functionalised dextran nanoparticles for drug delivery to ... · functionalised dextran...

i

Functionalised dextran nanoparticles

for drug delivery to the brain

IBEGBU, Madu Daniel

March, 2015

A thesis submitted in partial fulfilment of the requirements for the award of the degree of

Doctor of Philosophy of the University of Portsmouth.

Biomaterials and Drug Delivery Research Group

School of Pharmacy and Biomedical Sciences

University of Portsmouth

Abstract

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Abstract

Towards the development of drug carriers that are capable of crossing the Blood Brain

Barrier, the techniques of emulsion polymerisation and nanoprecipitation have been

utilised to produce nanoparticulate carriers from a systematic series of alkylglyceryl

dextrans (of two different average molecular weights, 6 kDa and 100 kDa) that had been

functionalised with ethyl and butyl cyanoacrylates. Also, zero length grafting of polylactic

acid to butyl, octyl and hexadecylglyceryl dextrans has allowed the preparation of

polylactic acid-functionalised nanoparticles. All materials and derived nanoparticles have

been characterised by a combination of spectroscopic and analytical techniques. The

average size of nanoparticles has been found to be in the range 100-500 nm. Tagging or

loading of the nanoparticles with fluorophores or model drugs allowed the preliminary

investigation of their capability to act as controlled-release devices. The effects of an

esterase on the degradation of one such nanoparticulate carrier have been studied.

Testing against bend3 cells revealed that all materials display dose-dependent

cytotoxicity profiles, and allowed the selection of nanocarriers that may be potentially

useful for further testing as therapeutic delivery vehicles for conditions of the brain.

Dedication

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Dedication

To God Almighty

Acknowledgements

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Acknowledgements

I would like to express my appreciations to my supervisors Dr Eugen Barbu and Dr John

Tsibouklis for their support and kind assistance through the period of my PhD.

I would like to acknowledge Dr Asme Boussahel for her assistance with cell work, I also

acknowledge Mrs Christine Hughes for SEM work, Val Ferrigan for logistical support; and

to my colleagues Ashleigh, Temmy and Fauzi for their friendly company.

I would like also to acknowledge TETFUND-Nigeria through University of Nigeria for

providing the financial support.

Declaration

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Declaration

Whilst registered as a candidate for the degree of Doctor of Philosophy, I have not been

registered for any other award. This work was sponsored by the Tertiary Education Trust

Fund (TETFUND) Nigeria, through University of Nigeria, Nsukka. The material contained

within this thesis is all my own work and has not been submitted for any other academic

award.

Ibegbu, Madu Daniel

Contents

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Contents

Contents

Abstract ............................................................................................................................................... ii

Dedication .......................................................................................................................................... iii

Acknowledgements ............................................................................................................................ iv

Declaration .......................................................................................................................................... v

Contents ............................................................................................................................................. vi

Abbreviation list .................................................................................................................................. x

Lists of Tables .................................................................................................................................... xii

Lists of Figures .................................................................................................................................. xiv

1. Introduction and aims ..................................................................................................................... 1

1.1 Drug delivery to the brain ......................................................................................................... 1

1.1.1 The blood-brain barrier ...................................................................................................... 2

1.1.2 Strategies for delivering drugs to the brain ........................................................................ 9

1.1.2.1 Invasive approaches ........................................................................................................ 9

1.1.2.2 Use of penetration enhancers .......................................................................................... 9

1.1.2.3 Prodrugs ........................................................................................................................ 10

1.1.2.4 Modifications of influx transporters.............................................................................. 10

1.1.2.5 Passive drug targeting ................................................................................................... 11

1.1.2.6 Active drug targeting .................................................................................................... 11

1.1.2.7 Carrier-mediated delivery (Colloidal drug delivery systems) ....................................... 11

1.1.3 Nanoformulated drugs currently in clinical trials ............................................................ 17

1.2 Polymeric drug delivery systems ............................................................................................ 18

1.2.1 Poly(alkyl cyanoacrylate)s ............................................................................................... 20

1.2.2. Poly lactic acid (PLA) ..................................................................................................... 22

1.2.3. Polysaccharides ............................................................................................................... 22

1.2.4. Polycyanoacrylates.......................................................................................................... 25

1.3. Methods for the preparation of nanocarriers ....................................................................... 26

1.4. Characterisation techniques .................................................................................................. 26

1.4.1. Elemental analysis........................................................................................................... 27

1.4.2. MALDI-TOF MS ............................................................................................................ 28

1.4.3. Nanoparticle Tracking Analysis (NTA) .......................................................................... 31

1.4.4. Zeta potential determination ........................................................................................... 32

1.4.5 TGA ................................................................................................................................. 32

1.4.6 SEM ................................................................................................................................. 33

Contents

vii

1.5. Aim of the study ..................................................................................................................... 35

2. Chemical modifications of dextran ............................................................................................... 36

2.1 Dextran .................................................................................................................................... 36

2.1.1 Chemical structure of oxiranes for synthesis of alkyl-glyceryl dextrans ......................... 38

2.2 Materials and instrumentation ............................................................................................... 38

2.3 Methods .................................................................................................................................. 39

2.4 Results and Discussion ............................................................................................................ 40

2.4.1 Synthesis of alkylglyceryl dextrans ................................................................................. 40

2.4.2 Characterisation of the alkylglyceryl dextrans ................................................................. 42

2.4.2.1 NMR and FTIR ............................................................................................................. 42

2.4.2 GPC .................................................................................................................................. 46

2.4.6 Discussion on synthesis and characterisation techniques ................................................ 56

2.5 Conclusions ............................................................................................................................. 57

3. Preparation and characterisation of nanoparticles ...................................................................... 58

3.1 Mechanism of ACA polymerisation ......................................................................................... 58

3.2 Materials and instrumentation ............................................................................................... 60

3.3 Methods .................................................................................................................................. 62

3.3.1 Attempted NP formulation with ethyl 2-cyano-3-ethoxyacrylate .................................... 62

3.3.1.1 Preparation of PECA-Dex100 nanoparticles ................................................................ 62

3.3.2 Preparation of poly (ethyl 2-cyanoacrylate)–alkylglyceryl-dextran NPs ......................... 63

3.3.3 Preparation of PLA15-Dex100G8-PECA ........................................................................ 64

3.3.4 Effect of filtration on size of PECA-Dex100G8 NPs ...................................................... 64

3.3.5 Preparation of PECA-Dex6G16 nanoparticles by nanoprecipitation ............................... 65

3.3.6 Preparation of PLA-derived nanoparticles by zero length crosslinking .......................... 65

3.3.7 Preparation of PBCA-Dex100G4 nanoparticles .............................................................. 66

3.3.8 Degradation study ............................................................................................................ 66

3.4 Results and discussion ............................................................................................................ 68

3.4.1 Preparation of nanoparticles of ethyl 2-cyanoacrylate ..................................................... 68

3.4.1.1 Preparation of PECA-Dex100 ....................................................................................... 68

3.4.1.2 Preparation of poly (ethyl 2-cyanoacrylate)–alkylglyceryl-dextran NPs ...................... 68

3.4.1.2.1 Preparation of PECA-Dex6G16 NPs by nanoprecipitation ....................................... 68

3.4.1.2.2 Effect of filtration on size of PECA-Dex100G8 NPs ................................................ 69

3.4.2 Elemental analysis results ................................................................................................ 72

3.4.3 MALDI-TOF-MS of PECA-alkylglyceryl dextran NPs .................................................. 74

3.4.4 Autotitration of PECA-alkylglyceryl dextran nanoparticles ........................................... 76

3.4.5 Preparation of PBCA-Dex100G4 nanoparticles .............................................................. 80

3.4.7 TGA and DSC thermograms of PLA-Dex100G8-PECA ................................................ 82

Contents

viii

3.4.8 Preparation of PLA-derived nanoparticles by zero length crosslinking .......................... 84

3.5 Enzymatic degradation of PECA-alkylglyceryl dextran nanoparticles ..................................... 86

3.6. Conclusions ............................................................................................................................ 90

4. Loading of nanoparticles with drugs, peptides and fluorophores ................................................ 92

4.1. Actives and fluorophores ....................................................................................................... 92

4.2 Methods and instrumentation ................................................................................................ 95

4.2.1. Loading of PECA-Dex, PECA-Dex100G4 and PECA-Dex100G8 NPs with Rhodamine

B ................................................................................................................................................ 95

4.2.2 PECA-Dex100G4 and PLA-Dex100G8PECA Nanoparticle-loading with Curcumin: the

EtOH/H2O (1:1) method ........................................................................................................... 96

4.2.2.3 Loading and release of Doxorubicin hydrochloride from PECA-Dex6G4 NPs and

PECA-Dex6G8 NPs by nanoprecipitation-solvent evaporation ............................................... 97

4.2.4 The preparation of PECA-Dex100G4-Dox ...................................................................... 98

4.2.5 Loading of Doxorubicin hydrochloride to PECA-Dex6G16 ........................................... 98

4.2.6 Determination of EGFP enhanced green fluorescence protein (EGFP) ........................... 98

4.2.7 Tagging of PECA-Dex100G8 with MIA ......................................................................... 98

4.2.8 Tagging of MIA to PECA-Dex16 and loading of Curcumin ......................................... 100

4.2.9 Tagging of Tetramethyl Rhodamine-5-carbonyl azide (TMRCA) to PECA-Dex6G12

nanoparticles ........................................................................................................................... 100

4.2.10 Evaluation of Evans blue retentive capacity ................................................................ 100

4.3 Results and discussion .......................................................................................................... 102

4.3.1 Rhodamine ..................................................................................................................... 102

4.3.2 Curcumin ........................................................................................................................ 103

4.3.3. Doxorubicin .................................................................................................................. 103

4.3.3.2 PECA-Dex6G16-Doxorubicin (PECA-Dex6G16-Dox) nanoparticles ....................... 104


PECA-Dex6G8 NPs by nanoprecipitation-solvent evaporation ............................................. 104

4.3.3.4 Loading of both Doxorubicin and Curcumin into PECA-Dex100G12nanoparticles: 106

4.3.3.5 PECA-Dex100G12-Dox TGA and DSC results ......................................................... 107

4.3.4 Other fluorescent markers and model drugs .................................................................. 108

4.3.4.1 Enhanced green fluorescence protein (EGFP) determination in NPs ......................... 108

4.3.4.2 N-methyl-isatoic anhydride (MIA) ............................................................................. 109

4.3.4.3 Tetramethyl Rhodamine-5-carbonylazide ................................................................... 110

4.4 Conclusions ........................................................................................................................... 112

5. In vitro studies - Cytotoxicity ...................................................................................................... 113

5.1 Methods and instrumentation .............................................................................................. 113

5.2 Results and Discussion .......................................................................................................... 114

5.3. Conclusions .......................................................................................................................... 119

Contents

ix

6. General Conclusions and suggestions for further work .............................................................. 120

7. References .................................................................................................................................. 122

8. Appendices .................................................................................................................................. 133

Abbreviations

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Abbreviation list

ACA= Alkyl cyanoacrylates

AFM= Atomic force microscopy

BBB= Blood-brain barrier

BCA= n-butyl cyanoacrylate

CNS= Central nervous system

DCM= Dichloromethane

Dex= Dextran

Dex6= Dextran 6 kDa

Dex100= Dextran 100 kDa

Dex6G4 = Dextran 6 kDa modified with butyl glycidyl ether

Dex6G8 = Dextran 6 kDa modified with octyl glycidyl ether

Dex6G12= Dextran 6 kDa modified with glycidyl lauryl ether

Dex6G14= Dextran 6 kDa modified with dodecyl /tetradecyl glycidyl ether

Dex6G16= Dextran 6 kDa modified with hexadecyl glycidyl ether

Dex100G4 = Dextran 100k Da modified with butyl glycidyl ether

Dex100G8 = Dextran 100 kDa modified with octyl glycidyl ether

Dex100G12 = Dextran 100 kDa modified with glycidyl lauryl ether

Dex100G14 = Dextran 100kDa modified with dodecyl /tetradecyl glycidyl ether

Dex100G16 = Dextran 100kDa modified with hexadecyl glycidyl ether

DLS= Dynamic light scattering

DMEM= Dulbecco's Modified Eagle Medium

DMSO= Dimethyl sulfoxide

DS= Degree of substitution

DSC=Differential scanning calorimetry

ECA= ethyl 2-cyanoacrylate

GPC= Gel permeation chromatography.

ICS= Cerebrospinal fluid

ISF= Interstitial fluid

M/Z= mass-to-charge ratio

MALDI/TOF MS = Matrix assisted laser desorption/ionisation time of flight mass

spectroscopy

Abbreviations

xi

MIA= N-Methylisatoic anhydride

MRP=Multi Drug resistance protein

MTT= 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NPs= Nanoparticles

NTA= Nanoparticle Tracking Analysis

OGE=Octyl glycidyl ether

PACA= Poly (alkyl cyanoacrylates)

PECA-Dex100G4(1:1)= prepared with equal ratio of ECA monomer to modified dextran

PECA-Dex100G16(1:6) =prepared with 6:1 ratio of ECA monomer to modified dextran

PBCA= Poly (butyl cyanoacrylate)

PDI= Polydispersity index

PECA= Poly (ethyl 2-cyanoacrylate)

PES=Polyethersulphone

PLGA= Poly(lactic-co-glycolic acid)

PLA= Poly (lactic acid)

SEM= Scanning electron microscope

t-BuOK= Potassium tert-Butoxide

TEM= Transmission electron microscope

TGA=Thermal gravimetric analysis

ZP= Zeta potential

Lists of tables

xii

Lists of Tables

Table 1.1: Representative examples of nanoformulations water insoluble drugs that are approved for clinical use or under clinical trial

Table 1.2: Classification of biodegradable polymers

Table2.1: The degree of substitution of the glyceryl dextrans

Table 2.2: Pullulan standard calibration data

Table 2.3: Equation calculated for GPC calibration curve

Table2.4: Mn, Mw and PDI of the dextrans and alkylglyceryl dextrans

Table 2.5: m/z fragment mass loss of the modified dextrans

Table 2.6: TGA results of first and second mass loss, and temperature of modified and unmodified dextrans

Table 3.1: Protocol for the degradation study of PECA-Dex100G4

Table 3.2: DLS Characterisation PECA-Dex6G16 nanoparticles

Table 3.3: Effect of filtration of NPs on size, PDI and zeta potential (±SD; n=3)

Table 3.4: Percentage elemental composition of PECA-alkylglyceryl dextran NPs (experimentally found and calculated)

Table 3.5: m/z fragment mass loss of PECA-alkylglyceryl dextran NPs

Table 3.6 Comparison of effects of pH on the size and zeta potential of titrated nanoparticles

Table 3.7: Size, PDI and zeta potential of PBCA-Dex100G4 nanoparticles

Table 3.8: Size and PDI of PLA15-Dex100G8-PECA NPs (±SD, n=3)

Table 4.1: Size, PDI and zeta potential of Rhodamine B-loaded, Polysorbate 80-coated PECA-Dex100 NPs

Table 4.2: The absorbance and concentration of Curcumin in the supernatant ( EtOH:H2O), (1:1) from the loading of NPs.

Table 4.3: Concentration of Curcumin content of dissolved Curcumin loaded NPs

Table 4.4: Size, PDI and zeta potential of PECA-Dex100G4-Dox nanoparticles

Table 4.5: Size, zeta potential and PDI characterisation of Doxorubicin-PECA-Dex16 (Dox-PD16) nanoparticles (DLS)

Table 4.6 percentage Doxorubicin loading to the nanoparticles(loading effeciency)

Table 4.7: Size, PDI and zeta potential of PECA-Dex6G12 nanoparticles loaded with Doxorubicin-Curcumin.

Lists of tables

xiii

Table 4.8: Determination of concentrations of EGFP in PECA-Dex100G8-EGFP NPs

Table 4.9: The effect of different liquid media on the size distribution of NPs of PECA-Dex100G8 that had been tagged with MIA (DLS)

Table 4.10: Fluorescence intensity of MIA-Curcumin nanoparticles

Table 4.11: DLS analysis of MIA-Curcumin NPs

Table 4.12: The size and PDI of PECA-Dex6G12-TMRCA nanoparticles

Table 5.1: cytotoxicity dose response of alkyl cyanoacrylate-alkylglyceryl dextran NPs against bEND3 cells

Lists of figures

xiv

Lists of Figures

Figure 1.1: The structure of the brain

Figure 1.2: Cellular interplay at the neurovascular unit (capillary level)

Figure 1.3: Tight junction

Figure 1.4: A simplified atlas of the BBB

Figure 1.5: Structures of heroin and morphine

Figure 1.6: Types of drug delivery systems

Figure 1.7: Schematic representation of liposomes and micelles

Figure 1.8: The structure of dendrimer

Figure 1.9: Schematic representation of the structure of nanosphere and nanocapsule for drug delivery

Figure 1.10: The structures of alkyl cyanoacrylate (R= methyl, ethyl, butyl, isobutyl, isohexyl, octyl etc.) (A) and poly(lactic acid) (B).

Figure 1.11: The chemical structure of dextran.

Figure 1.12: Schematic representation of an elemental analyser

Figure 1.13: Schematic representation of the MALDI instrument

Figure 1.14: Non-destructive vaporisation and ionisation of biomolecules by MALDI TOF

Figure 1.15: Schematic representation of the Nanoparticle Tracking Analysis (NTA)

Figure 1.16: Schematic representation of particles and surround charges

Figure 1.17: Representative TGA curve

Figure 1.18: The scanning electron microscope

Figure 2.1: The chemical structure of dextran

Figure 2.2: Chemical structure of alkyl glycidyl ether where: n=1, butyl; n=4,octyl; n=8, lauryl; n=12, hexadecyl.

Figure 2.3: Scheme for the synthesis of alkylglyceryl dextrans

Figure2.4: 1H-NMR spectrum of alkyl glyceryl dextran (Dex100G16)

Figure2.5: 1H-NMR spectrum of butyl alkyl glyceryl dextran (Dex100G4)

Figure2.6: 1H-NMR of native dextran

Figure2.7: FTIR of butyl glyceryl dextran (Dex100G4)

Figure2.8: FTIR of octyl glyceryl dextran (Dex100G8)

Lists of figures

xv

Figure 2.9: Pullulan standard calibration curve

Figure 2.10: GPC chromatogram of native dextran (Dex6)

Figure2.11: GPC chromatogram of native dextran (Dex100)

Figure2.12: GPC chromatogram of Dex6G4

Figure 2.13: GPC chromatogram of Dex100G16

Figure 2.14: MALDI TOF spectrum of Dex6G4

Figure 2.15 MALDI TOF spectrum of Dex100G4

Figure 2.16: TGA results for Dex6

Figure 2.17: TGA results of Dex6G4

Figure 2.18: First mass loss plot of percentage mass loss the chain length (Dex100 and derivatives)

Figure 2.19: First mass loss plot of percentage mass loss against the chain length (Dex6 and derivatives)

Figure 3.1: Mechanism for the anionic emulsion polymerisation of ACA, as initiated by

mild nucleophiles

Figure3.2: Structure of ethyl 2-cyano-3-ethoxyacrylate

Figure 3.3: Schematic representation of the protocol for the synthesis and characterisation of PACA-alkylglyceryl dextran.

Figure 3.4: Scheme for the synthesis of PLA-alkylglyceryl dextran nanoparticles

Figure 3.5: 1H NMR spectrum of PECADex100G8 nanoparticles

Figure3.6: 1H-NMR spectrum of pure PECA

Figure3.7: 13C-NMR spectrum of pure PECA

Figure 3.8 MALDI TOF spectrum of PECADex6G4

Figure 3.9: MALDI TOF spectrum of PECA-Dex6G12

Figure 3.10: Effect of pH on PECA-nanoparticles (0.25mg/mL) MPT-2 Titration-[0.005M/0.5 M HCl] (n=2, ±SD)

Figure 3.11: Effect of pH on PECA-Dex6G4 (1:1) nanoparticles (0.25 mg/mL); MPT-2 Titration (0.005/0.5 M HCl; n=3 ±SD)

Figure 3.12: Effect of pH on the characteristics of PECA-Dex6G4 (1:6) nanoparticles (0.25mg/mL); MPT-2 Titration (0.005/0.5 M HCl; n=3 ±SD).

Figure 3.13: Effect of pH on PECA-Dex6G16 (1:1) nanoparticles (0.25 mg/mL); MPT-2 titration (0.005/0.5 M HCl; n=3 ±SD).

Lists of figures

xvi

Figure3.14: Effect of pH on PECA-Dex6G16 (1:6) nanoparticles (0.25 mg/mL); MPT-2 Titration (0.005/0.5 M HCl; n=3 ±SD).

Figure 3.15: Size concentration intensity distribution of PBCA-dex100G4 NPs

Figure3.16 FTIR spectrum of PBCA-Dex100G4 NPs

Figure3.17: 1H-NMR spectrum of PLA15-Dex100G8-PECA (400 MHz).

Figure 3.18: TGA plots of PLA-Dex100G8-PECA NPs

Figure 3.19: DSC of PLA-Dex100G8-PECA NPs

Figure 3.20: 1H NMR spectrum of PLA15-Dex100G8 nanoparticles.

Figure 3.21: 13CNMR NMR of PLA15-Dex100G8 nanoparticles

Figure 3.22: 13CNMR NMR of PLA15-Dex100G8 nanoparticles

Figure 3.23: Effect of time on size and PDI of PECA-Dex100G4 NPs (n=3, ±SD) (Control, without enzyme)

Figure 3.24: Effect of constant enzyme concentration on size and PDI of PECA-Dex100G4 nanoparticles as a function of enzyme (n=3±SD).

Figure 3.25: Effect of enzyme concentrations on size and PDI of PECA-Dex100G4 nanoparticles as a function of enzyme (n=3 ±SD).

Figure3.26: Enzyme-mediated degradation of PECA-NPs during the first 4h of the extended study.

Figure 3.27: Enzyme degradation of PECA-Dex100G4

Figure 4.1: Chemical structure of Rhodamine B

Figure4.2: Chemical structure of Curcumin

Figure4.3: Chemical structure of Doxorubicin hydrochloride

Figure4.4: MIA reaction with PECA-Dex100G8

Figure 4.5: Covalent linkage of Tetramethyl Rhodamine to PECA-Dex6G12 nanoparticles

Figure 4.6: Size distribution of PECA-Dex, PECA-Dex100G4 and PECA-Dex100G8 loaded with Rhodamine B (n=3)

Figure 4.7: Cumulative release profile of Doxorubicin from Doxorubicin HCl, PECA-Dex6G4 and PECA-Dex6G8 (n=3, ±SD).

Figure 4.8 TGA plots from PECA-Dex100G12-Dox nanoparticles.

Figure 4.9: DSC of PECA-Dex100G12–Doxorubicin nanoparticles.

Figure 4.10: Chemical structure of Evans blue

Figure 5.1: Relative toxicity of PECA and PECA-alkylglyceryl dextran nanoparticles (1 mg/mL)

Lists of figures

xvii

Figure 5.2: Relative cytotoxicity of alkylglyceryl dextran (1 mg/mL) (p<0.05)

Figure 5.3: Relative viability as a function of dose response of alkyl cyanoacrylate NPs against bEnd3 cells, each incubated at specified concentrations (10 μg/mL, 25 μg/mL 50 μg/mL 100 μg/mL) for 24 h; MTT assay; Triton –X100 and media blank (n =3, ±SD; Triton-X100 positive control).

Introduction and aims

1

1

1. Introduction and aims

1.1 Drug delivery to the brain

In 2010 it was estimated that 38% of the European population suffered from a brain

disorder as compared with 27% in 2005 (1). These disorders include the

neurodegenerative diseases (dementia, epilepsy, multiple sclerosis) and mental disorders

(depression, schizophrenia, panic disorder, drug dependence and insomnia).

Epidemiological studies have shown that about one third of the world population may

suffer from some mental disorder at some stage in their lives (2). Schizophrenia,

depression, epilepsy, dementia, alcohol dependence and other mental, neurological and

substance-use (MNS) disorders constitute 13% of the global burden of disease, surpassing

both cardiovascular disease and cancer (3). A recent review has identified several CNS

(central nervous system) diseases that may be treatable with biological actives if

appropriate therapeutic delivery systems were to be developed (4).

Figure 1.1: The structure of the brain (5)

The human brain (Figure 1.1) is irrigated by capillaries the total length of which is

estimated at ca. 644 km; corresponding to surface area available for transport of about

20 m2 (6), and a thickness of the cerebral endothelial membrane of the order of 0.2-0.3

µm.


2

One of the major inhibiting factors to the efficient treatment of brain disorders is the lack

of universally applicable methods for transporting therapeutic agents across the blood-

brain barrier (BBB), which has evolved to separate circulating blood from the brain

extracellular fluid (BECF) in the central nervous system (CNS) such as to prevent potential

neurotoxins from reaching the brain (7).

