compound assay for solid phase screening of combinatorial libraries. Biopolymers 66 (2), 93-100 (2002).

142 Wang, L. X., Kristensen, J., and Ruffner, D. E., Delivery of antisense oligonucleotides using hpma polymer: Synthesis of a thiol polymer and its conjugation to water-soluble molecules. Bioconjugate Chem. 9 (6), 749-757 (1998).

159

tom's thesis submitted copy

Documents