The BBB, which is formed by capillary endothelial cells (ECs) that are connected by tight

junctions with an extremely high electrical resistivity (> 0.1 Ω m), allows the passive

diffusion of some gases and that of water and of some lipid soluble molecules, and also

the selective transport of certain molecules (glucose, amino acids) that are crucial to

neural function. Consequently, therapeutic approaches to circumvent the BBB without

altering its integrity are an area of intense activity in drug research and development.

1.1.1 The blood-brain barrier

The acknowledgement of the existence of the BBB is consequent to the work of Ehrlich,

Lewandowsky and Goldmann, in the late 1800s and early 1900s, who reported the

absence of bile acids, ferrocyanide or trypan blue in the brain and spinal cord following

intravenous administration of each of these agents (8-11). The BBB is a highly specialised

brain endothelial structure of the fully differentiated neurovascular system (8). It is

formed from brain capillary endothelial cells (12) and localized at the level of tight

junctions (TJ) between adjacent cells. It has been described as a multicellular vascular

structure that isolates the CNS from systemic blood circulation (13), inhibiting the

transport into the brain of plasma components, red blood cells and leukocytes. Thus the

BBB is crucial for preservation and regulation of the neural microenvironment; the

specialised TJs have been described as the core structures responsible for these functions.

The endothelial cells of the BBB are characterised by extremely low numbers of

transcytotic vesicles and by a restrictive paracellular diffusion barrier (13, 14). The barrier

functions by utilising carrier-mediated transport systems to exert a tight control over the

movement of nutrient molecules, ions, and oxygen, and to protect the brain from toxins

and pathogens (13). The barrier restricts the free flow of hydrophilic compounds, small

proteins and charged molecules but relatively small lipophilic molecules (<600 Da) that

can form fewer than nine H-bonds are normally capable of permeating the BBB (15, 16). It

has been argued that some therapeutic agents may reach the brain if they are capable of


3

becoming associated with specific transporters and/or receptors at the luminal side of

endothelial cells (9).

The main anatomical structure of the BBB (Figure 1.2) is the cerebral blood vessel formed

by the ECs, which is characterised by low concentrations of leukocyte-adhesion molecules

and consequently is responsible for the limited immune surveillance inherent to the CNS

(17). There is a body of experimental evidence which suggests that attempts to

compromise the integrity of the BBB for the purpose of delivering therapeutic agents to

the CNS may distort the balance of transport of molecules between the brain and the

blood, which in turn may lead to aberrant angiogenesis, vessel regression, brain

hypoperfusion, intracerebral haemorrhage, trauma, neurodegenerative processes,

inflammatory responses, or vascular disorder; these responses are capable of generating

toxic metabolites that could affect synaptic and/or neuronal functions (8, 18, 19, 20).

Figure 1.2: Cellular interplay at the neurovascular unit (capillary level) (13)

The BBB consists of a body of cell types that include pericytes, astrocytes, endothelial

cells and microglial cells (21). Brain capillary endothelial cells are characterised by narrow

tight junction, by low pinocytic activities and by high metabolic activity. They allow little

paracellular and no transcellular transport of high-molecular-weight molecules (22).


4

Pericytes are important in supporting BBB development since they influence the

differentiation and maturation of associated endothelial cells. Their interaction with

endothelial cells induces the formation of tight junctions. In addition, they serve as

partial foundation for the basement membrane. Also, pericytes play a significant role in

cytokines production and in antigens presentation, affect end-foot processes, and

support proper neuronal functions (13).

Astrocytes account for about 90% of the overall brain mass, they are involved in

regulation of brain homeostasis through K+ buffering, in the regulation of

neurotransmitter and growth-factor release, and in the regulation of brain immune

responses. Astrocytes produce Apolipoprotein E (ApoE), a molecule that has been shown

to be beneficial to brain homeostasis (23) and may be important in drug transport

through receptor mediated endocytosis (24).

The BBB is part of the Neurovascular Unit NVU, which presents as an elaborate

interplay of central and peripheral cells (13). It creates the extracellular fluid

compartment, the content of which is different to that of somatic extracellular fluid since

it accommodates both Cerebrospinal (CSF) and Interstitial (ISF) fluids. Also, the BBB acts

as neuroprotector in that potentially damaging xenobiotics and metabolites are

prevented entry or are removed from the organ through the action of specialised

transporters (25).

Tight junctions

TJs are located in the apical part of the paracellular space and contain transmembrane

proteins (occludin, claudins, and junctional adhesion molecule-1) and cytoplasmic

proteins (zonula occludens [ZO]-1, -2, -3 and cingulin) that are bound to the actin

cytoskeleton (10).


5

Figure 1.3: Tight junction(26)

TJs are domains of occluded intercellular clefts that are shaped either in grooves (E-face)

or in ridges (P-face) (Figure 1.3). The latter face exhibits higher electrical resistance and

lower permeability than the former. Particles that associate with tight junctions are found

in both faces (14). TJs serve a key function in the regulation of paracellular permeability

and in maintaining cell polarity (27). Intermingled with tight junctions are usually found

Adherens junctions. The paracellular route of drug delivery occurs via the intercellular

space and is mediated by alterations of the tight junction barrier of epithelial cells.

A number of signal pathways have been associated with tight junction regulation, which

involves but is not limited to G-proteins, serine, threonine and tyrosine-kinases, extra-

and intra-cellular calcium levels, cAMP levels, proteases and cytokines (14).


6

Bidirectional transport between brain and systemic circulation

The transcellular bidirectional transport across the BBB has been categorized into carrier-

mediated transport, ion transport, active efflux transport, receptor-mediated transport,

and caveolae-mediated transport of interstitial fluid to blood (28). The influx transporters

(mostly involved in nutrients import) and the efflux transporters (involved in removal of

metabolites and neurotoxic compounds from brain) both prevent the entry of xenobiotics

to the brain (29). An example is P-glycoprotein (P-gp; a 170 kDa protein member of the

ATP-binding cassette transporters), which is located at the luminal membrane of

endothelial cells. P-glycoprotein has been suggested to serve as an efflux transporter that

functions as a clearance system for metabolites and neurotoxic compounds produced in

the brain (28). It has been shown that P-gp is distributed throughout both the luminal and

abluminal membranes of the endothelium, and in astrocytes and pericytes, suggesting

that the pump may be involved in regulating drug transport processes over the entire CNS

and at both the cellular and subcellular levels (30).

The movement of nutrients to the brain (e.g. hexoses, amino acids and monocarboxylic

acids, nucleosides, amines and vitamins), which is facilitated by specialised carrier-

mediated transporters, is characterised by a concentration gradient of each nutrient as it

moves from blood to brain (Figure 1.4). The inflow of nutrients is regulated by the

metabolic needs of the brain, but is also affected by the availability of nutrients.

Since the brain depends mainly on glucose for its energy needs, Glucose transporter 1

(GLUT1) is crucial to brain function. The concentration of GLUT1 is higher at the abluminal

membrane as compared with that of the luminal membrane (29). Consistent with the

need for demand-regulated influx of glucose into the organ, the distribution of the same

transporter in the brain is asymmetric. Facilitative amino acid transport systems at the

BBB (e.g. L1, y+) provide the brain with all the essential amino acids, while sodium-

dependent amino acid transport systems (e.g. A, ASC) allow the movement of non-

essential amino acids. Sodium-dependent excitatory amino acid transporters are

responsible for the removal from the brain of potentially toxic acidic amino acids. Biotin,

pantothenic acid and lipoic acid are transported to the brain by means of sodium-

dependent transporter, while others vitamins (B1, B3, B5, and E) are transported by

specialised carriers.


7

Localised at the abluminal side of the brain, the sodium pump serves the sodium-

potassium (Na+-K+, ATPase; Figure 1.4) exchange in the brain. Also, the Na+-K+-2Cl- co-

transporter, which resides predominantly at the luminal side of the brain, facilitates the

control of sodium, potassium and chloride ions at the brain endothelium. The intracellular

pH of the endothelium is regulated by the co-operative actions of the sodium-hydrogen

exchanger and the chloride-bicarbonate exchanger, while calcium efflux is mediated by

the sodium-calcium exchanger (8, 31).

Monocarboxylate1 (MCT1) is involved in the regulated transport of ketone molecules to

the brain, which serve as energy sources, as do hexoses (8). The efflux of anionic

compounds is mediated by multi drug resistance-associated proteins (MRP) transporters,

which are known to be involved in the organ-specific efflux of molecules (e.g. breast-

cancer-resistance protein, BCRP) and family members of the organic anion transporting

polypeptide (OATP) and the organic anion transporter (OAT). These transporters have the

capability to work co-operatively to inhibit penetration of many drugs into the brain and

to increase their efflux from the brain.


8

Figure 1.4: A simplified atlas of the BBB (8)


9

1.1.2 Strategies for delivering drugs to the brain

Several strategies have been proposed towards overcoming the BBB-imposed limitations

to the delivery of some drugs and other actives to the brain. These strategies may be

categorised into invasive and non-invasive; the former requires specialised expertise,

which impacts on costs, while the latter may be readily accessible to patients.

1.1.2.1 Invasive approaches

Due to the limited access of drugs to the brain via the BBB, invasive procedures represent

the current method of choice for brain-specific therapeutic delivery. Most of these

methods are characterised by intraventricular drug infusion or by disruption of the BBB;

procedures that are both time-consuming and highly specialised (32, 33). Implantation of

a catheter into the ventricular system for the delivery of drugs directly to brain then

bypassing the BBB has been described as the most common invasive procedure for drug

delivery to CNS (34).

1.1.2.2 Use of penetration enhancers

Several penetration-enhancing approaches have been adopted towards increased

paracellular transport in brain capillaries. These include the administration of osmotic

solutions, the use of vasoactive substances, the utilisation of alkylglycerols (35) and the

application of physical stimuli (9) to induce an enhancement of drug transport across the

BBB (36).

Chemical stimuli and tight-junction modulations have been used for opening the BBB to

deliver anti-tumour agents to the brain. Although not without side effects, Mannitol has

been claimed to be preferable to Polysorbate 80 and to Bradykinin for this purpose (10).

The physical opening of TJs by means of electromagnetic radiation has been documented

(37); electromagnetic pulses have also been claimed to affect key TJ-related proteins,

including ZO-1, occludin, and claudin-5 (38). In attempts to open TJs, sodium caprate has

been utilised as have Claudin modulator (39) and junction Zonula Occludens toxin, Zot

(40).

However, disturbance of the BBB as a means for the delivery of therapeutic molecules to

the CNS is inhibited by the technological complexity of the approach, by risks of tumour

dissemination, neurotoxicity and by inadequate selectivity (10).


10

1.1.2.3 Prodrugs

The use of prodrugs represents another means of circumventing the BBB in the delivery

of actives to the brain (37, 38). This approach involves the chemical transformation of an

administered drug to generate an active precursor molecule that is capable of traversing

the target biological membrane, as is exemplified by the relationship between heroin and

morphine (Figure 1.5):heroin (a diacetyl ester congener of morphine) is capable of

penetrating the BBB and subsequently becoming metabolised to produce morphine,

which is a molecule that is not capable of penetrating the BBB (41).

Heroin Morphine

Figure 1.5: Structures of heroin and morphine

Amongst other strategies, the co-administration of drugs with P-gp modulators, the

utilisation of drug-loaded biopolymers, the use of drug-transporting peptides and the

structural modification of drugs have all been examined as means of overcoming the BBB

(33), as has nasal drug delivery (a route that is not impeded by the BBB).

1.1.2.4 Modifications of influx transporters

The formulation of drugs such that they exhibit structural resemblance to a substrate of

influx transporters to the brain has been proposed as a strategy towards increased

availability of a drug to the brain (28). However, an attempted use of a formulation of a D-

glucose-chlorambucil derivative failed to transport this active to the brain consequent to

the associated inhibition of the GLUT1 influx pump (42).


11

1.1.2.5 Passive drug targeting

Passive targeting occurs due to extravasation of the nanoparticles at the diseased site

where the microvasculature is often described as leaky (43).The molecular design of

nanoparticulate therapeutic vehicles is primarily determined by the need to overcome

the natural defence mechanisms mononuclear phagocyte system (MPS) that work

towards their elimination from circulation (43). Selective accumulation of nanocarriers

and drug then occurs by the enhanced permeability and retention (EPR) effect, The EPR

effect will be optimal if nanocarriers can evade immune surveillance and circulate for a

long period (44).

1.1.2.6 Active drug targeting

It has been suggested that epitopes or receptors that are overexpressed in certain

diseased states may be exploited in active drug targeting: if ligands are attached at the

surface of the nanocarrier for binding to specified receptors expressed at the target site,

targeted receptors may be expressed homogeneously on all targeted cells. Targeting

ligands are either monoclonal antibodies (mAbs) and antibody fragments or nonantibody

ligands. The binding affinity of the ligands influences the tumour penetration because of

the binding-site barrier. The ligand is selected to bind to a receptor that is overexpressed

by tumour cells or by tumour vasculature and not expressed by normal cells. The active

targeting is particularly attractive for the intracellular delivery of macromolecular drugs,

such as DNA, siRNA and proteins (44).

1.1.2.7 Carrier-mediated delivery (Colloidal drug delivery systems)

Commonly used nanocarriers for preclinical and clinical drug delivery studies include

liposomes (e.g. lipodox), dendrimers (Figure 1.6) and solid-lipid nanoparticles, since these

often combine effective drug delivery with high drug loading capacity (44).


12

Figure 1.6: Types of drug delivery systems (45)

Liposomes and micelles

Although the subject of considerable research efforts, liposome-mediated drug delivery is

often limited by: the inherent instability of liposomal dispersions, drug leakage, low

activity due to no specific tumour targeting, nonspecific clearance by the mononuclear

phagocytic system (MPS) and complications associated with the available large-scale

production methods (46). Nonetheless, liposome-loaded Doxorubicin (lipodox) has been

shown to be significantly more effective on resistant cell line than free Doxorubicin. Co-

administration of Doxorubicin and P-gp inhibitor indicated the capability of the liposomal

formulation to inhibit the efflux pump P-gp (47-49). Benefits to drug-delivery applications

have been shown by micellar formulations, as is exemplified by the capability of micelles

poly caprolactone-b-poly ethylene oxide (PCL-b-PEO) of Doxorubicin to shift the

accumulation of this active from the cytoplasm to the nucleus, thereby enhancing

efficacy.


13

In terms of structure, liposomes are small vehicles with an aqueous inner core which may

be enclosed by unilamellar or multilamellar phospholipid bilayers (50). Polymeric micelles

(Figure 1.7) are nanocarriers composed of amphiphilic multi-block copolymers capable of

forming a shell structure (50).

Figure 1.7: Schematic representation of liposomes and micelles (50).

Solid lipid nanoparticles

Solid lipid nanoparticles (SLN) are particles made from solid lipids and stabilised by

surfactants. The need for the use of SLNs in drug delivery arises from the inability of

highly ordered crystal lattices to accommodate large amounts of drug molecules. With

reference to parenteral application, SLNs offer physical stability, protection of

incorporated labile drugs from degradation, controlled drug release (fast or sustained,

depending on the adopted molecular design), good tolerability and the potential for site-

specific targeting. However SLN structures often suffer from sub-therapeutic loading

capacity, polymorphic transitions during storage that lead to the expulsion of the active

and relatively high water content in dispersions (70–99.9%); the removal of excess water

from SLN dispersions tends to impact upon particle size. Also, the formulation of SLNs

normally necessitates the use of high concentrations of surfactants and co-surfactants

(e.g. butanol), which in turn may be undesirable for regulatory purposes. Dependent

upon the drug/lipid ratio and solubility, the therapeutic content of SLNs may be

preferentially localised at the core of the particles or at the shell or be molecularly

dispersed throughout the matrix. The release profile of SLNs may be influenced by


14

chemical modifications at the lipid matrix, by the concentration of co-formulated

surfactant and by the physical parameters adopted during formulation. For therapeutic

applications, SLNs are normally injected (intravenously, intramuscularly, subcutaneously

or directly to the target organ). SLN formulations are suitable for therapeutic uses

requiring systemic body distribution since their small size minimises the risk of embolism

by blood clotting or particle aggregation. If administered subcutaneously or designed to

accumulate in the MPS, SLNs offer the opportunity to act as a sustained release depot of

the drug which allows the incorporated drug to be released over extended time scales

either by the erosion of the SLN matrix (e.g. by means of enzymic degradation) or by

diffusion from the particles (46).

Dendrimers

Dendrimer (Figure 1.8) nanocomposites are symmetric hyper-branched, star-shaped

structures that are designed such as to produce monodispersed formulations (51). The

repetitious nature of chain and branching afford a series of radially concentric layers with

increasing crowding, as is exemplified by poly(amidoamine)-based structures. The

classification of dendrimers according to generation reflects the exponential increase in

the number of branches in each layer: dendrimer growth is typically ca. 1 nm per

generation. Since dendrimers are typically symmetric around the core, their extended

forms in aqueous media are of spheroidal morphology. The loosely packed core and

tightly packed periphery of dendrimers often affords good drug loading capacity (52, 53)

but this is counterbalanced by little control over the release mechanism of loaded drugs.

Also, because of their branched nature, dendrimers are often amenable to conjugation

with pharmaceutically active moieties through functional groups. Notably, studies in

cultured cells (colon carcinoma) have shown that Dox-dendrimers are less toxic than free

Dox (54).

As compared with linear polymers, dendritic structures have “dendritic voids” that give

these molecules important and useful features. Dendrimers with a high surface charge

density due to ionisable groups are amenable to non-stoichiometric association with

therapeutic molecules through electrostatic interactions. Such dendrimer-drug


15

interactions may afford to actives enhanced solubility, increased stability and efficient

transport through biological membranes (55).

Figure 1.8: The structure of dendrimer(55)

Nanoparticulates

Since colloidal carriers, particularly biodegradable polymeric nanoparticles, are often

amenable to structural modifications that may bestow to them the capability to be

transportable through the BBB, many researchers regard these structures as promising

vehicles for the delivery of drugs to the brain. Many colloid-forming biopolymers (e.g.

dextran, PACA, PLA) have been considered for the purpose. The properties of

nanoparticles utilised for the in vivo transport and enhanced pharmacokinetic profile of

therapeutic compounds originate from their nano-scale size which furnishes them with

colloidal stability and allows them to penetrate tissues easily through capillaries and

epithelial linings. Amenability to functionalisation, both at the surface and at the core of

nanoparticles, has provided opportunities for applications in drug delivery and in

molecular imaging (56). The performance of drug-loaded nanocarriers for targeting

organs or tissues may be fine-tuned by adjusting the balance between particle size, size

distribution, surface charge, surface modification and hydrophobicity (57).


16

Nanoparticulate drug carriers are nanoscaled solid colloidal structures (nanospheres or

nanocapsules) that may be prepared from natural or from synthetic polymers (58-60).

Research efforts in nanoparticle-mediated drug delivery have been rationalised in terms

of their potential to effect: (i) long blood circulation time that is coupled with a capability

to enter the smallest capillaries; (ii) resistance to rapid phagocytic clearance; (iii)

capability to reach the target organ by penetrating cells and tissues; (iv) capacity to

exhibit controlled release properties that are inherent to the rate of biodegradability or

occur in response to a stimulus or a combination of stimuli (pH, temperature, ion

sensibility); and, (v) improved target-organ specificity (61).

For potential use in drug delivery nanoparticles must possess properties of

biocompatibility and biodegradability to non-toxic and non-immunogenic products (62).

Nanoparticles are differentiated from larger size congeners by their active surface area

which in turn impacts upon aspects of chemical and biological reactivity (63) through

effects at the solid–liquid interface and those at the contact zone with biological

substrates (64). Chemical composition, surface function, geometry, porosity, surface

crystallinity, size range, heterogeneity, roughness, and degree of hydrophilicity are all

considered important in determining the scope and extent of the interactions of

nanoparticles with biological systems (65). Of importance are the characteristics of the

surface layer (zeta charge, aggregation potential, dispersion state, stability, extent of

hydration) as influenced by the characteristics of the surrounding medium (ionic strength,

pH, temperature, and presence of organic molecules or surfactants) (66). The surface

chemistry of nanoparticles that are amenable to functionalisation offers the possibility to

optimise the behaviour of nanoparticles in biological environments (64). It has been

suggested that to be usefully applied in drug delivery to the brain (67), nanoparticles

must have the following characteristics: diameter around 100 nm, physical stability in

blood (no aggregation), non-susceptibility to the mononuclear phagocytic system (MPS),

prolonged blood residence time, capacity for BBB-targeted brain delivery (receptor-

mediated transcytosis across brain capillary endothelial cells), amenability to a scalable

and cost-effective manufacturing process, capacity to accommodate therapeutic agents

(small molecules, peptides, proteins, nucleic acids), chemical inertness (chemical

degradation/alteration, protein denaturation), and possible modulation of the drug


17

release profiles. There is experimental evidence to suggest that the size of NP for

effective brain drug delivery must be in the range 100-300 nm (68).

Dependent upon their method of formulation and constituent materials, nanoparticles

formulations may be divided into two broad types, namely: nanospheres and

nanocapsules. The method of choice for the formulation of nanoparticles of either type is

interfacial polymerisation (the mixing of an organic phase with an aqueous phase).

Differences in the two types of nanoparticle are manifested by their morphology and

architecture and also by the patterns characterising the drug distribution profile and the

rate of drug release. Drug molecules are localised at the central core of nanocapsules

whereas in nanospheres they are dispersed evenly throughout the matrix (Figure 1.9).

Nanocapsules offer the possibility for zero order release while nanospheres normally

exhibit first order release kinetics (69).

Figure 1.9: Schematic representation of the structure of nanospheres and nanocapsules for drug delivery (50).

1.1.3 Nanoformulated drugs currently in clinical trials

Amongst the first nanoformulated (Table 1.1) drugs to be employed in CNS-targeted

clinical trials were glutathione-decorated liposomes, drug-protein conjugates (e.g.

ANG1005) and polyglutamate paclitaxel (70). The initial promise of these formulations has

stimulated considerable research activities in the use of nanoparticles as drug carriers,

with polyester-based structures finding particular favour amongst researchers because of

their molecular-design-determined biocompatibility and tuneable degradation properties

(71).


18

Table 1.1 Representative examples of water- insoluble-drug nanoformulations that are approved for clinical use or are under clinical evaluation (72).

1.2 Polymeric drug delivery systems

Owing to their tuneable biodegradability, many polymers, especially polyesters, have

found applications in drug delivery and in tissue engineering. The archetypal example of

such a polymer is polycaprolactone, a biocompatible and biodegradable polyester (73).

(Polyesters are synthetic materials that are normally prepared through the

polycondensation of monomers containing two or more carboxylic acid groups with

monomers containing two or more hydroxyl functionalities or by the ring-opening

transesterification of lactones). The biodegradability of polyesters is the result of their

limited hydrolytic stability which over time results in depolymerisation to the precursor

monomers. Linked to the hydrolytic depolymerisation of esters is their biocompatibility

which can be designed into the molecular structure by the appropriate choice of the

alcohol- and carboxylic acid-functionalised monomers or co-monomers that condense to

form the macromolecular backbone.


19

The polymeric carriers used in drug delivery systems may be categorised into natural and

synthetic. The latter materials may be subdivided into biopersistent and biodegradable

(Table 1.2) polymers. The former comprise stable materials that maintain their

physicochemical features in the physiological environment; they are eliminated from the

body without undergoing metabolism (51) and their therapeutic payloads are released by

diffusion through the polymeric matrix. By contrast, biodegradable polymers undergo

metabolic transformation at the physiological environment, which renders the kinetics for

their release highly sensitive to their biodegradation pathway. As long as biodegradable

materials are not amenable to biodegradation into cytotoxic products, the use of such

polymers is considered preferable to that of biopersistent molecules, in that

biodegradable polymeric materials allow for the complete release of the active (51).

The use of biodegradable polymers in drug delivery avoids issues relating to the fate of

the body of the depleted drug delivery device. Polymeric nanoparticles are most often

prepared by poly(D,L-lactide-co-glycolide), polylactic acid, polycaprolactone, poly-alkyl-

cyanoacrylates, chitosan and gelatin (57). The loading of drugs to nanoparticles may

reduce the side effects inherent in most therapeutic agents (e.g. Doxorubicin-loaded

nanoparticles have been shown to be less toxic than the free drug), and may afford

control over the release and biodistribution of the active, increase specificity, prolong

bioactivity and inhibit opsonisation or rapid elimination from circulation. It has been

claimed that nanoparticles have the capacity to protect anticancer drugs from

biotransformation and to delay clearance from the host (74). The thermodynamic stability

of nanoparticles renders them preferable drug-host structures to liposomes, both in

terms of stability during storage and in biological fluids where they offer in vivo

protection from proteases (75). Neha et al. (76) have outlined the following potentially

useful characteristics of polymeric nanoparticles to applications in drug delivery:

- in terms of efficiency and effectiveness, they offer a significant improvement over

traditional oral and intravenous methods of administration;

- they deliver a high concentration of pharmaceutical agent to the desired location;

- Tuneable drug-release profiles render polymeric nanoparticles ideal candidate

vehicles for cancer therapy and for the delivery of vaccines, contraceptives and

targeted antibiotics;


20

- Polymeric nanoparticles can be easily incorporated into other technologies

related to drug delivery, such as tissue engineering;

- Readily accessible nanoparticle formulations are often capable of affording

increased stability to volatile pharmaceutical agents.

Table 1.2 Classification of biodegradable polymers(73)

1.2.1 Poly(alkyl cyanoacrylate)s

Alkyl cyanoacrylates (ACA) are low viscosity compounds that are formed by the

condensation reaction of alkyl cyanoacetates and formaldehyde. The first report of a

cyanoacrylate dates back to 1949, but the importance of this class of molecules as

adhesives was not realised until 1959 (77). The physicochemical properties of

cyanoacrylates are determined by the length of their alkyl side chains. Considering their

proven biocompatibility following extensive use as medical adhesives, alkyl

cyanoacrylates have been investigated for their ability to form nanoparticulate vehicles

for biomedical applications; especially for the delivery of cancer drugs (78) and for the


21

transport of therapeutic agents across the BBB (79). Poly(alkyl cyanoacrylate)s are

colourless brittle materials that are susceptible to environmental degradation unless

stabilised by acidic stabilizers (80). The ease of degradation is inversely proportional to

the length of the alkyl chain (81, 82). Methyl cyanoacrylate, ethyl 2-cyanoacrylate, n-butyl

2-cyanoacrylate, isobutyl cyanoacrylate, isohexyl cyanoacrylate and octyl cyanoacrylate,

have all been used in the formulation of nanoparticles (Figure 1.10A).

Figure 1.10: The structures of alkyl cyanoacrylate (R= methyl, ethyl, butyl, isobutyl, isohexyl, octyl etc.) (A) and poly(lactic acid) (B).

Alkyl cyanoacrylates have found diverse practical applications that include use as

adhesive products for household repairs and for wound closure (short-chain homologues

are preferred for use as adhesives; 77, 83), weed control (84) and detection of latent

fingerprints in crime investigations (85, 86). Alkyl cyanoacrylate monomers are highly

reactive. They polymerise at room temperature via initiation by nucleophiles

(environmental moisture) through a mechanism that involves chain propagation by the

repetitive addition of monomer units to the carbanionic end of the growing chain (87).

For the longer side-chain homologues, the rate of the polymerisation reaction is sensitive

to temperature. The polymerisation reaction is sensitive to pH, with neutral to basic pH

resulting in the agglomeration of monomers (83).

The pioneering work of Troster et al. (88), who reported that coating poly(methyl

methacrylate) nanoparticles with Polysorbate 80 increased the accumulation of these

nanoparticles into the rat brain, prompted much work towards nanoparticulate-assisted

drug delivery to the brain. Consequently, biocompatible PACA polymers have been

demonstrated to cross the BBB (89), as is exemplified by Polysorbate 80-coated n-butyl-2-

cyanoacrylate (79, 90-92). The initially proposed endocytotic mechanism of transport of

these nanoparticles, has however been disputed by Olivier et al. (93) who have argued

that the mechanism of drug transfer involves the induction of toxicity to the BBB.

Nonetheless, the mechanism of nanoparticle-mediated drug transport across the BBB is

generally accepted to involve an initial receptor-mediated endocytosis which is followed

R

n


22

by transcytosis into the brain or by drug release within the endothelial cells (94). The use

of PBCA nanoparticles that are coated with Polysorbate 80 has received considerable

attention in drug delivery across the BBB (41). Significantly, PBCA nanoparticles that had

been loaded with Doxorubicin and overlaid with the non-ionic surfactant Polysorbate 80

are reported to be capable of reaching to the brain intact and of releasing their

Doxorubicin content following endocytotic uptake by brain blood endothelial cells (91).

The use of Polysorbate 80 coatings has been rationalised in terms of earlier work by

Kreuter et al.(92), who, on the basis of electron microscopy and fluorescent studies, had

suggested that drug-loaded nanoparticles coated with Polysorbate-80 undergo

phagocytic uptake by brain blood vessel endothelial cells. However, the matter is still

subject to considerable debate since other findings suggest that the uptake of PBCA

nanoparticles by the brain is consequent to interaction with the LDL-receptor by

mimicking the LDL-protein following association with Apolipoprotein E from blood plasma

(95).

1.2.2. Poly lactic acid (PLA)

Poly (lactic acid) (Figure 1.10B), PLA, is amongst the most commonly used biodegradable

polymers in therapeutic delivery, especially that of vaccines. The main criticism this

polymer has received relates to the generation of acidic micro environments during

degradation. PLAs are linear aliphatic thermoplastic polyesters (96) that are commonly

synthesised from the condensation of α-hydroxy acids. The basic building block for PLA is

lactic acid. PLA is susceptible to degradation by simple aqueous hydrolysis and undergoes

thermal degradation above 200°C. The respective glass transition and melt temperatures

of the material are at 55°C and 175°C (97). Owing to its excellent biocompatibility and

biodegradability (98), PLA has found uses in drug delivery. PLA has been utilised widely as

a structural polymer or co-polymer in the preparation of nanoparticles. Its combination

with glycolic acid to form poly (lactic-co-glycolic acid) (PLGA) is well documented, as is its

zero-length grafting attachment to modified dextran and also its combination with poly Ԑ-

caprolactone.

1.2.3. Polysaccharides

Dextran, a hygroscopic polysaccharide, is an odourless and tasteless white amorphous

powder which is insoluble in ethanol and diethyl ether but gradually soluble in water. The

constitutional repeat unit of dextran is glucose, which forms linear bonds of α-1,6


23

glycosidic linkages with few branches at the α-1,2, α-1,3 and α-1,4 positions (99, 100).

Apart from its common use in density centrifugation (to remove vasculature of a given

homogenate) (70, 101); dextran is a useful matrix material for drug delivery applications

since it exhibits properties of biocompatibility, degradability and non-immunogenicity

(102). The same material is often employed as surfactant or as copolymer in

nanoparticles fabrication, especially those of ACA-based structures (103), where it

imparts increased hydrophilicity by means of the considerable –OH functionalisation of

the pyranose ring. Notably, the α-1,6 polyglucose linkages of dextran are not susceptible

to cleavage by most endogenous cellular glycosidases (104). It is a polysaccharide that is

present in certain microorganisms, especially bacteria. Dextran, which is primarily utilised

by microorganisms as a structural support material and as an energy store, is also integral

to the immune-response mechanisms.

m

O

OH

OH

OH

O

O

OH

O

OH

O

n

Figure 1.11: The chemical structure of dextran.

Dextran has been used for several decades as a plasma volume expander. This highly

water soluble substance can be readily attached to drugs either directly or through

linkers. Studies have shown that both the distribution and the elimination of dextran are

influenced by the combined effects of molecular weight and surface charge of the

polymer. The degree of water solubility of dextran decreases with increased branching, as

has been exemplified by the controllable degrees of hydrophobicity that have been

obtained through modifications with alkyl glycerols of systematically varied chain lengths

(105). An in vitro study involving modified dextran has shown that chemically modified

dextran exhibits reduced rates of dextranase-induced depolymerisation as compared with

the unmodified material (105).


24

The use of dextran in such applications has been extended to nanoparticulate

formulations that could involve co-formulation with alkyl cyanoacrylates. Native dextran

has been used extensively as a surfactant in the formulation of poly(alkyl cyanoacrylate)s-

containing molecular structures (90, 93, 103, 106). The combined use of these materials

has been rationalised in terms of the capability of dextran to impart to alkyl

cyanoacrylates flexural strength and an increased capacity to accommodate a therapeutic

load for drug delivery applications. However, the observation that PECA-Dex structures

are susceptible to aggregation and to becoming brittle over time has stimulated further

research activities towards the development of alternative materials with improved long-

term stability.

Polymeric nanoparticles prepared from alkyl cyanoacrylates and dextran have shown

promise as carrier vehicles for drug delivery to the brain, as is exemplified by the

observed delivery of drugs and peptides across the BBB by means of Polysorbate 80-

coated formulations of PACA and dextran. The capability of the biodegradable PACA

macromolecules to act as carriers for drugs and fluorophores alike has been integral to

their utilisation in drug delivery (107). It is common practice to incorporate covalently

bonded fluorophore end groups into the polymer structure by initiating the

polymerisation reaction by means of nucleophilic fluorophores (108). Alternatively, the

fluorescent labelling may be achieved by nanoprecipitation of the preformed polymer

(109).

Derived from chitin, chitosan is regarded as a nontoxic, biocompatible and biodegradable

cationic polysaccharide. Chitosan is comprised of β(1,4)-linked 2-acetamido-2-deoxy-β-D-

glucan and 2-amino-2-deoxy-β-D-glucan. The commercial preparation of chitosan is

through alkaline deacetylation of chitin. Chitosan nanoparticles are prepared mainly by

the ionic gelation of chitosan through the tripolyphosphate anions method (110, 111).

Chitosan has been utilised widely in the development of controlled-release drug delivery

systems (51,112). Interestingly, chitosan nanoparticles have been described as an

efficient vehicle for the delivery of insulin through the nasal mucosa (113).


25

1.2.4. Polycyanoacrylates

The chemical stability of PACA has been shown to be sensitive to the nature of the

initiator and the method employed for the polymerisation, and also by the length of alkyl

side chains. PACAs are susceptible to biodegradation both at the backbone C―C bond

(due to the presence of the strongly electron withdrawing cyanate group) and at the ester

linkage (by hydrolysis). The ease of hydrolytic degradation to the alkyl alcohol and to

poly(alkyl cyanoacrylic acid) has been observed to decrease with increasing length of the

side chain. Backbone (C―C) degradation occurs by the unzipping of the polymer chains in

a depolymerisation process that produces the precursor monomer (108).

The ease with which ACA monomers undergo polymerisation reactions to form PACA,

coupled with the biocompatibility of PACA and its biodegradability to innocuous products,

render this polymer a suitable structural material for the fabrication of drug carrier

structures (58). Conventionally ACA is polymerised in acidified water using a two phase

polymerisation process that affords control over particle size has been argued however

that this slow process and yields a particle size distribution that is broader than that

which can be obtained with ethanol/water systems (114). The easy accessibility and ready

availability of ECA renders this monomer the cyanoacrylate of choice for nanoparticle

formulation for anionic emulsion polymerisation reaction via initiation by the hydroxyl

group of water or the nucleophilic centre of other initiator molecules such as amines

(114). The amino group of proteins, has been utilised as an initiating moiety in the

polymerisation of ECA as exemplified by the reaction with Bovine serum albumin (BSA),

where the protein molecules are also claimed to serve as stabilizer surfactants (114).

Adjustment of the pH affords control over the size distribution of PACA nanoparticles

(115) whereas the hydrophobicity of the same nanoparticles may be tuned by adjusting

the length of the side chain such that control may be exerted over the capacity of

nanoparticles to swell in biological fluids (116). Transmission electron microscopy (TEM)

has shown that PECA nanoparticles are structured as a highly porous but dense polymeric

matrix with high surface area that facilitates the entrapment of wide range of drug

molecules (117). Clinical trials of formulations of PACA for drug delivery applications have

not unmasked any metabolites-related toxic effects of notable significance (118).

M


26

1.3. Methods for the preparation of nanocarriers

The variants of the two main methods of choice for the preparation of nanoparticles,

dispersion of preformed polymer and polymerisation of monomers, have been reviewed

by Rao and Geckeler (119). Nanoparticles formation by the dispersion of preformed

polymers may involve solvent evaporation, nanoprecipitation, salting out, dialysis, rapid

expansion of supercritical solution (RESS) and rapid expansion of supercritical solution

into liquid solvent (RESOLV). The formation of nanoparticles by the polymerisation of

monomers may utilise the techniques of micro-emulsion, mini-emulsion, surfactant-free

emulsion or interfacial polymerisation. The method of nanoparticle preparation is

determined by the chemical structure of the adopted matrix, which in turn is selected

according to the requirements of size and of the proposed use (120).

Other methods that have been utilised for the preparation of nanoparticles for drug

delivery include: (i) freezing-induced phase separation (121); (ii) emulsion solvent

evaporation (122); (iii) single emulsion–solvent evaporation technique, which has been

claimed to be the best general method of encapsulating hydrophobic molecules into

nanoparticles; and, (iv) miniemulsion, which has been utilised for the co-formulation of

paclitaxel and PBCA (123).

Bertholon et al. (124) in their studies of redox and anionic emulsion polymerisation of

nanoparticulate PACA have shown that low (highly acidic) pH leads to the formation of

higher molecular weight and higher average particle size than corresponding

nanoparticles prepared at higher pH. The same group of workers speculated that the

higher molecular weight polymers formed as a result of the highly acidic pH making fewer

OH groups available for the initiation of polymerisation.

1.4. Characterisation techniques

Integral to the characterisation of nanoparticles is the determination of size,

polydispersity index (PDI), zeta potential, texture morphology and thermal stability.

Particle size and its distribution influence such key properties as stability in suspension,

viscosity, surface area, and parking density. For biomedical applications, size also impacts

on the capability of therapeutic agents-loaded nanoparticles to penetrate deeply into

tissues through the narrow capillary route and further to penetrate cells; amongst other


27

techniques, the sizes of nanoparticles may be determined by DLS, TEM, NTA, SEM, and

AFM.

Zeta potential provides a measure of the stability of nanoparticles in a given medium.

Zeta potential values above 30 mV (-ve or +ve) are normally considered indicative of good

colloidal stability as it indicates that the electrostatic repulsion between neighbouring

nanoparticles is greater than the antagonistic van der Waals forces of attraction,

preventing aggregation or precipitation (125); zeta potential may be determined by

dynamic electrophoretic mobility. The preparation of nanoparticles of narrow size

distribution, as measured by the PDI, is prerequisite to the reproducible behaviour of

vehicles intended for systemic use.

1.4.1. Elemental analysis

The technique used for the determination of elemental C, H, and N and S is based on the

quantitative “dynamic flash combustion” method (Figure 1.12). The samples are held in a

tin capsule, placed inside the autosampler drum where they are purged by a continuous

flow of helium and dropped at pre-set intervals into a vertical quartz tube maintained at

900°C. When the samples are placed inside the furnace, the helium stream is temporarily

enriched with pure oxygen, the sample melts and the tin promotes a violent reaction

(flash combustion); under these conditions even thermally resistant substances are

oxidised fully. Quantitative combustion is then achieved by passing the mixture of gases

over a catalyst layer. The mixture of combustion gases is then passed over copper to

remove the excess oxygen and to reduce the nitrogen oxides to elemental nitrogen. The

resulting mixture is then directed to the chromatographic column where the individual

components are separated and eluted with the help of a thermal conductivity detector

(TCD) whose signal feeds the automatic EAGER300™ workstation as nitrogen, carbon

dioxide, sulphur dioxide and water. The instrument is calibrated with the analysis of

standard compounds using the K factors calculation or linear regression method

incorporated in the EAGER300™ software.


28

Figure 1.12: Schematic representation of an elemental analyser (adapted from analyser’s manual).

1.4.2. MALDI-TOF MS

MALDI-TOF MS, a technique developed by Hillenkamp and Karas in 1988 (126), is widely

utilised for the mass spectroscopic characterisation of proteins and other

biomacromolecules (127).

Principles of MALDI

Unlike conventional mass spectroscopy techniques (electron impact, chemical ionisation),

in MALDI-TOF MS (Figure 1.13) (a technique normally applied in the characterisation of

complex molecules) ionised molecules (cationic) or molecular fragments that have the

same energy travel at different velocities, with the implication that fragments of different

masses reach the detector at different times; larger ions travel with a lower velocity than

smaller cations. Since the time of travel depends on charge, mass and kinetic energy of

the ion, delay extraction (DE) has been shown to be very helpful in improving resolution

in the application of MALDI-TOF; DE is employed to delay the travel of ions by 150 ns

from formation, such that most of their excess energy is dissipated, before the

accelerating voltage is applied. However the resolution benefit of DE decreases with

samples of increasing molecular weight.


29

Sample preparation

The sample preparation stage is critical in the quality of the spectrum produced in MALDI-

TOF spectrometry. Amongst the methods of sample preparation, the dried-droplet

method has become the most widely utilised: a saturated matrix solution is mixed with

the analyte at the ratio of 5000:1, and an aliquot of about 0.5 µl is placed on the sample

receptor where is dried under vacuum before ionisation. Other approaches to sample

preparation include: thin and thick layer (127), fast-vaporisation, electrospray, matrix-

precoated layer, particle-doped (two phase liquid) and chemical-liquid (128) methods.

Figure 1.13: Schematic representation of the MALDI instrument (128).

The advent of MALDI has offered many advantages in the analysis of macromolecules as

compared with conventional chromatographic techniques. These include the capability to

determine absolute molecular weight irrespective of macromolecular structure, the ease

of use (the technique imposes few demands in terms of instrument- and sample-

preparation procedures), and the capability to characterise molecules of <20 kDa (128,

129). However, the technique is regarded as complementary to chromatographic

methods since it is not suitable for the study of macromolecules with PDI >1.6 or for the

analysis of complex mixtures of surfactants. Most importantly, MALDI is of little

usefulness in the analysis of macromolecules that do not possess chemical functionalities

that are capable of stabilising the prerequisite cationic structure at the gaseous phase

(128).


30

The vaporisation and ionisation technique is widely used in the characterisation of

biomolecules by MALDI-TOF. This is a non-destructive technique that vaporises and

ionises large and small biomolecules without promoting fragmentation (Figure 1.14 ). To

this end, the sample to be analysed is incoporated first into a matrix (e.g. 2,5

dihydroxybenzoic acid (130, 131), which is in excess of the analyte and functions by

absorbing strongly the energy of a laser source thereby facilitating for vaporisation of the

analyte. In doing this, it also serves as proton donor for the analyte thereby facilitating

ionisation and subsequent analysis by the detector (linear time-of-flight (TOF) analyzer,

TOF reflectron or fourier transformed mass analyzer).

Figure 1.14: Non-destructive vaporisation and ionisation of biomolecules by MALDI TOF (126)

The usefulness of mass spectrometry methods in the identification and characterisation

of unknown compounds is twofold, in that it allows the:

a. Determination of the molecular mass of the compound under study; mild direct

ionisation techniques allow the detection of the highest m/z ratio ion which often

corresponds to an isotopic form of the intact, ionised molecule, while chemical

ionisation techniques (e.g. ESI, MALDI) typically identify the protonated or

deprotonated form of the molecule.


31

b. Detection of the functional groups present in the molecule on the basis of specific

fragment ions and/or fragment ion series present in the mass spectrum, and

characteristic fragment ions formed by loss of neutral molecules from the intact

ionised molecule.

Synthesis of the spectral information regarding the molecular ion and the identified

molecular fragments allows the determination of the structure of the molecule under

study.

The use of high mass resolution instruments allows the minimisation of issues relating to

peak broadening and of those due to presence of any interfering or isobaric ions. The

technique advantages have been put at low cost, high sensitivity, large mass range and

recording a whole spectrum in a single acquisition step (132).

1.4.3. Nanoparticle Tracking Analysis (NTA)

Nanoparticle Tracking Analysis (NTA) was developed by Carr et al; in this technique,

particles under Brownian motion are recorded by software-guided video designed to

identify and track the centre of each particle on a frame-by-frame basis (Figure 1.15). The

image analysis software determines the distance moved by each particle, which in turn

allows the determination of the corresponding diffusion coefficient. The hydrodynamic

diameter (d) of particles is calculated (133) by inputting the temperature (T) and viscosity

( ) of the medium into the Einstein-Stokes equation (k = Boltzmann constant).

D

kTd

3 (eq. 1.1)

Figure 1.15: Schematic representation of the Nanoparticle Tracking Analysis (NTA)(133)


32

1.4.4. Zeta potential determination

There exist two layers around a particle that is suspended in an electrolytic solution,

namely: the Stern layer and inner region, where the ions are bound strongly; and the

outer, diffuse region, where the ions are bound less strongly (Figure 1.16). The net

surface charge of particles determines the distribution of ions surrounding it, effecting

the formation of an electrical double layer around each particle. Zeta potential is used to

determine the magnitude of the interaction between charges in a system, and provides a

measure of surface charge. Thus, zeta potential is the energy difference that exists at this

boundary. Colloidal systems with a zeta potential between +30 and -30 are normally

considered stable (134).

Figure 1.16: Schematic representation of particles and surround charges (134)

1.4.5 TGA

Thermogravimetric analysis TGA (135) is a method of thermal analysis in which changes in

the physical and chemical properties of materials are monitored either as a function of

increasing temperature (at constant heating rate), or as a function of time (at constant

temperature and/or constant mass loss). TGA is used to study the decomposition and

thermal stability of materials under specified conditions and to examine the kinetics of

the physicochemical processes occurring within a sample. The technique is commonly


33

employed to determine heat-induced changes that involve either mass loss or mass gain

due to decomposition, oxidation or loss of volatiles (such as moisture). Factors that

influence the nature of the TGA curve (Figure1.17) include sample mass, volume and

physical form, the shape and nature of the sample holder, the nature and pressure of the

atmosphere in the sample chamber, and the scanning rate. The principal applications of

TGA in polymers are the determination of the thermal stability of polymers,

compositional analysis, and identification of polymers from their decomposition pattern.

Figure 1.17 Representative TGA curve.

1.4.6 SEM

The SEM is a type of electron microscope that produces images of a sample by scanning it

with a focused beam of electrons. This complements optical images with the traditional

microscope in that it offers large depth of field (which allows more of a specimen to be in

focus at one time), and high resolution.


34

Figure 1.18 Scanning electron microscope (136)

The electron beam scans the surface line by line to produce the image (Figure 1.18); since

metals are conductive, their samples do not require pre-imaging treatment but non-

metals need treatment that renders them conductive; this may involve coating with gold,

graphite or other electrically conducting materials in a sputter coater. The sample to be

coated is fixed on a stub and placed under vacuum. The interaction of electron with the

sample produces many signals which include secondary electrons (SE), back-scattered

electrons (BSE), characteristic X-rays, light (cathodoluminescence) (CL), specimen current

and transmitted electrons. The signals result from interactions of the electron beam with

atoms at or near the surface of the sample. Secondary electrons and backscattered

electrons are commonly used for imaging samples.


35

1.5. Aim of the study

Amongst the approaches towards the delivery of therapeutic agents to the brain without

compromising the integrity of the BBB, the utilisation of polymeric nanoparticulate drug-

carrier vehicles – the focus of this work – has gained considerable following amongst

researchers. The objectives of this study are to:

i) synthesise amphiphilic dextran derivatives through functionalisation with alkyl glycidyl

ethers;

ii) use these modified dextrans in combination with alkyl cyanoacrylates (ACA) for the

preparation of well-defined nanoparticles;

iii) study the capability of these nanoparticles to act as carriers for fluorescent-marker

molecules and for small molecule drugs; and,

iv) investigate the biodegradability and the potential toxicity of these nanoparticles, and

assess any associated interactions for the purpose of evaluating promise in therapeutic

applications.

Chemical modification of dextrans

36

2

2. Chemical modifications of dextran

The chemical modification of dextrans has been rationalised in terms of the capability of

dextran to impart to alkyl cyanoacrylates flexibility and an increased capacity to

accommodate a therapeutic load for drug delivery applications. However, the observation

that PECA-Dex structures are susceptible to aggregation and to becoming brittle over

time has stimulated further research activities towards the development of alternative

materials with improved long-term stability. The functionalisation of dextrans with alkyl

glyceryl moieties represents one of the approaches towards that goal.

2.1 Dextran

Dextran is an odourless and tasteless hygroscopic white powder that is insoluble in

ethanol or diethyl ether but dissolves gradually in water. It is a polysaccharide that is

present in certain microorganisms, especially bacteria. Dextran, which is primarily utilised

by microorganisms as a structural support material and as an energy store, is also integral

to the immune-response mechanisms.

The chemical structure of dextran (Figure 2.1) is that of a neutral polysaccharide that has

glucose as its sole constitutional repeat unit. The glucose moieties form linear bonds at

the α-1, 6 glycosidic linkages (more than 95% of the chain) with few branches at the α-

1,2, α-1,3 and α-1,4 linkages (99, 100). Owing to its glucose-based structure, dextran is

characterised by biocompatibility, degradability and non-immunogenic properties which

render it a useful structural material for biomedical applications (102). The use of dextran

in such applications has been extended to nanoparticulate formulations that often involve

co-formulation with alkyl cyanoacrylates (24, 93, 137).


37

Figure 2.1: The chemical structure of dextran

The –OH functionalities render dextran a molecule that is readily amenable to structural

modification (138). Consequently, a number of modified dextrans have been prepared in

which aromatic rings or aliphatic chains are attached to the molecular structure (139-

144). For example, dextran that had been modified by esterification with benzoic acid and

valeric acid has been used in protein partitioning (145). In other examples, Rouzes et al.

(98) utilised hydrophobically modified dextran as a surfactant for the preparation of

nanoparticles with well-defined surface properties, while Sadtler et al. (146) reported its

use in the stabilisation of oil-in-water emulsions. Dextran derivatives with good chemical

stability at extreme pH environments have been achieved through functionalisation with

1,2-epoxydodecane (144). The chemical modifications of dextran have been extended to

attempts to obtain amphiphilic polymers which are capable of forming micellar structures

that can associate with organic solutes at the hydrophobic domain. Rotureau et al. (141)

have shown that lipophilic modification of dextran through linkage with aromatic or

aliphatic hydrocarbons results in macromolecular structures of controlled amphiphilicity.

The amphiphilic nature of modified dextrans renders these materials suitable

components for the fabrication of nano-sized structures of well-defined size, as is

exemplified by the work of Rouzes et al. (147) who utilised such materials in combination

with PLA to produce nanomaterials in the 150-200 nm range, and that of others who

utilised amphiphilic dextrans as surface-modification components of both nano- and

micro-sized drug delivery systems (147, 148). Native dextran has been used extensively as

α-1,6

α-1,3

...........

α-1,6

...........


38

a surfactant in the preparation of poly(alkyl cyanoacrylate)s-based structures (24, 90, 93,

103). The combined use of these materials has been rationalised in terms of the capability

of dextran to impart to alkyl cyanoacrylates flexibility and an increased capacity to

accommodate a therapeutic load for drug delivery applications. However, the observation

that PECA-Dex structures are susceptible to aggregation and to becoming brittle over

time has stimulated further research activities towards the development of alternative

materials with improved long-term stability. The functionalisation of dextrans with alkyl

glyceryl moieties represents one of the approaches towards that goal.

2.1.1 Chemical structure of oxiranes for synthesis of alkyl-glyceryl dextrans

Various chain lengths of oxiranes were used in the modification of dextran, these includes

butyl glycidyl ether, octyl glycidyl ether, glycidyl lauryl ether, tetradecyl glycidyl ether, and

hexadecyl glycidyl ether (Figure 2.2)

O On

Figure 2.2 Chemical structure of alkyl glycidyl ether where n=1, butyl; n=5,octyl; n=9, lauryl; n=11,tetradecyl; n=13 hexadecyl.

2.2 Materials and instrumentation

Dextrans from Leuconostoc spp. (MW 6 kDa and 100 kDa), dimethyl sulfoxide (DMSO,

anhydrous, ≥ 99.9)%), potassium tert-butoxide (t-BuOK; reagent grade > 97 %), butyl

glycidyl ether, octyl/decyl glycidyl ethers, glycidyl lauryl ether, tetradecyl glycidyl ethers

and hexadecyl glycidyl ether were all obtained from Sigma-Aldrich UK.

TGA analysis was carried out with TG 209 F1 Libra (TGA from Netzsch Instruments), with

temperature ranged from 25 to 500°C, heating at 10K/min. under Nitrogen atmosphere.

A Büchi Rotovapor (R-200) powered with a Sogevac Saskia PIZ 100 vacuum pump

equipped with a liquid nitrogen trap was used to remove solvents under reduced

pressure during the process of modified dextrans purification, Dialysis was performed

using Visking membrane tubing (Medicell International Ltd, UK) with cut-off either 12-14

kDa or 3.5 kDa in a discontinuous system (10.0 L deionised water (15.0 mΩ); exchanged 3


39

times/day). The alkylglyceryl derived dextrans were characterised by 1H- and 13C-NMR

spectroscopy using a JEOL Eclipse 400+ instrument (JEOL, UK; 400 MHz for 1H- and 100

MHz for 13C-NMR); samples were dissolved in either D2O or DMSO-d6 employing 0.2 %

tetramethylsilane (TMS) as a reference. The spectra were processed using the JEOL Delta

v 5.0.2 software or Advanced Chemistry Development (ACD) software. FT-IR spectra were

recorded using a Nicolet 6700 instrument (Thermo Scientific, UK) equipped with an ATR

Smart Orbit accessory with diamond crystal, at 64 scans and 4 cm-1 data spacing. The

analysis of spectra was performed using the Omnic Specta 8.0 software; for the FTIR

analysis, dried samples were used, and analysed by placing the sample on top of the

diamond crystal plate and reading taken with the Omnic Specta 8.0 software. The

molecular weights of alkylglyceryl dextrans were estimated using a GPC (waters 2000)

with a refractive index detector, under thermostatted conditions (temperature 30°C),

80/20 solution of water/methanol was used as eluent at a flow rate of 0.6 mL/min). The

calibration was performed with Pullulan standards (Shodex Denko) of MW 0.6 × 104, 1 ×

104, 2.17 × 104, 4.88 × 104 and 11.3 × 104, 21 × 104, 36.6 × 104, 80.5 × 104 g/mol.

MALDI-TOF MS experiments were performed on a Micromass MALDI MicroMX

instrument operating in positive reflectron mode in the m/z range of 400-1600; a 337nm

nitrogen laser used for ionisation (pulse setting 1800V; detector setting 2300V; flight tube

12Kv; reflectron 5.2Kv). α-Cyano-4-hydroxycinnamic acid (CHCA) solution at a

concentration of 10 mg/mL in acetonitrile:water (50:50) provided the matrix for the

experiment. CHCA was mixed in the 1:1 ratio with the sample (dissolved in 50:50 v/v

acetonitrile:water) and deposited onto the MALDI plate.

2.3 Methods

Dextran (2g; 12.35 mmol), dissolved in anhydrous DMSO (150 mL) , was allowed to stir

under nitrogen for 2 h before gradual addition of potassium tert-butoxide (t-BuOK) (1.385

g; 12.35 mmol) that had been dissolved in anhydrous DMSO (50 mL) (t-BuOK was

dissolved in DMSO by allowing it to stir for 30 min in anhydrous DMSO under Nitrogen

atmosphere at room temperature). Stirring was continued for another 2 h before the

gradual addition of a solution of specified alkyl glycidyl ether (G4, G8, G12, G14, G16) at

40°C and further stirring for 24 h. After this time, the reaction mixture was transferred

into a dialyzing membrane vessel (MWCO 3.5 or 12-14 kDa) and dialysed against


40

deionised water (10.0 L; exchanged 3x per day) over five days. The content of the dialysis

vessel (ca. 400 mL) was washed with diethyl ether or with DCM (3× 150 mL) to remove

impurities and any unreacted ether. Traces of volatile solvents were removed using the

rotary evaporator (reduced pressure, 50°C) and the sample was again subjected to the

dialysis procedure (48 h) before lyophilisation with a Virtis freeze dryer (<-85°C, for 48h)

to yield the corresponding alkylglyceryl dextrans, which were characterised using FTIR,

1H- and 13C-NMR spectroscopy, GPC, MALDI-TOF MS, SEM and TGA. Yields were in the

range 40-60%.

2.4 Results and Discussion

2.4.1 Synthesis of alkylglyceryl dextrans

The modification of dextrans with alkyl glycidyl ethers (Figure 2.3) was carried out by

attaching alkylglyceryl side chains to the hydroxyl groups through two-step reactions

which involved the conversion of the hydroxyl groups of the pyranose rings of dextran

into the more reactive alcoholates and subsequent reaction with specified alkyl glycidyl

ethers (149). The synthesis was carried out, at 40°C, in anhydrous DMSO and under a

nitrogen atmosphere for 24 h; similar modification of dextran are well documented in the

literature (139, 141, 144, 150). The reactivity of the –OH pyranose ring of the glucose unit

is in the order 2>4>3.


41

Figure 2.3: Scheme for the synthesis of alkylglyceryl dextrans

Native dextrans with a molecular weight of either 6 kDa or 100 kDa were modified by

covalent attachment of alkylglyceryl pendent chains to their hydroxyl groups. This was

carried out by means of a series of commercially available alkyl glycidyl ethers of different

chain lengths: butyl glycidyl ether (G4; 1H NMR, 13C-NMR; Appendix 1), octyl glycidyl ether

(G8), glycidyl lauryl ether (G12), tetradecyl glycidyl ether (G14; 1H-NMR, 13C-NMR;

Appendix 2), and hexadecyl glycidyl ether (G16; 1H-NMR, 13C-NMR; Appendix 3). The

reactions produced a range of alkylglyceryl dextrans: butylglyceryl dextrans (Dex100G4 or

Dex6G4), octylglyceryl dextrans (Dex100G8 or Dex6G8), laurylglyceryl dextrans

(Dex100G12 or Dex6G12), tetradecylglyceryl dextrans (Dex6G14 or Dex100G14) and

hexadecylglyceryl dextrans (Dex100G16 or Dex6G16); the numbers 6 and 100 represent

the molecular weight of the dextrans while 4, 8, 12, 14, 16 represent the chain lengths of

alkyl glycidyl ethers used for the modification of dextrans.

Hexadecyl glyceryl dextran was prepared in two-step reactions which involved conversion

of OH group to more reactive alcoholate with t-BuOK and subsequent reaction with the

specified glycidyl ether; other dextran derivatives were synthesised through the same

procedure.

t-BuOKN2 atm. 24 h.

m=3; G4m=7; G8m=11; G12m=13; G14m=15; G16

m=3; DexG4m=7; DexG8m=11; DexG12m=13; DexG14m=15; DexG16

m

R = H or m


42

2.4.2 Characterisation of the alkylglyceryl dextrans

2.4.2.1 NMR and FTIR

The modified dextran structures were confirmed by FTIR and NMR spectroscopy. The

modification of the dextran was assessed by comparing the 1H-NMR spectrum of the

modified dextrans (examples in Figures 2.4 and 2.5) with that of the native material

(Figure 2.6): the presence of resonances at 0.87 ppm, 1.3 ppm and 1.4 ppm confirmed the

successful functionalisation of dextran. Similar results were obtained for butyl, octyl,

lauryl and tetradecyl dextrans. Table 2.1 summarises the DS% of the modified dextrans.

The successful synthesis of alkylglyceryl dextran was consistent with FTIR spectroscopy

data: the grafting of alkyl glyceryl chains resulted in the formation of a new secondary

alcohol. The band at 1012 cm−1 is assigned to the vibrational mode of C –O. In crystalline

dextran, two absorption peaks at 851 and at 914 cm−1 were observed, respectively

assigned to (C–C) and (C–H) bending modes. It has been proposed that the absorption

bands in the spectral range between 1200 and 1500 cm−1 are mainly due to CH

deformation vibrations and (COH) bending vibrations (151). The bands at 2921 cm-1 and

at 3215 cm-1 are respectively consistent with (C–H) and (O–H) stretching vibrations

(Figure 2.7 and Figure 2.8).

SEM images of native dextran and of Dex100G4 visualised the morphology of these

materials (Appendix 13).

The enumeration of the degree of substitution (Table 2.1), as expressed by the

percentage of substituted saccharide units carrying an alkylglycerol group, was achieved

by 1H-NMR through the comparison of the ratio of integral of the intensity of the terminal

methyl group of alkyl glycerol (IA) with that of the anomeric proton of the glucopyranose

ring (IB); (equation 2.1; Table 2.1).


43

P3069JT_PROTON-3.jdf

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

Chemical Shift (ppm)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

alized Inte

nsity

Figure 2.4: 1H-NMR spectrum of alkyl glyceryl dextran (Dex100G16)

P4039EB_PROTON-3 EA4.esp

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Norm

alized Inte

nsity

Figure 2.5: 1H-NMR spectrum of butyl alkyl glyceryl dextran (Dex100G4)

b

a

a b


44

P3054JT_PROTON-3.esp

5.0 4.5 4.0 3.5 3.0 2.5


0

0.05

0.10

0.15

0.20

0.25

0.30

Norm

alized Inte

nsity

Figure 2.6: 1H-NMR of native dextran

100*3

%B

A

I

IDS ) (eq. 2.1)

Table 2.1: The degree of substitution of the synthesised glyceryl dextrans.

DexnGm DS (%)

Dex6G4 87.70

Dex100G4 68.49

Dex6G8 114.94

Dex100G8 64.91

Dex6G12 54.35

Dex100G12 67.56

Dex6G16 55.56

Dex100G16 144.92


45

Figure 2.7: FTIR of butyl glyceryl dextran (Dex100G4)


46

Figure 2.8: FTIR of octyl glyceryl dextran (Dex100G8)

2.4.2 GPC

GPC was used to determine the molecular weight distribution profile of some of the

synthesised alkylglyceryl dextrans and that of the starting material (native dextrans, Dex 6

kDa and Dex 100 kDa). Owing to differences in the experimental protocol, the average

molecular weights of commercial dextrans that served as starting materials were higher

than those given by the manufacturer (Table 2.4). It was found that Dex6G4 had an

average molecular weight that was lower than that of the native precursor dextran

(Dex6), while Dex100G16 had an average molecular weight that was higher than that of

its precursor macromolecule (Dex100). Since longer chain length should influence the

GPC-determined average molecular weight, a study was conducted to compare

Dex100G16 with its progenitor molecule, Dex100, (Table 2.4). The calibration curve of

pullulan standard, calibration data and the equation of the calibration curve are

respectively presented in Figure 2.9, Table 2.2 and Table 2.3, while the chromatograms of

the analysed dextran and alkylglyceryl dextran are reproduced in Figures 2.10-2.13.


47

Figure 2.9 GPC calibration curve (Pullulan standards)

Table 2.2: GPC calibration data using Pullulan standards

Retention Time /min

Elution Volume /mL

Mol Wt Log (Mol Wt) Calculated Weight

% Residual

Standard Type

Relative Weight

1 9.549 9.549 805000 5.905796 790018 1.896 Narrow 1.00

2 10.413 10.413 366000 5.563481 383256 -4.502 Narrow 1.00

3 11.281 11.281 210000 5.322219 206578 1.657 Narrow 1.00

4 12.219 12.219 113000 5.053078 109750 2.961 Narrow 1.00

5 13.295 13.295 48800 4.688420 49454 -1.322 Narrow 1.00

6 14.188 14.188 21700 4.336460 21831 -0.602 Narrow 1.00

7 14.865 14.865 10000 4.000000 10136 -1.338 Narrow 1.00

8 15.271 15.271 6000 3.778151 5914 1.459 Narrow 1.00

Table 2.3: Equation calculated for the GPC calibration curve

R R^2 Standard Error Equation

1 0.999902 0.999804 1.407507e-002 Log Mol Wt = 2.24e+001 - 3.87e+000 T^1 + 3.08e-001 T^2 - 8.84e-003 T^3

Log M

ol W

t

3.00

4.00

5.00

6.00

Retention Time

9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00


48

Figure 2.10 GPC chromatogram of native dextran (Dex6)

Figure 2.11: GPC chromatogram of native dextran (Dex100)

dw

t/d(logM

)

Cum

ula

tive %

0.00

0.20

0.40

0.60

0.80

1.00

0.00

20.00

40.00

60.00

80.00

100.00

Slice Log MW

2.503.003.504.004.505.00

Mn=

4223

Mz=

23547

Mz+

1=

50405

Mw

=9811

MP

=5925

dwt/d(logM)

Cumulative %

dw

t/d(logM

)

Cum

ula

tive %

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.00

20.00

40.00

60.00

80.00

100.00

Slice Log MW

3.003.504.004.505.005.506.00

Mn=

24919

Mz=

291485

Mz+

1=

522793

Mw

=109399

MP

=42332

dwt/d(logM)

Cumulative %


49



dw

t/d(logM

)

Cum

ula

tive %

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0.00

20.00

40.00

60.00

80.00

100.00

Slice Log MW

2.803.003.203.403.603.804.004.204.40

Mn=

3640

Mz=

7756

Mz+

1=

10240

Mw

=5469

MP

=4330

dwt/d(logM)

Cumulative %

dw

t/d(logM

)

Cum

ula

tive %

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

7.50

8.00

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Slice Log MW

5.165.185.205.225.245.265.285.305.325.345.365.385.405.42

Mn=

185810

Mz=

190943

Mz+

1=

193698

Mw

=188308

MP

=175450

dwt/d(logM)

Cumulative %


50

Table 2.4: Mn, Mw and PDI of the dextrans and alkylglyceryl dextrans

sample Mn Mw PDI

Native dextran 6 kDa

4223 9811 2.3

Native dextran 100 kDa

24919 109399 4.4

Dex6G4 3640 5469 1.5

Dex100G16 186810 188308 1.0 Where: Mn = number average molecular weight

Mw = weight average molecular weight

PDI = Polydispersity index

2.4.3 MALDI-TOF MS

Figure 2.14: MALDI TOF spectrum of Dex6G4.

Peak-to-peak mass differences of 130.2 and 146.2, observed between the main peaks

that form the spectral pattern, are likely due to fragmentation at the O-C bond level (as

suggested in Figure 2.14, insets); this is indicative of a fragmentation pathway that is

characterised by the loss of C7H14O2 and C7H14O3 (MW 130.18 and 146.18 Da,

respectively), which in turn confirms the successful chemical grafting of native dextran

with butylglyceryl pendent chains; no cross-ring fragments could be identified.

OO

OHOH

OO

O

OH

C7H

11O

6

191.0556 Da

C7H

15O

2

131.1072 Da

146.2

130.2


51

The isotopic ratios (discernible in Figure 2.15) are indicative of primarily singly charged

ions. Since the CHCA matrix used (a strong acid in the gaseous phase) tends to induce

extensive fragmentation (Karas et al. 1995, Harvey 1999;(152, 153), the detection of

molecular ions was not possible. This is consistent with suggestions that 2,5-

dihydroxybenzoic acid (2,5-DHB) is a better matrix for the MALDI-TOF MS study of high

molecular weight polysaccharides (such as dextran) that require a high matrix-to-analyte

ratio to give enhanced signals (153, 154). The mass/charge (m/z) fragment loss and

nature of fragment loss are presented in Table 2.5.

Table 2.5: Identified m/z fragment mass loss in the MALDI-TOF spectra of the modified dextrans.

sample m/z fragment loss

Nature of fragment loss identified

Dex6G4 130.1 C7H14O2

Dex100G4 130.1; 146 C7H14O3

Dex100G8 ~186 C11H22O2

Dex6G12 242.2 C15H30O2

Dex100G12 242.3 C15H30O2

Figure 2.15 MALDI TOF spectrum of Dex100G4

Similar spectra have been obtained for the grafted 100kDa or 6kDa dextran (Appendix 7).

130.2

O

OOH

OH

O

O

O

OH

C7H

11O

6

191.0556 Da

C7H

15O

2

131.1072 Da

Molecular Formula = C14

H26

O8


52

2.4.5 TGA

TGA was carried out to determine the thermal stability of samples; the thermograms of

analysed samples are collated in Appendix 6.

Figure 2.16: TGA results for Dex6

Figure 2.16 is the TGA plot from a sample that had been heated under nitrogen to 500°C.

There are 2 mass loss steps: the first of these corresponds to a mass loss of -8.91% and

exhibits a maximum mass loss rate (peak in DTG curve) at 82.5°C, while the second step

(maximum mass loss rate at 315.0°C) is associated with a -74.25% loss relative to the

original mass.


53

Figure 2.17: TGA results of Dex6G4

Figure 2.17 shows the TGA profile of the sample that was heated under nitrogen

atmosphere to 500°C. The thermogram is again associated with two mass-loss steps, the

first of which corresponds with a mass loss of -9.01% and is characterised by a mass-loss

rate maximum at 66.6°C; the second step (mass loss -86.6%) exhibits its maximum mass

loss rate at 327.1°C.

The thermograms of other samples are presented in Appendix 6: the data of the

thermograms in appendices are summarised in Table 2.6; the tabulated temperature

values were determined from the first derivative (point of inflection) of the corresponding

thermogram.


54

Table 2.6: TGA data of first and second mass loss for modified and unmodified dextrans

First mass loss/temperature, °C

Second mass loss/temperature, °C

Total mass loss %

sample % mass loss Temperature /°C

mass loss Temperature /°C

Dex6 -8.90 82.5 -74.25 315.0 -83.2

Dex6G4 -9.01 66.6 -86.58 327.1 -95.6

Dex6G8 -3.54 69.0 -77.81 388.0 -81.4

Dex6G12 -5.36 59.7 -86.17 319.8 -91.5

Dex6G14 -4.39 61.7 -86.49 305.9 -90.9

Dex6G16 -2.85 58.1 -87.70 252.6 -90.6

Dex100 -9.38 84.2 -70.63 304.6 -80.0

Dex100G4 -9.70 66.0 -76.63 326.5 -86.3

Dex100G8 -6.41 61.6 -77.30 328.8 -83.7

Dex100G12 -4.95 59.1 -90.60 335.6 -95.6

Dex100G14 -4.82 74.4 -86.00 309.2 -90.8

Dex100G16 -3.12 24.3 -90.08 332.6 -93.2

All thermograms exhibit two decomposition stages: the first mass loss, which occurs

below 100°C, is attributed to water loss from the sample while the second mass loss,

which occurs in the temperature range 200-350°C, is due to the degradation of the

alkylglyceryl dextran structure (155, 156).

To determine the trend of water content loss across the range of materials, percentage

mass losses were plotted against chain lengths.


55

Figure 2.18: first-stage mass loss as a function of chain length (Dex100 and derivatives)

Figure 2.19: first-stage mass loss as a function of chain length (Dex6 and derivatives)

In accord with expectation, the first mass loss (loss of water) varies with chain length,

with more hydrophilic samples exhibiting correspondingly higher percentage mass loss

(Figures 2.18 and 2.19): the longer the chain length, the more hydrophobic the sample

and the lower its water content. Accordingly, for samples of Dex100 kDa (Figure 2.18), the

plot reveals the gradual decrease in water content. A similar behaviour was observed for

samples of Dex6kDa (Figure 2.19), with Dex6G8 presenting the only anomaly.

-12

-10

-8

-6

-4

-2

0Dex100 Dex100G4 Dex100G8 Dex100G12 Dex100G14 Dex100G16

%m

ass

loss

chain length

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0Dex6 Dex6G4 Dex6G8 Dex6G12 Dex6G14 Dex6G16

%m

ass

loss

chain length


56

2.4.6 Discussion on synthesis and characterisation techniques

Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) has been

developed as a soft ionisation and as a sensitive method that allows the rapid detection

of macromolecules and biopolymers (157). MALDI can generate singly or multiply charged

ions ([M+nH]n+), the prevalence of which depends on the nature of the matrix, the laser

intensity and/or the voltage used. While it is generally accepted that the MS analysis of

polysaccharides is of lower sensitivity than that of peptides and proteins, it has been

shown that MALDI-TOF MS may be usefully applied to the measurement of the molecular

mass of carbohydrates up to 106 Da (158); it has been recently acknowledged however

that this becomes more difficult for higher molecular weights as neutral polysaccharides

show poor ionisation efficiency (154). Figure 2.15 presents a typical MALDI TOF spectrum

of the modified dextran polymers considered in this study. The spectra are characterised

by the lack of a molecular ion and of patterns that are characteristic of natural glycans,

and a series of glycosidic ions. The observed pattern is attributed to the loss of

butylglyceryl chains, which is consistent with the successful chemical modifications of

dextran.

The mechanism for the attachment of alkyl glycerol chains to dextran involves the t-BuOK

-mediated deprotonation of the alcohol moieties of dextran to the strongly nucleophilic

butoxide ion, which in turn attacks the strained glycidyl ether ring (149).

Since the viscosity of the reaction medium, which is influenced by both the length of the

alkyl chains and the concentration of t-BuOK, appeared to impact upon the fate of the

reaction, preliminary experiments were conducted in order to determine optimised

mixing ratios for the reactants. An attempt to reduce the viscosity of the reaction medium

by increasing the amount of solvent (DMSO) by 30% did not produce the desired effect.

By contrast, modulation of the ratio of t-BuOK to alkyl glycidyl ether increased the extent

of the functionalisation. For optimal functionalisation, the ratio 1:3:4 (dextran: t-BuOK:

alkyl glycidyl ether) was used for the shorter chain butyl- and octyl- glycidyl ethers; the

corresponding ratio of 1:2:1 was selected for the modification of dextrans with lauryl

glycidyl ether; the ratio of 1:1:1 was used for the reaction involving the tetradecyl glycidyl

and hexadecyl glycidyl ethers.


57

As expected, GPC experiments showed that the higher the degree of substitution or the

chain length of the sample the higher the corresponding molecular weight. All other 1H-

and 13C-NMR signals were in good agreement with those expected on the basis of

published work (159).

2.5 Conclusions

Alkylglyceryl dextrans have been synthesised by the reaction of commercially available

dextrans and a range of alkyl glycidyl ethers in the presence of a strong base (t-BuOK) in

anhydrous DMSO and under an atmosphere of nitrogen. The crude products were

washed with organic solvent, subjected to dialysis, and freeze-dried to a final yield of 40-

60% before characterisation with NMR and FTIR. NMR spectroscopic investigations

showed that the degree of substitution was in the range 50 - 145%.

Thermograms of the alkylglyceryl dextrans have shown a two-step mass loss profile that

corresponds to the initial loss of water and the higher-temperature thermal

decomposition of the dextran core. TGA measurements revealed that the water content

of each dextran and modified dextrans is influenced by the length of the hydrophobic side

chain.

GPC data showed that butylglyceryl dextran (Dex6G4) has an average molecular weight

that is lower than that of the 6kDa native dextran. By contrast, Dex100G16 exhibits an

average molecular weight that is higher than that of the native dextran (Dex100), which is

attributed to the contribution of the pendent alkylglycerol chain.

Due to extensive fragmentation of the modified dextrans and to the absence from the

spectra of molecular ion peaks, MALDI-TOF MS did not prove suitable for the

determination of the molecular weight of samples.

Preparation and characterisation of nanoparticles

58

3

3. Preparation and characterisation of nanoparticles The preparation of nanoparticles of poly(alkyl cyanoacrylate) was carried out by anion-

propagated emulsion polymerisation and solvent-displacement nanoprecipitation,

whereas the preparation of nanoparticles of PLA-alkylglyceryl dextran was by means of

the dialysis method.

3.1 Mechanism of ACA polymerisation

The method of choice for the chemical synthesis of alkyl cyanoacrylates (ACA) is the

Knoevenagel condensation reaction (73). ACA molecules polymerise readily at room

temperature in the presence of any nucleophile, base or even trace moisture to yield

PACA polymers. In solution, PACA polymers are highly susceptible to rapid degradation

into species of lower molecular weight, which is witnessed as anomalous solution-

viscosity behaviour (160). The low ceiling temperature of PACA has been linked to retro-

Knoevenagel depolymerisation (161).

The polymerisation of ACA has been examined in depth by Ryan and McCann, who have

made the following observations: (i) the ACA monomer and polymer are in rapid, dynamic

equilibrium; (ii) a very high concentration of monomer relative to that of the initiator

favours the production of long chains; (iii) chain-termination occurs by the anionic end-

group becoming protonated by water or other acid functionalities to form a species that

is not capable of carrying the reaction; (iv) the use of base effects a deprotonation of this

dormant chain end, allowing the reactivation of the polymerisation reaction; (v) the

monomer that is present during the equilibrium adds rapidly to any nucleophiles present

to initiate new-chains formation; (vi) since the concentration of the monomer in the new

equilibrium is much lower than that during polymerisation, the new chains that form have

drastically lower molecular weights (implying that longer-chain formation is favoured by

the distortion of the monomer to polymer ratio through the continuous addition of

monomer into the polymerisation vessel); and, (vii) the addition of acid into the reaction

mixture inhibits the deprotonation of the chain ends, preventing the establishment of the

equilibrium (160).


59

The most widely used alkyl cyanoacrylate monomer is ethyl cyanoacrylate (ECA). As with

other alkyl cyanoacrylates, anionic species or a Lewis base are both capable of initiating

the rapid polymerisation of this monomer to give a high molecular weight polymer;

amines and phosphines are adequately nucleophilic substances for the purpose. The

degree of substitution in amines affects reactivity, with tertiary amines initiating an

exothermic polymerisation that results in the formation of high molecular weight

polymers; primary and secondary amines give polymers of lower molecular weights (162).

The high reactivity of alkyl cyanoacrylates is consequent to the electron withdrawing

effect of the ester and nitrile functionalisation of the monomer unit. The effective

utilisation of alkyl cyanoacrylates in nanoparticles formulation involves polymerisation,

which requires a nucleophile or base for the reaction initiation; water, amines, alcohols

and phosphines are employed commonly. The combination of the monomer with the

initiating nucleophile results in the formation of a chain-carrying species that is stabilised

by the ester and nitrile moieties, while the associate polarisation of the double bond

activates further nucleophilic attack. Chain propagation occurs by the repetitive addition

of electron deficient monomer to the anionic end of the growing chain and further to the

biomedically useful polymer (Figure 3.1). The hydrogen bonds between ammonium and

cyano groups that become established during the polymerisation of ACA coupled with

van der Waals interactions represent the forces of attraction that are responsible for the

cohesion of PACA nanoparticles that are important in their use as biomedical materials

(163).


60

Figure 3.1: Mechanism for the anionic emulsion polymerisation of ACA, as initiated by mild nucleophiles (164).

3.2 Materials and instrumentation

Ethyl-2-cyanoacrylate, dextrans (100 and 6 kDa), ethyl-2-cyano-3-ethoxyacrylate (Sigma-

Aldrich), butyl cyanoacrylate (donated by Henkel Ireland) and dimethyl sulfoxide (DMSO;

anhydrous, ≥ 99.9 %) were sourced from Sigma-Aldrich, UK. Dicyclohexylcarbodiimide

(DCC, ≥ 99 %,) was obtained from Fluka. Poly(lactic acid) with free carboxyl group (PLA,

MW 15 kDa) was obtained from Purac, Netherlands. Ultrapure water (18.2 mΩ) was

obtained using a Triple Red instruments. All reagents were used as obtained. The dialysis

purification step of PLA-alkylglyceryl-dextrans was performed in a discontinuous system


61

(deionised water, 15.0 mΩ, 10.0 L; exchanged 3 times/day) using Visking membrane

tubing (Medicell International Ltd, UK; cut-off either at 12-14 kDa or at 3.5 kDa).

Alkylglyceryl dextran of different chain lengths. Low speed centrifugation was performed

by means of a Rotofix 32A Hettich (Zentrifugen-Germany). Ultracentrifugation

experiments utilised a Beckman Ultracentrifugation XL-90 instrument. The Z-average

diameter (nm), PDI and zeta potential (mV) were determined by Dynamic Light Scattering

experiments (Malvern Zetasizer Nano instrument, Worchester, UK) using Whatman filter

paper of specified pore size for filtration. Freeze drying involved the use of either a Virtis

(Advanced Sentry 2.0 Controller for Virtis® Benchtop(TM) or a 'K' Freeze Dryer (SP

Industries-Warminster, USA) instrument. Sonication was by means of a Grant ultrasonic

bath XB3 (Farnell, UK).

TGA analysis was carried out with TG 209 F1 Libra (TGA from Netzsch Instruments), with

temperature ranged from 25 to 500°C, heating at 10K/min. under Nitrogen atmosphere.

Nanoparticle Tracking Analysis (NTA) was carried out at 25 °C using a Nanosight

instrument that was equipped with a thermostatted LM-14 unit and with a 532 nm

(green) laser. The image capture (length 60 s) was by means of a CCD Marlin camera, data

were analysed using NTA 3.0 software.

The hydrodynamic diameter of the nanoparticles was determined with DLS using a

Malvern Zetasizer Nano instrument, UK, equipped with a 633 nm He-Ne laser (173° back

scattering angle detection; Zetasizer software v.7.01 software). Samples analysis was

done in triplicate (25 °C, 2 min equilibration) using clear polycarbonate disposable

cuvettes. Data are presented as Z averages (Z-av.) and the associated polydispersity index

(PDI).

The autotitrations of colloidal material were performed to simultaneously determine Z-

average diameter (Z-av) in (nm), and ZP (mV) as a function of pH; an MTP-2 (Multi-

Purpose Titrator-2, Malvern, UK) equipped with a solvent degasser was employed. The

sample (10 mL) was titrated automatically against aqueous NaOH solutions (5 mM or 50

mM) and against aqueous HCl solutions (5 mM or 50 mM). Prior to each experiment,

solutions were filtered through 0.2 μm PES filters (Whatman).


62

3.3 Methods

3.3.1 Attempted NP formulation with ethyl 2-cyano-3-ethoxyacrylate

Since there are few reports regarding the capability of ethyl 2-cyano-3-ethoxyacrylate

(Figure 3.2) to undergo co-polymerisation to nanoparticulate structures, an attempt was

made to formulate nanoparticles by means of an anionic emulsion polymerisation

protocol (24, 93). To this end, ethyl 2-cyano-3-ethoxyacrylate (1 g) was dispersed in

acetone/water (1:1 v/v; 1 mL) and added dropwise to a solution of dextran (100 kDa; 1%

w/v, 100 mL) in KH2PO4 buffer (pH 2.5). The mixture was allowed to stir overnight (room

temperature), after which time it was neutralized with NaOH solution (1 N), filtered

through 0.8 µm filter paper and centrifuged (90000 g, 1 h).

Figure 3.2: Structure of ethyl 2-cyano-3-ethoxyacrylate

Since no solid product could be isolated, attempts were made to repeat the experiment

at increased pH, in water-free acetone and in the absence of phosphate buffer. All these

attempts failed to produce polymeric structures, suggesting that this molecule is not

amenable to polymerisation under the selected experimental conditions. Hence, ethyl 2-

cyano-3-ethoxyacrylate was excluded as a co-monomer in further nanoparticle-formation

experiments, limiting the choice of cyanoacrylates to the ethyl and butyl homologues.

3.3.1.1 Preparation of PECA-Dex100 nanoparticles

Method: PECA-Dex100 was prepared using a standard literature method (24,93). Ethyl 2-

cyanoacrylate (1 mL) was added dropwise into dextran solution (1% (w/v), 100 mL, pH

2.5; 100 KDa) and the solution was allowed to stir at room temperature overnight. After

this time, the mixture was neutralised with NaOH solution (1 N), centrifuged (Rotofix;

3500 rpm, 20 min), filtered (0.8 µm Whatman filter paper), isolated by

ultracentrifugation (18000 g, 30 min; Beckman ultracentrifuge), rinsed with deionised

water and re-suspended in deionised water (5 mL; sonication in water bath, 10 min). The


63

size and zeta potential of the thus suspended nanoparticles was determined using a

Malvern instrument before the water was removed (freeze dryer, 24 h). The synthesis of

PECA repeated in phosphate buffer medium resulted in the formation of NPs that were

characterised by poor yield, low zeta potential and high conductivity.

3.3.2 Preparation of poly (ethyl 2-cyanoacrylate)–alkylglyceryl-dextran NPs

Method 1 (24, 93): Ethyl 2-cyanoacrylate (ECA) (600 µL) was added dropwise to

alkylglyceryl-dextran (DexnGm; 100 mg; n=6 or 100; m=4, 8, 12, 16) dispersed in water

(100mL; pH 2.5, HCl) and stirred at room temperature for 4 h (Figure 3.3). After this time

the solution was neutralised with NaOH solution (1 N), centrifuged for 15 min (3000 rpm;

Rotofix) and further ultra-centrifuged for 30 min at 12,200 rpm (18000 g; Beckman

ultracentrifuge). The isolated sample (pellet) was rinsed, dispersed in water and sonicated

for 10 min before freeze drying to a batch-dependent yield in the range 20-40%.

Figure 3.3: Schematic representation of the protocol for the synthesis and characterisation of PACA-alkylglyceryl dextran.

IR

HCl(aq.) pH 2.5

SEM

MALDI-TOF-MS

TA-DSC

EA

Characterization

4h

Modified-dextran-PACA-NPs


Modified-dextran-PACA1.1N NaOH

2.Centrifugation

3.Dispersion

Freeze drying

Characterization

DLS

NTA

NMR

AFM

High speed stirringACA


Modified-dextran

Supernatant

Pellet

Re-dispersion

Characterization

DLS

NTA




64

Method 2 (69): ECA (600 µL) was dissolved in acetone (100 mL) and gradually added

(room temperature) to a stirring solution of alkylglyceryl dextran (DexnGm; 100 mg) in

water (100 mL); stirring was continued overnight at room temperature to allow the

evaporation of most of the acetone. Following centrifugation at 40000 rpm for 30 min,

the pellet was rinsed, re-suspended in water and subjected to sonication for 10 min

before isolation by freeze drying to a batch-dependent yield in the range 50-70%.

Method 3 (24): ECA (1 mL) was added dropwise to DexnGm (1% w/v; pH 2.5, HCl) and was

allowed to stir for a minimum of 4 h at room temperature, before neutralisation with

NaOH solution (1 N), filtering (P3 glass filter), and isolation by centrifugation (12200 rpm,

30 min). The crude product was rinsed, dispersed in water (10 mL), sonicated (10 min)

and freeze dried to a yield of 30-40%. SEM image of PECA-Dex100G4 is in Appendix 4.

3.3.3 Preparation of PLA15-Dex100G8-PECA

Alkylglyceryl dextran, PLA and ECA were all incorporated into a single multi-block

structure through the simple polymerisation procedure of nanoprecipitation solvent

displacement.

Dex100G8 (0.05 g) was dispersed (by sonication) into an acetone (50 mL) solution of ECA

(0.265 g) and PLA (0.265 g). This mixture was then added dropwise in water (100 mL; 18.2

mΩ) under continuous stirring. After overnight stirring at room temperature, the mixture

was dialysed in a discontinuous manner (10 L container; 3x a day for 2 days), filtered

(Whatman paper, 90 mm; 1 qualitative circle) placed over sintered glass (P3) and the

filtrate was freeze dried before being subjected to characterisation by DLS (Table 3.8),

TGA (Figure 3.18), DSC (Figure 3.19) and 1H-NMR (Figure 3.17).

3.3.4 Effect of filtration on size of PECA-Dex100G8 NPs

ECA (100 mg) in acetone (100 mL) was gradually added to stirring Dex100G8 (100 mg)

that had been dispersed in water (100 mL) and was allowed to stir at room temperature

overnight. After this time, NaOH solution (2 mL, 0.1 N) was added to the resulting mixture

and allowed to stir for a further 30 min before filtration over sintered glass (P3),

centrifugation (40000 rpm, 30 min), rinsed and re-dispersion in water (10 mL).

Following filtration (yield, 15-25%) the isolated product was characterised using DLS

(Table 3.3) and NMR (Figure 3.5).


65

3.3.5 Preparation of PECA-Dex6G16 nanoparticles by nanoprecipitation

ECA (600 µL) in acetone (100 mL) was added gradually to stirring Dex6G16 (100 mg) in

water (100 mL; 18.2 mΩ). Stirring was continued overnight (room temperature) after

which time the product was isolated by centrifugation (12200 rpm, 30 min), rinsed with

deionised water, dispersed in water (10 mL) and sonicated (ultrasonic bath).

3.3.6 Preparation of PLA-derived nanoparticles by zero length crosslinking

Three different alkylglyceryl dextrans were employed for the preparation of PLA-

alkylglyceryl dextran derived nanoparticles (Figure 3.4; Scheme 1) by zero length grafting

of PLA to alkylglyceryl dextran with N,N’-dicyclohexylcarbodiimide (DCC) (165, 166) and

DMAP. PLA15 respectively grafted onto Dex100G4, Dex100G8 and Dex6G16 produced

PLA15-Dex100G4, PLA15-Dex100G8 and PLA15-Dex6G16.

The apparatus was dried (vacuum) and flushed with Nitrogen before use. DCC (0.045 g,

0.2 mmol) dissolved in anhydrous DMSO (10 mL) was added with stirring to a solution of

PLA (1.75 g, 0.11 mmol) in anhydrous DMSO (100 mL) contained within a round-bottomed

flask that had been equipped with a Nitrogen inlet tube. After 30 min, the solution was

transferred into a stirring solution (Nitrogen atmosphere) of octylglyceryl-dextran

(Dex100G8, 0.85 g) and 4-(dimethylamino)-pyridine (DMAP; 0.026 g, 0.22 mmol) in

anhydrous DMSO (100 mL) that had been stirred for an hour. The reaction mixture was

allowed to stand at room temperature for 24 h, after which time it was filtered through

Whatman paper over P3 sintered glass (to remove dicyclourea (a byproduct of the

reaction) and dialyzed for 48 h (MWCO 12-14 kDa) against deionised water, before freeze

drying. The dried powder was dispersed in acetone (50 mL), filtered through Whatman

filter paper (to remove unreacted PLA) and the isolated pellet was dried under vacuum at

room temperature. The thus dried pellet was washed with water, dispersed in deionised

water (100 mL) and freeze dried to yield (20-30%) of the product. This was used to purify

PLA15Dex100G8.

Purification (method 2): The preparation of PLA15-Dex100G16 and PLA15Dex100G4

nanoparticles followed the protocol presented in Figure 3.4. The zero-length grafting (165,

166) of PLA to alkylglyceryl dextran was carried out with N,N’-dicyclohexylcarbodiimide

(DCC). After dialysis and freeze drying, the powder was dispersed in acetone (100 mL),


66

spun at 22,000 rpm for 30 min (to remove unreacted PLA) and the pellet was dried

overnight under vacuum. The dried pellet was suspended in deionised water (300 mL) and

placed on a shaking platform overnight (room temperature) before freeze drying to yield a

pellet of the material labelled PLA15-Dex100G16. To the supernatant (acetone solution

containing mostly unreacted fraction of PLA) was added water (500 mL) to precipitate any

unreacted PLA, which was removed by filtration. The water-acetone mixture was allowed

to stir gently at room temperature for 48 h (to remove excess acetone), and the solid mass

that formed (yield 50-70%) was labelled PLA15-Dex100G16-acetone fraction.

Figure 3.4 scheme for the synthesis of PLA-alkylglyceryl dextran nanoparticles

3.3.7 Preparation of PBCA-Dex100G4 nanoparticles

BCA (200 mg) was added gradually into a stirred suspension of Dex100G4 (200 mg) in

water (100 mL) and the mixture was allowed to stir for a further 4 h at room temperature.

After this time, the mixture was neutralised with NaOH solution (1 N), filtered through

sintered glass, centrifuged (40000 rpm, 30 min) and the nanoparticles that separated

were rinsed with water, sonicated, dispersed in water (20 mL) and freeze dried to a yield

of 26%. This product was characterised with DLS and NTA.

3.3.8 Degradation study

The esterase-induced study of the degradation of PECA-Dex100G4 was conducted

according to a standard literature method (167, 168): ECA (2.5 g, 2.36 mL) was added

m m

m=3; DexG4

m=7; DexG8

m=15;DexG16

R=H or

25oC

N2.atm. 24h

DMAPDCC

PLA

m=3; PLA-DexG4

m=7; PLA-DexG8

m=15; PLA-DexG16

z

z


67

dropwise to water (250 mL; 18.2 mΩ, pH 3) that contained Dex100G4 (250 mg; 0.1%

w/v) and maintained at 25°C (water bath) and stirred for 3 h. After this time, specified

concentrations (1-5 mg/mL) of an esterase solution in phosphate buffer (pH7) were

added to each NPs suspension (3.5 mL) contained in 30 mL of Ringer’s solution. The

resulting mixtures were incubated at 37°C under shaking (50 cycles/min). Aliquots were

withdrawn after 0.5 h, 1 h, 2 h, 3 h and 4 h and size and PDI were determined using DLS.

The same procedure was repeated with samples at a constant esterase concentration of 2

mg/mL and also with an esterase-free mixture (which served as control).

The effect of increased enzyme concentration on the degradation pattern of PECA-

Dex100G4

The methods adopted for the formulation of nanoparticles and for the study of their

degradation were identical to those described previously (167, 168); with the only

variations for the latter study being the duration of the experiment, which was prolonged

and the concentration of enzyme that was increased (Table 3.1). A mass of 31 mg of

PECA-Dex100G4 contained in a 3.5 mL volume of each suspended population of

nanoparticles was used as substrate for the degradation study. A stock solution of the

enzyme (180 mg in 15 mL; equivalent to 12 mg/mL) was made and distributed amongst

the samples under test as indicated in Table 3.1. Samples were incubated at 37°C (Grant

OLS 200 orbital/linear shaking water bath) under shaking (50 rpm/min); the extraction of

aliquots at premeditated time points allowed the measurement of size and the

determination of PDI by DLS.


68

Table 3.1: protocol for the degradation study of PECA-Dex100G4 NPs

sample A B C D E F

Enzyme (mg) - 7.06 12.94 24.71 36.47 71.75

Units of Enzyme - 120 220 420 620 1220

Enzyme Vol. (mL;

conc. 12mg/mL)

- 0.588 1.078 2.059 3.04 5.98

Ringer’s Solution

(mL)

30 30 30 30 30 30

Nanoparticles

(mL)

3.5 3.5 3.5 3.5 3.5 3.5

3.4 Results and discussion

3.4.1 Preparation of nanoparticles of ethyl 2-cyanoacrylate

3.4.1.1 Preparation of PECA-Dex100 NPs

The yield of PECA-Dex100 NPs (2.41%), was very poor as compared with that reported by

Han et al. (108) who, using a similar method, reported a yield of over 93%. Three batches

of NPs were prepared, all of which produced nanoparticles of reproducible size in the Z-

average diameter range 250-400 nm and PDI of 0.364.

3.4.1.2 Preparation of poly (ethyl 2-cyanoacrylate)–alkylglyceryl-dextran NPs

3.4.1.2.1 Preparation of PECA-Dex6G16 NPs by nanoprecipitation

This method combines the emulsion polymerisation, solvent evaporation and

nanoprecipitation methods into a one-pot synthesis. As the acetone evaporates the

acetone-soluble ECA is allowed to react via an anionic mechanism leading to

instantaneous nanoprecipitation. The presence of acetone appears to play an important

role in regulating the size and size distribution of the formed nanoparticles (Table 3.2),

which is very small (153.3 nm) as compared with that of nanoparticles obtained from

conventional anionic polymerisation.


69

Table 3.2: DLS Characterisation PECA-Dex6G16 nanoparticles (n=1)

Parameter value

Zeta Av. diameter 153.3±0.4 nm

PDI 0.132±0.006

Zeta potential -27.6±0.7 mV

3.4.1.2.2 Effect of filtration on size of PECA-Dex100G8 NPs

A study was conducted in an effort to evaluate the effect of filtration on the size, PDI and

zeta potential of the nanoprecipitation-solvent-displacement-method formulated PECA-

Dex100G8; nanoparticles were characterised by DLS (Table 3.3) and NMR (Figure 3.5).

To evaluate the effect of filtration, poly(ethersulfone) (PES) filter membrane (0.45 µm)

was used for the filtering of a portion of the dispersed sample before DLS analysis (Table

3.3) – no significant differences were observed between the filtered and unfiltered

samples.

Table 3.3: Effect of filtration on NPs size, PDI and zeta potential (±SD; n=3)

parameter Un-filtered Filtered

Size (Z-av.d)nm 120.94±30.57 110.77±29.12

PDI 0.22±0.02 0.18±0.00

Zeta (mV) -38.37±1.12 -31.39±0.90


70

Figure 3.5: 1H NMR spectrum of PECADex100G8 nanoparticles

NMR spectrum PECA-Dex100G8 (Figure 3.5) shows that at least two of the characteristic

resonances of PECA are well defined, namely those at 1.3 ppm and at 4.2 ppm. The

spectral features of alkylglyceryl dextran are less pronounced (Figure 3.5), as is consistent

with the high ratio of PECA to alkylglyceryl dextran. This confirms data from elemental

analysis determinations, which estimated the ratio of PECA to alkylglyceryl dextran at ca.

90:10. The 1H- and 13C-NMR spectra of pure PECA are respectively presented in Figures

3.6 and 3.7.

f

d

c

fe

R= H or

e c

a


71

Figure 3.6: 1H-NMR spectrum of pure PECA

Figure 3.7: 13

C-NMR spectrum of pure PECA

a

bc

d

e

a

bc

d

e

d e

b

a

c

c

b


72

3.4.2 Elemental analysis results

The method for the formation of nanoparticles appeared to be of little influence on the

elemental composition and hence to the ratio of PECA to alkylglyceryl dextran in the

nanoparticulate formulation. The nanoparticles evaluated for elemental composition and

ratio of PECA to alkylglyceryl dextran had been formulated by: (i) anionic emulsion

polymerisation (samples 1-8); or (ii) the nanoprecipitation-solvent-diffusion method

(samples 9-10; Table 3.4).

Elemental analysis enabled the determination of the ratio of PECA to alkylglyceryl-dextran

in complex nanoparticles (Table 3.4). The percentage ratio of alkylglyceryl dextran and

PECA of the polymer obtained in the experiment was deduced by considering their

percentage composition of carbon, nitrogen, oxygen, and hydrogen following the

equation presented in Appendix 11. Experimental elemental compositions were in

agreement with theoretically calculated values. Across the range of synthesised

nanoparticles, PECA was the higher percentage constitutional unit (over 90% in some

samples) while alkylglyceryl-dextran accounted for a small percentage (3.9% to 19.62%)

of the structural component of nanoparticles (Table 3.4).

Elemental analysis reported for the respective composition percentage weight per weight

(w/w) of dextran and poly (isobutyl cyanoacrylate), PIBCA, in a polymer obtained from the

emulsion polymerisation of isobutyl cyanoacrylate (IBCA) is 22% and 78% (169), which is

consistent with current findings in that the ratio of dextran to alkyl cyanoacrylate is

consistently low. This observation is in agreement with that from comparative

(alkylglyceryl-dextran to PECA) NMR experiments which were characterised by the low

intensity of signals from alkylglyceryl-dextran moieties as compared with those from alkyl

cyanoacrylate groups (Figure 3.5). Since it has been observed that sequential washing and

centrifugation of nanoparticles from dextran and BCA reduces the PBCA:dextran ratio to

9:1, it may be reasonably assumed that a significant amount of dextran is simply

physisorbed onto the nanoparticles (170).


73

Table 3.4: Percentage elemental composition of PECA-alkylglyceryl dextran NPs (experimentally found and calculated)

S/No Material g %DS X %PECA (w) %Dex-G (w)

Found (av%C)

Cal C.% Found (av%H)

Cal. (H%)

Found (av%N)

Cal.(%N) av%O(100-CHN)

1 PECA Dex6G4

4 87.7 26.29 92.25 7.75 55.94 57.22 5.77 5.83 10.33 10.33 27.96

2 PECA Dex100G4

4 68.49 26.66 92.99 7.01 56.07 57.17 5.81 5.79 10.41 10.41 27.71

3 PECA Dex6G8

8 114.94 10.21 80.38 19.62 56.31 58.03 6.34 6.51 8.65 8.65 28.70

4 PECA Dex100G8

8 64.91 46.35 95.92 4.079 56.22 57.51 5.73 5.78 10.67 10.67 27.38

5 PECA Dex6G12

12 54.35 31.04 94.34 5.66 56.31 57.61 5.89 5.88 10.41 10.41 27.39

6 PECA Dex100G12

12 67.56 49.07 96.09 3.91 56.35 57.69 5.82 5.83 10.63 10.63 27.19

7 PECA Dex6G16

16 133.33 19.84 88.09 11.91 56.95 59.36 6.45 6.61 9.13 9.133 27.47

8 PECA Dex100G16

16 144.92 17.32 86.07 13.93 57.22 59.78 6.58 6.80 8.78 8.78 27.42

9 PECA Dex10G4A

4 68.49 36.92 94.84 5.159 56.81 57.29 5.67 5.75 10.62 10.62 26.90

10 PECA Dex100G16A

16 144.92 18.85 87.05 12.95 57.99 59.63 6.35 6.72 8.94 8.94 26.73

g = no of C atoms in the alkyl group present in the pendent chain (in substituted dextran) %DS = the degree of substitution of dextran with pendent chains (as calculated by NMR) X = molar ratio (ECA monomer : dextran glucopyranose units) ECA C6H7NO2 Dex C6H10O5 G C(g+3)H(2g+6)O2 (equations in Appendix 11)


74

For the 1H-NMR determination of the percentage degree of substitution (%DS) of

modified dextran used for nanoparticles formulation, the ratio of the intensity of the

resonance integral of the terminal methyl of the alkylglycerol chain (A) was divided by

three-times (corresponding to the ratio of attaching protons) the intensity of the integral

of the signal due to the anomeric proton of the pyranose ring of the glucose unit of

dextran (equation 3.1). As expressed, %DS is defined as the number alkylglycerol chains

attaching per 100 glucose units of dextran (171) normalised with reference to the

maximum theoretical percentage of 300 for the fully –OH substituted pyranose groups

structure.

100*3

%B

ADS (eq. 3.1)

3.4.3 MALDI-TOF-MS of PECA-alkylglyceryl dextran NPs

Figure 3.8 MALDI TOF spectrum of PECADex6G4

PECA-Dex6G4 NPs Peak to peak mass differences between neighbouring peaks was ca.

130 Da, which may be explained in terms of the mass loss corresponding to the PECA

OO

OH

O O

O

O

OHO

O

N

C14

H25

O8

321.1549 Da

C6H

7NO

2

125.0477 Da

114.2

130.1

130.1


75

fragment. The spectrum exhibits the characteristic features of carbon-oxygen bond

fragmentation (Figure 3.8).

Figure 3.9: MALDI TOF spectrum of PECA-Dex6G12

PECA-Dex6G12 NPs characteristic peak-to-peak mass differences was ca. 125 Da,

indicating that PECA was eliminated by means of carbon-oxygen bond fragmentation

(figure 3.9). Other spectra were appended in (Appendix 8).

The most characteristic fragment mass loss was that of PECA, which is consistent with the

80-90% proportion of PECA that was calculated from elemental analysis data. This large

percentage of PECA structural units in nanoparticle structures may account for the mass

loss of 125 Da observed for most nanoparticles except those from PECA-Dex100G4 and

PECA-Dex6G4 that had peak differences of 130 Da (Table 3.5) and those from

PECADex6G8 (Table 3.5) which did not have consistent characteristic of 125 Da peak-to-

peak fragment mass difference (172).

OO

OHO O

O

O

OH

O

O N

C24

H47

O8

463.3271 Da

C6H

8NO

2

126.0555 Da

CH2

N

COOEt

Molecular Formula = C6H

7NO

2

Formula Weight = 125.12528

125.1

16.0


76

Table 3.5: m/z fragment mass loss of PECA-alkylglyceryl dextran NPs

sample m/z fragment mass

Nature of fragment loss

PECA-Dex6G4 130.1,114.2 C7H14O2

PECA-Dex100G4 130.2 C7H14O2

PECA-Dex6G8 125.1 C6H7NO2


PECA-Dex6G12 125.1,16 C6H7NO2

PECA-Dex100G12

125.1 C6H7NO2


PECA-Dex100G16

125.1 C6H7NO2

3.4.4 Autotitration of PECA-alkylglyceryl dextran nanoparticles

To evaluate the stability of the nanoparticles, there was carried out an automatic pH

titration of PECA and PECA-alkylglyceryl nanoparticles (Figures 3.10-3.14) against aqueous

HCl (0.005/0.05M). Data show that the zeta potential of PECA nanoparticles and PECA-

alkylglyceryl dextran nanoparticles decreases with decreasing pH.

PECA-alkylglyceryl dextran nanoparticles (PECA-Dex6G4 (1:1), PECA-Dex6G4 (1:6), PECA-

Dex6G16 (1:1), PECA-Dex6G16 (1:6); respectively presented in Figures 3.11; 3.12, 3.13

and 3.14) and PECA nanoparticles (Figure 3.10) were all assessed for their stability at

different pH environments. The Z-averaged diameter (nm) and zeta potential (mV) were

determined (MPT-2 Malvern instruments) as a function of pH over the range of ca. pH 3-8

using HCl solutions as titrants (0.005 M and 0.05 M). Protonation/deprotonation

processes may explain the slight variations in the size of nanoparticles under different pH

conditions.

The observed pH-induced decreases in the size of PECA-NPs (Figure 3.10) and PECA-

Dex6G4 (1:1) NPs (Figure 3.11) are not statistically significant (p<0.05, ANOVA; Table 3.6).

Similarly, the average sizes of PECA-Dex6G4 (1:6) NPs (Figure 3.12), PECA-Dex6G16 (1:1)

NPs (Figure 3.13) and PECA-Dex6G16 (1:6) NPs (Figure 3.14) were all little influenced by

pH. The pH-induced differences in the measured zeta potentials of PECA NPs (Figure 3.10)

and PECA-Dex6G4 (1:1) were not statistically significant (p<0.05) but those of other NPs

exhibited considerable variation (Table 3.6).


77

Figure 3.10: Effect of pH on PECA-nanoparticles (0.25mg/mL) MPT-2 Titration-[0.005M/0.5M HCl] (n=2, ±SD)

Figure 3.11: Effect of pH on PECA-Dex6G4 (1:1) nanoparticles (0.25mg/mL); MPT-2 Titration (0.005/0.5M HCl; n=3 ±SD)

-50

-40

-30

-20

-10

0

10

0

100

200

300

400

500

600

700

800

7.44 6.99 6.65 5.83 5.46 5.09 4.67 4.16 3.71 3.2

Zeta potential (mV) Z.av.diameter (nm)

pH

size zeta

-40

-35

-30

-25

-20

-15

-10

-5

0

0

100

200

300

400

500

600

7.78 7.42 7.02 6.6 6.19 5.5 5.15 4.61

Zeta potential (mV) Z.av.diameter(nm)

pH

size zeta


78

Figure 3.12: Effect of pH on the characteristics of PECA-Dex6G4(1:6) nanoparticles (0.25mg/mL); MPT-2 Titration (0.005/0.5M HCl; n=3 ±SD).

Figure 3.13: Effect of pH on PECA-Dex6G16(1:1) nanoparticles (0.25mg/mL); MPT-2 Titration (0.005/0.5M HCl; n=3 ±SD).

-35

-30

-25

-20

-15

-10

-5

0

100

200

300

400

500

600

700

7.47 7.13 6.66 5.88 5.19 4.88 4.61 4.19 3.68 3.21

Zeta potential (mV) Z-av.diameter (nm)

pH

size zeta

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

0

50

100

150

200

250

300

350

400

7.63 7.26 6.71 6.33 5.68 5.39 4.94 4.49 3.97 3.53 3.04

Zeta potential (mV) Z.av.diameter (nm)

pH

size zeta


79

Figure3.14: Effect of pH on PECA-Dex6G16 (1:6) nanoparticles (0.25mg/mL); MPT-2 Titration (0.005/0.5M HCl; n=3 ±SD).

Table 3.6: Comparison of effects of pH on the size and zeta potential of titrated nanoparticles

Sample

Size Zeta

potential

ANOVA (p)

1 PECA 0.262 0.193

2. PECA-Dex6G4(1:6) 0.785 0.022

3. PECA-Dex6G4(1:1) 0.056 0.184

4. PECA-

Dex6G16(1:1)

0.685 0.001

5. PECA-

Dex6G16(1:6)

0.158 0.000

(p < 0.05 significant)

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

0

50

100

150

200

250

300

350

400

450

8.1 7.47 7.17 6.73 6.05 5.41 4.91 4.65 4.36 3.86 3.36

Zeta potential (mV) Z.av.diameter(nm)

pH

size zeta


80

3.4.5 Preparation of PBCA-Dex100G4 nanoparticles

PBCA-Dex100G4 nanoparticles were characterised for size, PDI and zeta potential with

DLS and NTA (Table 3.7), NTA (Figure 3.15) and further with FTIR (Figure 3.16). The

functional group of interest were noted as: C=O stretch at 1743.88 cm−1; C–O stretches in

the range 1300 - 1000 cm−1; hydrocarbon functionalities CH, CH2 and CH3, respectively at

2954, 1450 and 1374 cm−1; OH absorption at 3500–3200 cm−1 (168); and CN band at 2242

cm−1 (173).

Table 3.7: Size, PDI and zeta potential of PBCA-Dex100G4 NPs (n=1)

DLS NTA

Z-av.diameter(nm) 149.8±1.2 120.3±35.5

PDI 0.394± 0.018

Zeta potential

(mV)

-47.8± 2.0

Figure 3.15: Size concentration intensity distribution of PBCA-dex100G4 NPs


81

Figure3.16 FTIR spectrum of PBCA-Dex100G4 NPs

3.4.6 Preparation of PLA15-Dex100G8-PECA

Alkylglyceryl dextran, PLA and ECA were all incorporated into a single multi-block

structure through the simple polymerisation procedure of nanoprecipitation solvent

displacement.

PLA-Dex100G8-PECA was prepared (n=3) in nanoparticulate form with narrow size

distribution (Table 3.8).

Table 3.8: Size and PDI of PLA15-Dex100G8-PECA NPs (±SD, n=3)

The combination of alkylglyceryl dextran, PLA and ECA was used in an attempt to

formulate PLA15-Dex100G8-PECA nanoparticles that are both stable in suspension and

flexible. ECA was employed as the crosslinker that connects covalently the polymer

chains. Since available OH groups in both PLA and alkylglyceryl dextran could serve as the

Parameter Value

Z-av.diameter(nm) 143.13±1.70

PDI 0.19±0.10


82

initiating site for the polymerisation of ECA, the covalent bond formed by PECA served as

a link between the alkylglyceryl dextran chain on one side and the PLA on the other.

Other intermolecular forces (H-bond and van der Waals interactions) are however

assumed to be of significance in maintaining the nanoparticulate structure in suspension.

The NMR spectrum of PLA15Dex100G8PECA (Figure 3.17) shows the characteristic methyl

and methylene resonances of PLA at 1.45 ppm and 5.19 ppm, respectively; the 1.2 ppm

signal in the spectrum of PECA is attributed to methyl protons while that at 4.2 ppm is

assigned to the methylene proton of PECA. The anomeric proton of the pyranose ring of

dextran is manifested at 4.67 ppm. Protons corresponding to the alkylglyceryl chain

methyl group are observed at 0.87 ppm.

Figure 3.17: 1H-NMR spectrum of PLA15-Dex100G8-PECA (400 MHz).

3.4.7 TGA and DSC thermograms of PLA-Dex100G8-PECA

Figure 3.18 shows the TGA plots of a sample of PLA-Dex100G8-PECA which had been

heated to 600°C under a nitrogen atmosphere and then subjected to an oxidative

treatment (synthetic air) to 1000 °C.

d

c

a

b

z

fe

R= H or

c d

b e

a

f


83

In the temperature range up to 600 °C there are at least three mass loss steps. The major

mass loss (51.4%) occurs at a rate (peak in DTG curve) at 168.6 °C. This step is marked by

a shoulder (at 192.4°C) in the DTG signal, which indicates the complexity of the

mechanism of degradation. The second step is associated with a mass loss of 42.4% and is

characterised by a rate maximum at 333.3 °C. The final (third) step exhibits a mass loss of

5.5 %. Exposure of the sample to synthetic air causes a further mass loss of 1.2%.

Figure 3.18: TGA plots of PLA-Dex100G8-PECA NPs

DSC results for the sample of PLA-Dex100G8-PECA.

In (Figure 3.19) the sample was heated from -100 °C up to 100 °C at a heating rate of 10

K/min. Indicative of a glass transition, the first heating cycle (blue curve) identifies a

thermal event at 54.2 °C (midpoint) which is accompanied by a change in specific heat

capacity of 0.444 J/g*K. The shift in heat capacity is partially overlapped by a relaxation

event that is centred at 60.1 °C. The same plot shows a small endothermic effect, which is

most likely associated with the onset of evaporation or that of oxidative degradation of

some sample components.

The second heating cycle (green curve) confirms the glass transition temperature of the

sample at 49.6 °C, with a change in the specific heat capacity of 0.232 J/(g*K). The


84

endothermic relaxation effect observed during the first heating was reproduced during

the second heating run.

Figure 3.19: DSC of PLA-Dex100G8-PECA NPs

3.4.8 Preparation of PLA-derived nanoparticles by zero length crosslinking

P4041EB_PROTON-3 .esp

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0


0

0.5

1.0

1.5

2.0

2.5

Norm

alized Inte

nsity

Figure 3.20:

1H-NMR spectrum of PLA15-Dex100G8 nanoparticles.


85

Figure 3.21: H/H-COSY NMR of PLA15-Dex100G8 nanoparticles.

Figure 3.22:

13C-NMR of PLA15-Dex100G8 nanoparticles

X : p a r t s p e r M i l l i o n : 1 H

5 . 0 4 . 0 3 . 0 2 . 0 1 . 0

Y : p a r t s p e r M i l l i o n : 1 H

5 . 0

4 . 0

3 . 0

2 . 0

1 . 0

( M i l l i o n s )

1 . 0 2 . 0

p r o j e c t X

( M i l l i o n s )

1 . 0

2 . 0

p r o j e c t Y

P4041EB_CARBON-3 e223.esp

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20


0

0.05

0.10

0.15

Norm

alized Inte

nsity


86

The grafting of PLA to alkylglyceryl dextran (PLA-Dex100G8) has been successful in that it

yielded nanoparticles that are readily dispersible in water. The nanoparticles were

characterised with NMR spectroscopy, 1H (Figure 3.20), H/H-COSY (Figure 3.21) and 13C-

NMR (Figure 3.22). The mixing of alkylglyceryl dextran and PLA in the 1:2 ratio results in

insufficient crosslinking as witnessed by the significant amounts of unreacted PLA that are

removed during purification. Consequently the mixing of these materials in the 1:1 ratio,

which will result in considerably lower loses of PLA during purification, was suggested for

subsequent reactions.

PLA15-Dex100G16(or4) nanoparticles were synthesised successfully, but their purification

required several steps (166) and was conducted in a way that two fractions of the material

were obtained. While both fractions of nanoparticles exhibited the characteristic 13C- and

1H-NMR resonances of glyceryl-dextran and PLA, it was the 1H NMR spectra of the

acetone fraction that showed PLA and alkylglycerol chain resonances that were of notably

higher intensity than that of dextran, as is desirable. The acetone fraction was associated

with the dominant yield of 50-70%.

The chemical shifts of 13C-NMR and 1H-NMR of dextran were in agreement with published

data (159, 172, 174-177), with characteristic features in their ranges 65 ppm to 99 ppm for

13C NMR, and 3 ppm to 5.2 ppm for 1H NMR. Slight variations in the observed chemical

shifts are attributed to solvent effects (178). The chemical shifts of PLA both in 13C- and

1H-NMR were consistent with the structure of PLA, exhibiting 13C-NMR multiplets at

169.53-169.75 ppm (–CO), 69.23 ppm (–CH), and 17.00 ppm (–CH3), and 1H-NMR

resonances at 5.19 ppm (CH) and 1.46 ppm (CH3); the observed chemical shifts correlate

well with published data (179, 180).

3.5 Enzymatic degradation of PECA-alkylglyceryl dextran nanoparticles

The degradation study of PECA-Dex100G4 nanopaticles, along with that of a control

sample, was conducted at constant enzyme concentration and variable enzyme

concentration. The use of heat to stop the degradation process resulted in the formation

of cloudy solutions, which could not be utilised effectively for further analysis.


87

The measurement of size and PDI by DLS did not reveal any statistically significant

differences in the degradation behaviour of control and enzyme-containing PECA-

Dex100G4 nanoparticles (Figure 3.23, 3.24 and 3.25). It is possible that the amount or

activity of the enzyme utilised was not sufficient to bring about the significant

degradation of the nanoparticles within the timescale of the experimental protocol,

especially since the degradation of PECA-Dex100G4 NPs was found to be dependent on

both time and enzyme concentration: the longer the time of the experiment or the higher

the concentration of the enzyme, the faster the degradation. Notably, PECA-nanoparticles

are known to be susceptible to surface degradation (181).

Figure 3.23: Effect of time on size and PDI of PECA-Dex100G4 nanoparticles (n=3, ±SD; control, no enzyme)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0

50

100

150

200

250

300

350

400

450

500

0.5 1 2 3 4

PDI Z-av.diameter(nm)

Time (h)

SIZE PDI


88

Figure 3.24: Effect of constant enzyme concentration on size and PDI of PECA-Dex100G4 nanoparticles (n=3, ±SD).

Figure 3.25: Effect of enzyme concentrations on size and PDI of PECA-Dex100G4 nanoparticles (n=3, ±SD).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0

100

200

300

400

500

600

2 2 2 2 2

PDI Z.av.diameter (nm)

Enzyme conc. mg/mL

SIZE PDI

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0

50

100

150

200

250

300

350

400

1 2 3 4 5

PDI Z.av.diameter (nm)

Enzyme conc. mg/mL

size PDI


89

The effect of increased enzyme concentration on the degradation pattern of PECA-

Dex100G4 NPs

Figure 3.26: Enzyme-mediated degradation of PECA-Dex100G4 nanoparticles during the first 4h of the extended study (0, 1 and 4 h time points) for different enzymatic activity (A= 0 units; B= 120 Units; C= 220 Units; D= 420 units; E=620 units; F=1220 units).

Consideration of the data for the first 4 h of the 84 h degradation study, for all the sample

sets there was an apparent decrease in size during the first hour of the experiment

(Figure 3.26), and also between aliquots of A, B and C sampled at the first- and fourth-

hour periods; this is in agreement with published data on PBCA nanoparticles (168).

Aliquots of D, E and F sampled at four hours from the onset of the experiment exhibited

increased sizes relative to those sampled at one hour. The increases in the size of D, E,

and F after four hours of incubation with the enzyme were witnessed as initiation of

aggregation. The increase appears to be directly related to enzyme concentration. In

accord with the findings of Muller et al. (181), who reported the NaOH-induced surface

degradation of PECA-nanoparticles, the degradation pattern observed with samples A, B

and C could be consequent to surface degradation whereby PECA becomes depleted over

time; the increase in size observed for D, E and F after the expected initial decrease may

be explained in terms of agglomeration that is consequent to the complete degradation

0

200

400

600

800

1000

1200

A-size B-Size C-size D-size E-size F-size

Z.av.diameter(nm)

0

1

4


90

of the nanoparticles (Figure 3.27); considering the high concentration of the enzyme, it is

possible that depleted NPs plays a significant role in this process. The esterase enzyme

degradation study showed that the degradation of nanoparticles is directly proportional

to the amount of enzyme, which is in agreement with published data (182), and that the

higher the enzyme concentration the faster the degradation.

Figure 3.27: Enzyme degradation of PECA-Dex100G4 over time, for different enzymatic activity (A= 0 units; B= 120 Units; C= 220 Units; D= 420 units; E=620 units; F=1220 units).

3.6. Conclusions

In our hands, ethyl 2-cyano-3-ethoxyacrylate on its own could not be formulated into

stable nanoparticles. The synthesis of PECA in phosphate buffer resulted in the formation

of NPs that were characterised by poor yield, low zeta potential and high conductivity.

PBCA-Dex100G4 NPs was prepared at an average size of 149.8 nm and zeta potential of -

47.8 mV. FTIR studies confirmed the presence of the cyanoacrylate group since the

spectra exhibit the characteristic stretching vibrations of C=O (1743.88 cm−1), CN (2242

cm-1) and C-O (1300 - 1000 cm-1).

The dispersion of nanoparticles in water and its subsequent filtration through a PES

membrane showed that the filtering process does not influence the DLS-determined

characteristics of nanoparticles.


91

Alkylglyceryl dextran was successfully reacted with PLA through the zero-length grafting

of PLA to alkylglyceryl dextran in the presence of DCC. As determined by 1H NMR, the

degree of grafting of PLA to alkylglyceryl dextran was 163.9%.

An esterase-induced degradation study of samples of PECADex100G4 showed that the

higher the enzyme concentration the faster the degradation, as evidenced by the rate of

the agglomeration process that accompanies nanoparticle depletion.

Loading of nanoparticles with drugs peptide and fluorophores

92

4

4. Loading of nanoparticles with drugs, peptides and fluorophores

The labelling of drug delivery vehicles with fluorescent dyes is rationalised in this work in

terms of the capability to track the progress of the label through the host environment by

means of the many fold array of fluorescence-detection technologies. To facilitate cell

interaction studies, fluorophores, peptides and fluorescence drugs were loaded or tagged

to the synthesised NPs.

4.1. Actives and fluorophores

Rhodamine B (Figure 4.1) is an organic dye that has been used traditionally as a tracer dye

in water to determine the rate and direction of flow. Owing to its highly fluorescent

nature and solubility in water, methanol and ethanol, Rhodamine B has found extensive

use as a tracer molecule in biomedical research.

Figure 4.1: Chemical structure of Rhodamine B

Curcumin, found in rhizomes of curcuma longa or tumeric, is a low molecular weight

diphenol-functionalised bis–α,β-unsaturated β-ketone that exists in equilibrium with its

enol tautomer (183). Curcumin (structure in Figure 4.2) is soluble in both polar and non-

polar organic solvents. Although highly insoluble in water (11 ng/mL), it is soluble in

alkaline or extremely acidic aqueous solvents; the pH-dependent solubility is assumed to

be due to ionisation of the phenolic or enolic groups and/or due to degradation. This is

Cl-


93

witnessed by the solvent-dependent UV-vis absorption profile of Curcumin. Toluene

solutions of the material fluorescence strongly, exhibiting maximum absorption at 418

nm (184). The main degradation products of Curcumin (185) are ferulic acid,

feruloylmethane, vanilline and acetone. Curcumin is known to possess many therapeutic

effects but is rapidly eliminated from systemic circulation (186).

Figure 4.2: Chemical structure of Curcumin

It has been suggested that the low bioavailability of Curcumin limits its capability to reach

target tissues where it can exert its claimed pleiotropic effects. Hence, the covalent

attachment of Curcumin to nanoparticles represents one approach towards overcoming

this limitation.

Because of its low aqueous solubility and its amphiphilic nature, the fluorescence

intensity of Curcumin decreases with increasing water volume and also decreases as the

acidity of its liquid host varies from neutral to extremely acidic or to basic (pH >8.0) (185).

The absorption and fluorescence capacity of Curcumin may be used to both locate and

quantify the molecular distribution in normal and tumour cells (187). The fluorescence of

the molecule allows the visualisation of the distribution of Curcumin while UV absorption

measurements provide a means for the quantification of this molecule within a specified

sample size, rendering the molecule suitable for the assessment of the fate of Curcumin-

loaded nanoparticles for delivery through the BBB. However, the determination of the

fate of Curcumin in living tissues is limited by its amphiphilic nature and susceptibility to

degradation: deprotonation of Curcumin effects spectral changes, both in terms of the

extinction coefficient and in terms of the spectral profile (184) while degradation is

manifested by colour change. Furthermore, the steady-state absorption and


94

fluorescence characteristics of Curcumin are sensitive to the immediate environment due

to keto-enol isomerism. The loading of Curcumin amphiphilic NPs may therefore lead into

structural changes that need to be taken into consideration in attempts to follow the fate

of such nanoparticles. These potential complications are balanced by the capability of

Curcumin-labelled nanoparticles to fluoresce in the blue laser channel, which renders

those nanoparticles amenable to direct visualisation under the optical microscope. Also,

the use of Curcumin fluorophores is complementary with the concomitant use of DAPI

(4’6-diamino-2-phenyl indole) nuclear stain. Although the spectral characteristics of

Curcumin are sensitive to solvent polarity (188), the molecule displays an intense

absorption band in the region 350-480 nm in all media of interest for this work.

Figure 4.3: Chemical structure of Doxorubicin hydrochloride

Doxorubicin (Figure 4.3) has been described as a very potent anti-cancer agent (189, 190)

that has great potential for CNS chemotherapy but lacks the ability to cross the BBB

because it is a substrate for the ATP-dependent efflux pump P-glycoprotein (P-gp) at the

base of the BBB (191, 192). Prerequisite to the transport of Doxorubicin to the brain is the

development of a suitable carrier that can cross or circumvent this barrier (193). Studies

with P-gp suppressors (e.g. Cyclosporine A) have shown that significant amounts of

Doxorubicin may be capable of penetrating the BBB following administration (191, 194).

Also, PACA nanoparticles have been associated with the transport of Doxorubicin to the

brain in significant amounts (91, 195). In another example, co-administration of

Doxorubicin and Curcumin that had been loaded into PACA/chitosan nanoparticles has

been suggested to increase the penetration of Doxorubicin across the BBB (196). An

additional benefit associated with the use of nanopolymeric PACA carriers is the claimed

lowering of the cardiotoxicity of Doxorubicin, which is a known side effect that limits its

.HCl

2


95

use for therapeutic purposes (193, 197, 198). The change in the biodistribution profile of

Doxorubicin NPs may be responsible for the relatively low cardiotoxicity of this agent.

The ratio of copolymer to PECA in Doxorubicin nanoparticles is reported to influence the

initial burst release of the drug from the nanoparticulate matrix, as is exemplified by the

observation that a structure of PECA-PLGA in the 1:1 ratio exhibits reduced losses during

the initial burst release of Doxorubicin (199). The burst release behaviour seen in this

study may be due to the adopted ratio (1:6) of alkylglyceryl dextran to PECA. Notably,

emulsifier-free PBCA loaded with Doxorubicin is reported to release its drug content very

slowly, exhibiting a half-life of over 100 h in PBS (200).

4.2 Methods and instrumentation

Dialysis membrane (10000-MWCO) and PBS tablets were sourced from Sigma-Aldrich.

Other equipment used included a: Perkin Elmer UV-Vis spectrophotometer, Grant water

bath, Varian Cary Eclipse Fluorescence spectrophotometer, Falcon tubes (50 mL). The

Perkin Elmer UV-Vis spectrophotometer was zeroed before use, with the wavelength set

between 400-750nm. Samples were measure individually by placing the sample-

containing cuvette against that of the reference sample. A Varian Cary Eclipse

spectrophotometer recorded fluorescence measurements after placing the sample-

containing cuvettes in the pre-zeroed sample reading chamber; the wavelength was set

according to absorbance wavelength of the sample to be measured (excitation-emission)

and data were processed using Cary Eclipse software. Mini-spin Eppendorf centrifuge.

4.2.1. Loading of PECA-Dex, PECA-Dex100G4 and PECA-Dex100G8 NPs with Rhodamine B

Rhodamine B solution (10 µl, 10 µg/µl) was added to a stirred suspension of PECA-Dex,

PECA-Dex100G4 and PECA-Dex100G8 nanoparticles each (1 mL, 20 mg/mL) and stirring

was continued for 3h at room temperature. After this time, the nanoparticles where

separated by centrifugation (18000 g) and re-suspended in deionised water (5 mL)

characterised by DLS (Figure 4.6).

For the preparation of Polysorbate 80-coated nanoparticles, Polysorbate 80 (100 µl, 10%

w/v) was added into the stirred suspension of PECA-Dex100 nanoparticles (tagged with

Rhodamine B) to a final concentration of 1% (w/v) and stirring was continued for a further


96

30min. The particles were separated by centrifugation using the Eppendorf centrifuge

(12200 rpm, 30 min) and re-dispersed following rinsing with deionised water and re-

suspension in deionised water (5mL) under sonication (10 min) and characterised with

DLS (Table 4.1); the NTA fluorescing video record is placed in (Appendix 5).

Monitoring the release of Rhodamine B

PECA-dex, PECA-Dex100G4 and PECA-Dex100G8 nanoparticulate samples (2 mg) that had

been loaded with Rhodamine B were each dispersed in PBS (10 mL, pH 7.4), and

sonicated for 10 min before 1.5 mL of each solution was introduced into an 1.5 mL

Eppendorf tube and incubated in a water bath (Grant thermostat temperature shaker,

thermostatted at 37°C). Samples were removed sequentially at specified time intervals

and centrifuged (12200 rpm, 15 min). To a specified volume of the supernatant (1 mL)

was added to PBS (2 mL) and the absorbance was recorded over the UV range 650 nm to

450 nm using a Elmer Perkin instrument; the variation in the intensity of the absorption

maximum (564.76 nm) was monitored at specified time intervals. Since the release of

Rhodamine B from nanoparticles was found to be very limited, this study was

discontinued.

4.2.2 PECA-Dex100G4 and PLA-Dex100G8PECA Nanoparticle-loading with Curcumin: the

EtOH/H2O (1:1) method

Curcumin (0.1 mg) was loaded onto PECA-Dex100G4 NPs and PLA-Dex100G8PECA NPs

(100 mg) by stirring (overnight, room temperature) nanoparticles in ethanol-water (1:1

v/v). After this time, the samples were spun at 12200 rpm for 15 min using the mini-spin

Eppendorf and the absorbance of the supernatants (1 mL aliquot to which 2 mL of DMSO

had been added) were taken to determine the degree of loading. Concentration was

determined using the equation of the calibration curve of Curcumin (Appendix 12).

4.2.2.1 Determination of Curcumin content of PECA-Dex100G4 NPs and PLA-Dex100G8-

PECA NPs

Freeze-dried nanoparticles (20 mg) of PECA-Dex100G4 and PLA-Dex100G8-PECA that had

been loaded with Curcumin were each dissolved in DMSO (3 mL) and their absorbance

were measured to allow the determination of the concentration of released Curcumin,

which corresponds with that loaded onto the specified weight of nanoparticles. Again,

loading was determined with reference to the standard plot of Curcumin in DMSO.


97

4.2.2.2 Loading of Curcumin into PECA-Dex100G4 nanoparticles: cross linking with acetic

acid and TPP (Tripolyphosphate)

The loading of PECA-Dex100G4 NPs with Curcumin was conducted according to the

method of Rejinold et al. (201) in which acetic acid and Tripolyphosphate (TPP) were

employed to promote entrapment to the nanoparticles. A release study was conducted in

which a portion (0.008 g) of the pellet was dispersed in PBS (10 mL) before being

distributed into 9 Eppendorf tubes that were subsequently incubated at 37oC in a Grant

water bath. Aliquots were withdrawn at specified time periods and each sample was spun

for 5 min at 5000rpm; the method was not suitable because the released Curcumin was

either sticking to the container or washed off during the process of supernatant removal.


PECA-Dex6G8 NPs by nanoprecipitation-solvent evaporation.

Doxorubicin was loaded by nanoprecipitation using acetone as the organic solvent for

ECA.

Preparation of the NPs was by the monomer polymerisation method. ECA (150 µl) in

acetone (25 mL) was gradually added to a stirring mixture of Dex6G4 or Dex6G8 (25 mg)

in water (25 mL, 18.2mΩ) followed by Doxorubicin hydrochloride (2 mg) in acetone

(concentration 2 mg/mL). The reaction mixture was allowed to stir overnight (room

temperature) and the colloid that formed was separated by centrifugation at 40000 rpm

for 30min. The amount of unloaded Doxorubicin hydrochloride in the supernatant was

determined fluorimetrically. The pellet was dispersed in deionised water (10 mL) and

after sonication (10 min) an aliquot (5 mL) of each sample was extracted and used for the

release study. To this end, the aliquot (5 mL) of either PECA-Dex6G4-Dox or PECA-

Dex6G8-Dox was placed in a dialysis membrane bag and immersed in 50 mL capacity

falcon tubes containing PBS (20 mL). The calculated amount of the free Doxorubicin

hydrochloride equivalent contained in the nanoparticles was used as control. The samples

were incubated in water bath (37°C) at an oscillating speed of 100 rpm. At specified time

intervals, an aliquot (1 mL) of each sample was withdrawn and replaced with fresh media.

Fluorescence intensity was measured using a Varian Cary Fluorescence

spectrophotometer (Excitation and Emission wavelengths respectively at 480 nm and 590

nm) and the amount of drug released at each time point was quantified by comparison

against the Doxorubicin hydrochloride calibration curve (Appendix 14).


98

4.2.4 The preparation of PECA-Dex100G4-Dox

Method: The preparation of PECA-Dex100G4-Dox was by the anionic emulsion

polymerisation method, which is widely employed for the formulation of cyanoacrylate

monomers into nanoparticles. ECA (100 µl) was gradually added to Dex100G4 (100 mg) in

water (50 mL; 18.2 mΩ, pH 2.5) containing Doxorubicin hydrochloride (1.5 mg) and

allowed to stir at room temperature for 4 h before neutralisation with aqueous NaOH (1

N). The colloid that formed was filltered through P3 sintered glass. An aliquot of specified

volume was taken and subjected to centrifugation (40000 rpm, 30 min). The pellet was

dispersed in water (10 mL, 18 mΩ) and sonicated and thereafter characterised by DLS.

Drug loading was calculated by subtraction following the determination of the drug

content of the supernatant by UV-Vis measurements (400 nm-750 nm; Doxorubicin

exhibits its absorbance maximum at 480nm). The Doxorubicin loading effeciency was

determined to be 68.7%.

4.2.5 Loading of Doxorubicin hydrochloride to PECA-Dex6G16 NPs

The monomer polymerisation method via nanoprecipitation by solvent evaporation was

used. To a flask (stirring) containing Dex6G16 (100 mg) and Doxorubicin (1.4 mg) in

deionised water (100 mL) was added gradually and in sequence a solution of ECA (ethyl 2-

cyanoacrylate; 600 µl) in acetone (100 mL). Stirring was continued overnight (room

temperature), after which time the mixture was spun (40000 rpm, 30 min; XL-90 Beckman

Ultracentrifuge) and the pellet was dispersed in deionised water, sonicated (10 min) and

freeze dried.

4.2.6 Determination of EGFP enhanced green fluorescence protein (EGFP)

PECA-Dex100G8-EGFP NPs were dispersed (1573 µg/mL) in PBS (pH7.4) and diluted to

specified concentrations before the measurement of absorbance at each dilution to

determine the concentration of EGFP through comparison with a BSA calibration curve

(Appendix 15).

4.2.7 Tagging of PECA-Dex100G8 with MIA

The tagging of MIA to NPs was accomplished according to a published procedure (202). To

a dispersion of PECA-Dex100G8 NPs (ca. 20 mg) in ethanolic sodium borate buffer (5 mL)

was added under sonication/vortexing a solution of MIA (20 mg) in ethanol (0.2 mL); a UV

lamp facilitated the detection of MIA (Figure 4.4; the MIA excitation and emission

wavelengths are at 350nm and 445nm respectively).The mixture was dialysed (72 hr) and


99

freeze dried. Nanoparticle size was determined (DLS and Nanosight). MIA attachment to

NPs was assessed further through visualisation by confocal microscopy (Appendix 9).

Figure 4.4: MIA reaction with PECA-Dex100G8

Purification of MIA-tagged nanoparticles with Sephadex

Column chromatography (Sephadex 50 medium) was used in an effort to increase the

purity of MIA-tagged NPs. After packing, the column was washed repeatedly with water

(18.2 mΩ), (the eluent) before the sample was introduced. Fractions were collected in

6mL aliquots. The second and third fractions were identified as those containing the

fluorophore-tagged NPs, with the second fraction containing the highest proportion.

Further fractions (up to nine 6mL aliquots) did not contain any fluorophore, as indicated

with aid of a UV lamp.

3

3

R= H or

EtOH

pH 8.5

Borate buffer


100

4.2.8 Tagging of MIA to PECA-Dex6G16 NPs and loading of Curcumin

Method: PECA-Dex6G16 NPs (200 mg) was dispersed in sodium borate/ethanol (1:1;

40mL) before being added to MIA (100 mg) in ethanol (20 mL). After sonication, the

mixture was shaken in Bio-Rad (2h, room temperature) before purification by dialysis (72

h). To the dialysed product was added Curcumin (1 mg) in acetone (2 mL) and stirring was

continued for 3h (room temperature). After filtering (P3 sintered glass), the

determination of Curcumin and MIA content in MIA-Curcumin-tagged nanoparticles was

carried out by utilising the dual wavelength capability of the fluorimeter (Cary Eclipse,

Varian Fluorescence Spectrophotometer). The excitation-emission wavelength of

Curcumin was set at 420nm-480nm and that of MIA was set at 350nm-445nm.

4.2.9 Tagging of Tetramethyl Rhodamine-5-carbonyl azide (TMRCA) to PECA-Dex6G12

nanoparticles

Method: PECA-Dex6G12 nanoparticles (50 mg) that had been dispersed in anhydrous

toluene (20 mL) and mixed with TMRCA (0.6 mg) were allowed to stir at 80°C for 5h under

an atmosphere of nitrogen (Figure 4.5) (203-205). After this time, the mixture was placed

in a dialysis-membrane vessel (3500MWCO) and first dialysed against DMF (HPLC grade,

Fisher) for 72 h before further dialysis against double-distilled water for 4 days. The

aqueous-medium-derived Tetramethyl Rhodamine-nanoparticles were freeze dried.

4.2.10 Evaluation of Evans blue retentive capacity

To evaluate the retentive capacity of the nanoparticles for the dye, after loading Evans

Blue to PECA-Dex6G4 NPs, the colloidal mixture that formed was centrifuged twice and

after each centrifugation cycle, the absorbance of supernatant was measured. After the

second centrifugation, the degree of loading was evaluated at 0.016% and the

entrapment efficiency at 2.034%. An attempt to load Evans blue into PECA-Dex6G12

nanoparticles yielded similar results; the calibration curve for Evans blue is appended

(Appendix 16).


101

Figure 4.5: Covalent linkage of Tetramethyl Rhodamine to PECA-Dex6G12 nanoparticles

-

+ -

+

(( )3

2 3)2

-

+

(( )3

2 3)2

-

+

()32 3)2

5h

N2.

atm.

(

R= H or


102

4.3 Results and discussion

4.3.1 Rhodamine

4.3.1.1 Rhodamine B loading into nanoparticles

The size distribution of nanoparticles tagged with Rhodamine B coated with Polysorbate

80 was within the range 400-500nm and exhibited low zeta potential (see example for

Rhodamine B-loaded PECA-Dex100 NPs in Table 4.1). The size distribution of NPs tagged

with Rhodamine B alone were within the size range 300 nm to 500 nm (Figure 4.6).

Table 4.1: Size, PDI and zeta potential of Rhodamine B-loaded, Polysorbate 80-coated PECA-Dex100 NPs

parameter value

Size (Z average) /nm 402.9±3.70

PDI 0.35±0.012

Zeta potential /mV -18.0±0.1

Figure 4.6: Size of Rhodamine B-loaded nanoparticles prepared from PECA-Dex, PECA-Dex100G4 and PECA-Dex100G8 (n=3)

0

100

200

300

400

500

600

700

800

PECA-Dex PECA-Dex100G4 PECA-Dex100G8

Size

(n

m)


103

4.3.2 Curcumin

4.3.2.1 PECA-dex100G4 and PLA-dex100G8PECA Nanoparticle-loading with Curcumin: the

EtOH/H2O (1:1) method

In accord with expectation due to the very low solubility of Curcumin in water, Curcumin

loading into nanoparticles was highly efficient (Table 4.2).

Table 4.2: The absorbance and concentration of Curcumin in the supernatant ( EtOH:H2O), (1:1) from the loading of NPs.

Sample Absorbance Concentration (µg/µl)

PLA-Dex100G8-PECA-Curcumin

0.47308 0.00285

PECA-Dex100G4-Curcumin

0.52779 0.00319

4.3.2.2 Determination of Curcumin content of PECA-Dex100G4 Nps and PLA-Dex100G8-

PECA NPs

Freeze-dried nanoparticles of PECA-Dex100G4-Curcumin NPs and PLA-Dex100G8-PECA-

Curcumin were evaluated for their Curcumin content (Table 4.3).

Table 4.3: concentration of Curcumin content of dissolved Curcumin loaded NPs

Sample Absorbance Concentration (µg/µl)

PLA-Dex100G8-PECA Curcumin

0.64525 0.00392

PECA-Dex1004G4 Curcumin

0.45075 0.00271

4.3.3. Doxorubicin

4.3.3.1 The preparation of PECA-Dex100G4-Dox

The preparation of PECA-Dex100G4-Dox by the anionic emulsion polymerisation method

yielded nanoparticles of low PDI and high zeta potential (Table 4.4).

Table 4.4: Size, PDI and zeta potential of PECA-Dex100G4-Dox nanoparticles

sample Size PDI Zeta potential

PECA-

Dex100G4-Dox

281.0±6.7 nm 0.153±0.022 -35.7±2.7 mV


104

4.3.3.2 PECA-Dex6G16-Doxorubicin (PECA-Dex6G16-Dox) nanoparticles

To assess the effects of purity on size, PDI and zeta potential, the characterisation of

PECA-Dex6G16-Dox was carried out by DLS both before (as prepared) and after

centrifugation (by resuspending the pellet and sonicating for 10 min). As compared with

their as-prepared congeners, the purified NPs exhibit smaller size, lower PDI and higher

zeta potential (Table 4.5), all of which highlight the importance of the purification step.

Table 4.5: Size, zeta potential and PDI characterisation of Doxorubicin-PECA-Dex16 (Dox-PD16) nanoparticles (DLS)

Sample Size (nm) PDI Zeta Potential (mV)

Before centrifugation 268.2±2.9 0.540±0.021 -26.2±0.5

Dispersed pellet 254.0±4.6 0.389±0.047 -39.4±3.3


PECA-Dex6G8 NPs by nanoprecipitation-solvent evaporation

The chain length of the Dex6G4 or Dex6G8 used as copolymer in the formation of

nanoparticles of PECA-alkylglyceryl dextran that were loaded with Doxorubicin

hydrochloride appears to affect loading capacity. Dex6G4, which has a shorter chain

length as compared with Dex6G8, had a smaller amount of Doxorubicin hydrochloride

loaded to its nanoparticulate formulation (PECA-Dex6G4) than Dex6G8 (PECA-Dex6G8).

The more hydrophobic nature of Dex6G8 could have promoted the adsoption of the

Doxorubicin hydrochloride [Doxorubicin base is hydrophobic whereas its HCl salt is water

soluble (206)]. The drug is assummed to be entrapped into PECA-Dex6G8 NPs in its

hydrophilic Doxorubicin.HCl form, which probably enables efficient diffusion from the

nanosphere matrix. Of the two nanoparticulates investigated, those of PECA-Dex6G8 NPs,

which are associated with the higher loading capacity for Doxorubicin.HCl, were seen to

exhibit the faster rate of release of this active.


105

Figure 4.7: Cumulative release profile of Doxorubicin from Doxorubicin HCl, PECA-Dex6G4 and PECA-Dex6G8 (n=3±SD).

The freeze dried nanoparticles drug content (Table 4.6) was determined by dissolving the

NP-drug in DMSO and the values were determined from the calibration curve (presented

in Appendix 14). Samples of nanoparticles that had been subjected to freeze drying,

contained drastically reduced amounts of entrapped drug relative to those that had not

been subjected to a freeze drying protocol. This was particularly pronounced with PECA-

Dex100G4 NPs. The release of the Doxorubicin content of PECA-Dex6G8 or PECA-Dex6G4

nanoparticles is influenced by polymer-chain hydrophilicity, which in turn is influenced by

the liquid environment and by the amount of loaded drug that is retained in the

nanoparticle matrix. The complex binding and releasing mechanisms are yet to be

deconvoluted (207).

Doxorubicin release from PECA-Dex6G8-Dox nanoparticles was very fast (similar to that of

the free Doxorubicin from the dialysis-membrane bag; Figure 4.7). PECA-Dex6G4-Dox

nanoparticles had about 40% of their drug content released within about 8 h from the

start of the release study, which also saw about 95% of the drug released from PECA-

Dex6G8 nanoparticles within the same timescale (Figure 4.7). The release profile (Figure

4.7) of Doxorubicin from the nanoparticulate matrix, especially that of PECA-Dex6G8-Dox,

-10

10

30

50

70

90

110

130

0 2 4 6 8 10

cumulative drug release

(w/w) %

Time (h)

Free-Dox PD4-Dox PD8-Dox


106

suggested that almost all loaded Doxorubicin had been released within few hours. It is

possible that Doxorubicin release from these nanoparticles may have been better

controlled if structures containing different ratios of the constitutional polymers had

been used, or by the use of Doxorubicin in its free base form. The release profiles

demonstrated that the nanoparticles exhibited limited affinity for the drug at the

temperature and pH of the adopted experimental protocol.

Table 4.6: Percentage Doxorubicin loading to the nanoparticles (loading efficiency)

sample % drug loaded

PECA-Dex100G4 45.8%

PECA-Dex100G8 58.3%

4.3.3.4 Loading of both Doxorubicin and Curcumin into PECA-Dex100G12nanoparticles:

The monomer polymerisation method provided the means for the loading of PECA-

Dex100G12 nanoparticles with equal proportions of Curcumin and Doxorubicin (208).

Since for both molecules OH functionalities may become involved in the initiation of the

polymerisation of ECA, the formulation cannot be regarded as a mere combination of

Doxorubicin and Curcumin but rather as a molecular complex of these actives with the

nanoparticles. Accordingly, release studies involving this nanoparticulate complex have

shown that Doxorubicin is released from the complex at a faster rate than Curcumin. In

the absence of detailed spectroscopic characterisation data the relative contributions of

the binding forces that operate between the individual components of the complex

cannot be deconvoluted. However, the observed release patterns suggest that these

nanoparticles may be amenable to employment for therapeutic purposes; Table 4.7

summarises the DLS characterisation data of Doxorubicin-Curcumin nanoparticles.

Table 4.7: Size, PDI and zeta potential of PECA-Dex6G12 nanoparticles loaded with Doxorubicin-Curcumin.

Size (nm) PDI Zeta potential (mV)

DLS NTA Mode

PECA-

Dex100G12-

Dox-Cur

211.4±0.1 152±66 120 0.249±0.022 -30.40±0.40


107

4.3.3.5 PECA-Dex100G12-Dox TGA and DSC results

TGA performed under an atmosphere of nitrogen indicated that the main degradation

step for PECA-Dex100G12-Dox is characterised by a mass loss of 91.4% (Figure 4.8), with a

maximum rate of mass loss at 192.0 °C. The second step of the same thermogram is

associated with a mass loss of 7.7% and exhibits a peak mass loss rate at 327.5 °C.

Exposed to a synthetic air atmosphere the sample exhibited an additional mass loss of

1.1%.

Figure 4.8: TGA plots from PECA-Dex100G12-Dox nanoparticles.

In (Figure 4.9) the first heating curve (blue curve) of this sample, there is observed a glass

transition at 52.4 °C (midpoint) which is accompanied by a change in specific heat

capacity of 0.059 J/g*K. Immediately after the glass transition, a broad endothermic event

is observed which is assumed to be associated with the onset of evaporation or with the

oxidative degradation of a components of the sample. The second heating curve (green

curve) confirms the glass transition at 52.3 °C and enumerates the change in specific heat

capacity at 0.041 J/g*K. Notably, the endothermic event did not occur during the second

heating cycle, which is indicative of a possible relaxation effect in the polymer structure.


108

Figure 4.9: DSC of PECA-Dex100G12–Doxorubicin nanoparticles.

Since the extraction of Doxorubicin-free base with methanol and triethylamine has been

shown to be an effective means of increasing Doxorubicin entrapment in the PECA-

DexnGm nanoparticles (199), it is likely that the use of Doxorubicin as its hydrochloride

salt would have affected the extent of entrapment to PECA-DexnGm nanoparticles. The

interactions between PACA and Doxorubicin have been indicated to involve the

establishment of hydrogen bonds between the ammonium N-H function and the cyano

group, while the cohesion of PACA nanoparticles have been indicated by the same

technique to be due to the combined effects of electrostatic forces, H-bonds and dipolar

interactions (163).

4.3.4 Other fluorescent markers and model drugs

4.3.4.1 Enhanced green fluorescence protein (EGFP) determination in NPs

Determination of EGFP at various concentrations of PECA-Dex100G8-EGFP NPs

EGFP loading was studied over a range of concentrations of nanoparticles that had been

dispersed in PBS. This was to confirm whether the fluorescence protein has been loaded

unto the nanoparticles. The various concentrations of the NPs-loaded EGFP confirmed the


109

presence of the fluorescence marker in the NPs (Table 4.8). The nanoparticles were

further analysed with SEM. SEM image of PECA-Dex100G8-EGFP loaded nanoparticles is

appended in (Appendix 10)

Table 4.8: Determination of concentrations of EGFP in PECA-Dex100G8-EGFP NPs

Initial Conc. of NPs (µg/mL) Absorbance (nm) Conc. of EGFP

(µg/mL)*

100 0.0558 1.294

200 0.0770 13.760

300 0.1084 32.240

400 0.3335 164.558

500 0.3481 173.240

4.3.4.2 N-methyl-isatoic anhydride (MIA)

Dispersion of freeze dried MIA-NPs in a range of media demonstrated the medium-

influenced variation in the size of nanoparticles (Table 4.9).

Table 4.9: The effect of different liquid media on the size distribution of NPs of PECA-Dex100G8 that had been tagged with MIA (DLS)

Liquid medium Z-av.d (nm) PDI

water 141.4±1.0 0.241±0.006

NaCl (10mmol) 133.2±1.6 0.224±0.009

PBS (7.4) 226.0±5.3 0.467±0.011

PBS (filtered 0.2µm) 134.6±0.4 0.221±0.007

In an experiment designed to establish the optimum method of co-loading MIA and

Curcumin, it was observed that the tagging of MIA before loading the sample with

Curcumin was more efficient than that involving loading with Curcumin before tagging

with MIA (Table 4.10); DLS results of sizes and zeta potentials are presented in (Table

4.11).


110

Table 4.10: Fluorescence intensity of MIA-Curcumin nanoparticles

sample Tagged with MIA before

loading with Curcumin

Loaded with Curcumin

before tagging with MIA

Fluorescence

intensity (Curcumin)

56.9538 20.5744

Fluorescence

intensity(MIA)

507.3941 547.0530

Table 4.11: DLS analysis of MIA-Curcumin NPs

Loaded with Curcumin before tagging with MIA

Tagged with MIA before loading with Curcumin

Z-Av (nm) 634.5±16.1 Z-av (nm) 430.1±8.0

PDI 0.159±0.023 PDI 0.248±0.009

Zeta potential (mV)

-27.6±0.1 Zeta potential (mV)

-23.0±0.5

4.3.4.3 Tetramethyl Rhodamine-5-carbonylazide

The synthesis of Tetramethyl Rhodamine-nanoparticles by covalent bond formation

involved reaction between the hydroxyl group of constituent macromolecules and the

isocyanate group formed from the acyl azide functionality of TMRCA on heating at 80°C.

The conjugation of these molecules was evidenced by NTA, and by the difference

between the fluorescence intensities of the blank and Tetramethyl Rhodamine

nanoparticles dissolved in DMSO as measured with Microplate Reader. Proton NMR

(205) was used to determine the molar ratio of the attached Tetramethyl Rhodamine to

the nanoparticles by comparing the signal intensity or the peak area of its methyl proton

to the anomeric proton of the pyranose ring of the dextran or, the methylene protons (or

methyl protons) of the alkyl glyceryl group attaching to the dextran.

Evidence for the conjugation of Tetramethyl Rhodamine to the nanoparticles was

obtained by considering the difference in fluorescence intensities of nanoparticles with

reference to a blank sample, which had been dissolved in DMSO and measured

(Microplate Reader) at the respective excitation and emission wavelengths of 544 nm and

590 nm; fluorescence intensities obtained were 21902 for Tetramethyl Rhodamine tagged

nanoparticles and 1952 for empty nanoparticles. The linking of Tetramethyl Rhodamine

to the nanoparticles was further evidenced with NTA, in that fluorescing Tetramethyl


111

Rhodamine nanoparticles could be observed when the filter was shut. The sizes of tagged

nanoparticles, as measured by DLS and NTA, are presented in (Table 4.12). Since the size

distribution of nanoparticles was high, as reflected by the associated PDI, the separation

of nanoparticles to narrower size samples was attempted by filtration.

Table 4.12: The size and PDI of PECA-Dex6G12-TMRCA nanoparticles (n=1)

DLS NTA(nm)

Size (nm) PDI Size Mode

NP-TMRCA 879.6±42.8 0.525±0.030 735±264 752

4.3.4.4 Evans Blue

Loading of PECA-Dex100G4 NPs with Evans blue

Evans blue dye (structure in Figure 4.10) was loaded into the nanoparticles by means of

the monomer polymerisation method. The degree of loading and the efficiency of

entrapment were determined by measuring the Evans blue content of the supernatant.

The system exhibited a poor loading efficiency and an associated low degree of loading.

Evans blue is a negatively charged dye; it is possible that the negative zeta potential of

PECA-DexnGm nanoparticles is responsible for the poor affinity of this dye for these

nanoparticles.

Figure 4.10: Chemical structure of Evans blue

-

-

2

2

-

-

4 Na+


112

4.4 Conclusions

Rhodamine B was successfully loaded into the nanoparticles but since the release of

Rhodamine B from nanoparticles was poor this study was discontinued.

To promote the entrapment of Curcumin into nanoparticles, the loading of PECA-

Dex100G4 NPs with Curcumin was carried out in the presence of acetic acid and TPP.

However, inherent to this method was the inefficient study of the release profile.

The study of the release of Doxorubicin hydrochloride indicated that nanoparticles of

PECA-Dex6G4 NPs can sustain the release of Doxorubicin hydrochloride more efficiently

than those formulated from longer chain homologues (e.g. PECA-Dex6G8 NPs).

Towards the optimisation of methods of tagging MIA to nanoparticles, it was observed

that the tagging of MIA prior to loading with Curcumin was preferable to the tagging of

MIA after Curcumin had been loaded to the nanoparticles.

EGFP was successfully loaded into the nanoparticles as confirmed by absorbance

measurements at specified concentrations.

The synthesis of Tetramethyl Rhodamine-containing nanoparticles by covalent bond

formation was achieved through reaction between the hydroxyl group of constituent

macromolecules and the isocyanate group formed from the acyl azide functionality of

TMRCA. The successful tagging of TMRCA to the nanoparticles was indicated by NTA and

by the difference in fluorescence intensities of nanoparticles with reference to those of

the blank as determined using the microplate reader.

Evans blue had very low affinity for the nanoparticles as evidence by the ease with which

the dye washed away from their nanoparticulate hosts.

In vitro studies

113

5

5. In vitro studies - Cytotoxicity

The cytotoxicity of nanoparticulate formulations was assessed by measuring the viability

of mouse brain endothelial cells (bend3) following incubation with selected

nanoformulations.

5.1 Methods and instrumentation

The cytotoxicity evaluation protocol involved the following materials and equipment:

incubator (37°C, 5% CO2), Plate reader PolarStar Optima (BMG Labtech) (570nm

Absorbance filter), 96-well plate (Greiner bio-one) Fisher, inverted microscope,

haemocytometer, pipettes (1 mL, 100 µl, 50 µl, 20 µl), class II safety cabinet, sterile

pipette tips, sterile pipette (10mL, 3mL) disposable, sterile 0.4 µm polyethersulfone (PES)

filter, electric pipette (Fisher), T25 culture flask (Fisher), centrifuge, alkyl cyanoacrylate-

alkylglyceryl dextran NPs, alkylglyceryl dextrans, MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-

diphenyltetrazolium bromide), DMEM (media), trypan blue, Triton-X100, PBS, Tryple

Express, mouse-brain endothelial cells (bend3), DMEM (500 mL), L-Glutamine (5 mL),

Sodium pyruvate (5 mL), 2-mercaptoethanol (0.5 mL), NEAA (5 mL), FBS (50mL),

Pen/Strep (5 mL).

The cytotoxicity study was conducted by means of the MTT (3-(4, 5-dimethylthiazolyl-2)-

2, 5-diphenyltetrazolium bromide) assay (209, 210): MTT metabolism is known to show a

good correlation between cell viability and conversion of MTT tetrazolium salt (yellow) to

MTT formazan (purple) since only metabolic active cells have the capacity to effect the

conversion of this salt to its purple metabolite.

Various alkyl cyanoacrylate-alkylglyceryl dextrans NPs and alkylglyceryl dextrans were

assessed at 1 mg/mL concentration.

To assess the concentrations of nanoparticles that induce cytotoxicity to bend3 cells, a

dose-response study employing a MTT assay were conducted for PECA-Dex100G4 NPs

(1:1), PECA-Dex100G4 NPs (1:6) and PBCA-Dex100G4 NPs samples at four specified

concentrations (10 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL).

In vitro studies

114

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay protocol

The MTT working solution was prepared by allowing a solution of MTT in DMEM (5

mg/mL; kept in the dark at 2-8°C until needed) to reach 37°C and a steady state

concentration of 5% CO2 before dilution (1:5) with DMEM. Bend3 cells (20,000) were

seeded (in triplicate) into each of a 96 well plate containing 200 µl of normal media

(DMEM containing 10% fetal bovine serum, FBS) and incubated for 24h. After this time, in

each well were added 20 µl of each nanoformulation and of alkylglyceryl dextran

formulations dispersed in PBS at specified concentrations prior to incubation for 24h at

37°C (5% CO2). After this time, to each well was added MTT working solution (50 µl) and

the mixture was incubated for 2h at 37°C (5% CO2). The media was then removed from

each well, replaced with 100 µl of DMSO and this mixture was agitated gently until the

formazan crystals had dissolved. Each plate was inserted into the plate reader and the

absorbance was read at 570 nm. The viability of cells was calculated (Equation 5.1) as

percentage relative to PBS (negative control); Triton-X100 provided the positive control.

100*.

.%..Re

controlA

testAviabilitycelllative (eq. 5.1)

5.2 Results and Discussion

Tested against bend3 cells at a relatively high concentration of 1 mg/mL (MTT assay),

PECA and PECA-alkylglyceryl dextran nanoparticles exhibited significant cytotoxicity

(p<0.05) as compared with the PBS control (Figure 5.1).

In vitro studies

115

Figure 5.1: Relative toxicity of PECA and PECA-alkylglyceryl dextran nanoparticles (1mg/mL; p<0.05, n=3)

Figure 5.2: Relative cytotoxicity of alkylglyceryl dextrans (1mg/mL; p<0.05, n=3)

0

20

40

60

80

100

120

viability against PBS control[%]

0

20

40

60

80

100

120

140

160

PBS Dex100G4 Dex100G16 Dex6G4 Dex6G16

viability against PBS control[%]

In vitro studies

116

To account for these results, alkylglyceryl dextrans were also evaluated using the same

assay, and the results (Figure 5.2) exhibited variable behaviour in that Dex100G4 was

cytotoxic relative to the PBS control (p<0.05) while Dex100G16, Dex6G4 and Dex6G16 did

not display any statistically significant differences at the p<0.05 level with reference to

the same control. It appears that a decrease in the molecular weight of dextran reduces

cytotoxicity, though the degree of substitution with alkylgyceryl chains is clearly another

contributing factor.

Figure 5.3: Relative viability as a function of dose response of alkyl cyanoacrylate NPs against bend3 cells, each incubated at specified concentrations (10 μg/mL, 25 μg/mL 50 μg/mL 100 μg/mL) for 24 h; MTT assay; Triton –X100 and media blank (n =3; ±SD; Triton-X100 positive control).

To identify a range of safe concentrations, a dose response study has been carried out;

the apparent toxicity induced by PECA was also compared to that of poly(butyl

cyanoacrylate), by testing nanoparticles that were prepared from the same materials but

where ECA was replaced by BCA. We found that PBCA-Dex100G4 NPs at concentrations

of up to 50 µg/mL were well tolerated by bend3 cells (Figure 5.3, Table 5.1) while NPs of

PECA-Dex100G4 (1:1) and PECA-Dex100G4 (1:6) began to exhibit toxic effects at

concentrations >25 µg/mL.

In vitro studies

117

Table 5.1: Cytotoxicity dose response of alkyl cyanoacrylate-alkylglyceryl dextran NPs against bEND3 cells

Sample viability (Abs) percentage viability

SD

Media 1.8002 100.0000 0.0294

PBCA-10 1.6036◊ 89.0808 0.2972

PBCA-25 1.5912◊ 88.3901 0.2988

PBCA-50 1.4338◊ 79.6504 0.3314

PBCA-100 0.5609* 31.1613 0.0096

PECA(1:1)10 1.5958◊ 88.6475 0.1781

PECA(1:1)25 1.3793◊ 76.6211 0.2434

PECA(1:1)50 0.4315* 23.9714 0.2047

PECA(1:1)100 0.3808* 21.1569 0.0213

PECA(1:6)10 1.4633◊ 81.2891 0.2183

PECA(1:6)25 1.3477◊ 74.8657 0.1566

PECA(1:6)50 0.3850* 21.3902 0.0421

PECA(1:6)100 0.3507* 19.4811 0.0292

0.1%T-X100Media 0.2697 14.9853 0.0295

0.1%T-X100(water) 0.2046 11.3654 0.0030

1%T-X100(media) 0.2716 15.0890 0.0045 *significant difference (P<0.05) compared to media; ◊ significant difference (P<0.05) compared to 0.1% TritonX100 Legend:

PBCA-10 =PBCA-Dex100G4 NPs at dose of 10 µg/mL PBCA-25 =PBCA-Dex100G4 NPs at dose of 25 µg/mL PBCA-50 =PBCA-Dex100G4 NPs at dose of 50 µg/mL PBCA-100 =PBCA-Dex100G4 NPs at dose of 100 µg/mL PECA(1:1)10=PECA-Dex100G4,(equal ratio of PECA:Dex100G4) at dose of 10 µg/mL PECA(1:1)25=PECA-Dex100G4,(equal ratio of PECA:Dex100G4) at dose of 25 µg/mL PECA(1:1)50=PECA-Dex100G4,(equal ratio of PECA:Dex100G4) at dose of 50 µg/mL PECA(1:1)100=PECA-Dex100G4,(equal ratio of PECA:Dex100G4) at dose of 100 µg/mL PECA(1:6)10=PECA-Dex100G4,( ratio 1:6 of Dex100G4:PECA) at dose of 10 µg/mL PECA(1:6)25=PECA-Dex100G4,( ratio 1:6 of Dex100G4:PECA) at dose of 25 µg/mL PECA(1:6)50=PECA-Dex100G4,( ratio 1:6 of Dex100G4:PECA) at dose of 50 µg/mL PECA(1:6)100=PECA-Dex100G4,( ratio 1:6 of Dex100G4:PECA) at dose of 100 µg/mL

Literature reports indicate that, when tested against a L929 fibroblast cell culture, ethyl

and isobutyl cyanoacrylate-derived nanoparticles were found to be more toxic than the

methyl derivative, which showed intermediate toxicity, while isohexyl cyanoacrylate was

the least cytotoxic of the materials tested (81). A further study involving cyanoacrylate

nanoparticles (PMCA,PECA,PBCA and PIBCA) and mouse peritoneal macrophages showed

identical cellular damage for all tested cyanoacrylates particles (211), indicating that

contrary to earlier reports, chain length influences toxicity. Nemati et al. have reported

that the toxicity of cyanoacrylates on P388 cells is time dependent and that the rate and

In vitro studies

118

amount of uptake is determined by the cell types against which the evaluation is

conducted (212).

The cytotoxicity of poly(cyanoacrylate) nanoparticles seems to be more complex than the

direct contributions of each of the released degradation products, stimulation of

inflammation mediators and cell adhesion (107, 213): concentration and time appear to

play an important role. For example, literature indicates that PBCA nanospheres did not

induce any cytotoxicity at a concentration of 75 µg/mL while membrane damage was

observed at a concentration of 150 µg/mL (107). This in turn indicates that PBCA may

become cytotoxic at a specified concentration >75 µg/mL. In another example,

poly(isobutyl cyanoacrylate) nanospheres at 100 µg/mL exhibited no cell toxicity within

the 7 h timescale but showed notable mortality beyond 24 h (81). Other reports on the

cytotoxicity of cyanoacrylates are available in the literature, but those are limited to the

concentration level of 0.1 mg/mL (214).

Although retro-Knoevenagel to poly(acrylic acid) and the corresponding alcohol is

assumed to be the major degradation pathway of cyanoacrylate polymers, the cellular

cytotoxicity of cyanoacrylate polymers has been shown to be directly related to the

release of formaldehyde (215), which is produced though a minor pathway during the

degradation of PACA (214). The rate of degradation is primarily determined by the length

of the side chain, with shorter chain homologues degrading more readily (presumably due

to the steric hindrance effects exerted by the longer-chain materials; (216). Due to its

relative high propensity to adhere to cells (81, 107) PECA would be expected to exhibit

higher toxicity than its shorter-chain homologue PMCA. Contrary to expectation, when

tested against fibroblasts, PMCA was found to be more toxic than PECA (214). A study of

the cytotoxicity of PBCA and POCA (10 µg/mL) against human foreskin fibroblasts showed

that the former material is more toxic (217).

As compared with the PBS control, the PECA and PECA-derived nanoparticles evaluated in

this study at concentration of (1 mg/mL) exhibited marked toxic effects against all cells

tested (bend.3) (Figure 5.1). Complementary dose-response experiments that included

PBCA-derived nanoparticles have demonstrated that the respective toxicities of PBCA-

Dex100G4 NPs, and PECA-Dex100G4 (both 1:1 and 1:6 ratio samples) nanoparticles are

notable at the 100 μg/mL and the 50 μg/mL levels, respectively (Figure 5.3 and Table 5.1).

With reference to the PBS control, Dex100G4 appeared significantly more toxic but no

In vitro studies

119

significant difference in toxicity could be identified (P<0.05) with Dex100G16, Dex6G16 or

Dex6G4 (Figure 5.2).

Consistent with the findings of Wiegand et al. who reported the molecular-weight-

dependent toxic effects of chitosan on human keratinocyte cell lines (218), the

percentage viabilities (214) determined for alkylglyceryl dextran samples of different

molecular weights imply that similar molecular-weight-dependent effects operate with

the latter class of molecules. Notably however, tested against CT-26 cells, dextran derived

micelles did not appear to exhibit a significant molecular weight-related effect (219).

5.3. Conclusions

Since complementary studies have indicated that the chain length of PACAs influences

their rate of degradation, it is assumed that there is a relationship between chain length

and toxicity: the shorter the chain, the faster the degradation and the more toxic the

parent structure. It is notable however that the toxicity of degradation products of PACA

nanoparticle is reported, through the evaluation of data from clinical trials, not to induce

any adverse effects of therapeutic significance (220). The molecular weight of native

dextran and the degree of substitution with alkylglyceryl chains also appear to influence

the cytotoxicity of the nanoparticulate formulations tested.

General Conclusions

120

6

6. General Conclusions and suggestions for further work The delivery of therapeutic agents to the brain is often limited by the BBB. Research

findings have indicated that this barrier may be overcome by the use of nanoparticulate

drug delivery vehicles which incorporate the therapeutic molecule that is not capable of

crossing the BBB. To achieve this goal, it is essential that nanocarriers that do not alter

the integrity of the BBB are developed.

This study designed and evaluated one such class of drug-carrying polymeric

nanoparticles. To this end, dextran derivatives have been prepared through attachment

of alkylglycerol of systematically varied chain length to the dextran core. These

macromolecular structures were then combined with a widely used class of

biocompatible moieties, the alkyl cyanoacrylates, to formulate nanoparticles.

Alkylglycerols of systematically varied chain lengths were utilised to generate alkylglyceryl

derivatives of two native dextrans with average molecular weights of 6 kD and 100 kD.

These alkylglyceryl dextrans were characterised by MALDI-TOF MS, TGA, GPC, FTIR and

NMR. The degree of substitution of each of the synthesised alkyl glyceryl dextrans, as

determined using 1H-NMR spectroscopy, ranged between 30% and 200%. It was found

that the longer the chain length of the progenitor alkylglycerol the lower the

hydrophilicity of the alkylglyceryl dextran. GPC results demonstrated that the

polydispersity index of alkylglyceryl dextrans was lower than that of the precursor

dextran irrespective of average molecular weight (6 kDa or 100 kDa).

In the chemical attachment of alkylglycerol to dextran the carbon at position 2 was seen

to be regioselectively favoured, as is consistent with its expected higher reactivity. The

synthesised alkylglyceryl dextrans were readily amenable to formulation into

nanoparticles through combination with alkyl cyanoacrylates (ethyl or butyl); the

techniques of nanoprecipitation and emulsion polymerisation were both usefully

employed for this purpose. Nanoparticles were characterised by DLS, NTA, elemental

(CHN) analysis, NMR, FTIR, MALDI-TOF MS, SEM, DSC and TGA. Elemental analysis

calculations determined that the ratio of alkylglyceryl dextran to alkyl cyanoacrylate in

General Conclusions

121

the formulated nanoparticles was in the respective, formulation-dependent, range of 3-

19% and 80-95%; this was in agreement with NMR data.

The sizes of nanoparticles were of the order of 100-500 nm. Studies involving the

systematic variation of the alkylglyceryl chain length indicated that there is no direct

relationship between chain length and nanoparticle size. Notably, nanoparticles that had

been prepared from alkylglyceryl dextrans exhibited consistently higher negative zeta

potentials than their congeners that had been formulated from native dextrans.

Autotitration experiments revealed that empty NPs exhibit decreasing zeta potentials

with decreasing pH. The average sizes of nanoparticles appeared relatively stable across

the pH range considered, while the sizes decreased with decrease in pH for some of the

tested nanoparticles.

The nanoparticles were amenable to tagging with fluorophores and to loading with a

range of model drugs (MIA, EGFP, Curcumin, Doxorubicin, Evans blue, TMRCA).

Experiments involving nanoparticles that had been loaded with Curcumin demonstrated

that the longer the alkylglyceryl chain length the smaller the size of nanoparticles.

Studies of the release of loaded Evans blue have shown the rapid release of this agent

from the nanoparticulate matrix, presumably due to the incompatibility in the surface

charges of the negatively charged Evans blue and the negative-zeta-potential

nanoparticles. The release of Doxorubicin or that of Curcumin from their nanoparticulate

host was notably slower than that observed with Evans blue.

Evaluated for cytotoxicity against bend3 cells, nanoparticles exhibited dose-dependent

toxicity profiles: PBCA-Dex100G4 NPs were found to be more biocompatible than PECA-

Dex100G4 NPs.

Integral to the future development of the technology towards clinical applications is an in

vitro study that evaluates nanoparticles for their capability to permeate modelled BBB

structures.

References

122

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Appendices

133

8

8. Appendices

APPENDIX 1 1H and 13C NMR spectra of butyl glycidyl ether

APPENDIX 2 1H AND 13C NMR spectra of tetradecyl glycidyl ether

APPENDIX 3 1H AND 13C NMR spectra of hexadecyl glycidyl ether

APPENDIX 4 SEM image of PECA-Dex100G4

APPENDIX 5 Snapshot of video measurement for PECA-NP loaded Rhodamine B

and coated with Polysorbate 80

APPENDIX 6 Thermograms of modified dextrans

APPENDIX 7 MALDI TOF spectra of modified dextrans

APPENDIX 8 MALDI TOF spectra of nanoparticles

APPENDIX 9 PECA-Dex100G8 tagged with MIA in PBS (confocal microscope)

APPENDIX 10 SEM image of PECA-Dex100G8 EGFP NPs

APPENDIX 11 Equations for elemental analysis determination

APPENDIX 12 Calibration curve for Curcumin

APPENDIX 13 SEM images of native dextran and Dex100G4

APPENDIX 14 Calibration curve for Doxorubicin

APPENDIX 15 Calibration curve for BSA

APPENDIX 16 Calibration curve for Evans blue

Appendices

134

APPENDIX 1

1H- and 13C-NMR spectra of butyl glycidyl ether

Appendices

135

APPENDIX 2

1H- AND 13C-NMR spectra of tetradecyl glycidyl ether

Appendices

136

APPENDIX 3

1H- AND 13C-NMR spectra of hexadecyl glycidyl ether

Appendices

137

APPENDIX 4

SEM image of PECA-Dex100G4

Appendices

138

APPENDIX 5

Video snapshot of PECA-NP loaded Rhodamine B and coated with Polysorbate 80

Appendices

139

APPENDIX 6 Thermograms of modified dextrans

Figure A6.1: TGA results of Dex6G8


Appendices

140



Appendices

141

Figure A6.5: TGA results of Dex100


Appendices

142



Appendices

143



Appendices

144

APPENDIX 7 MALDI TOF spectra of modified dextrans

Figure A7.1 MALDI- TOF Spectrum of Dex100G4

Figure A7.2: MALDI TOF spectrum of Dex6G8

O

OOH

OH

O

O

O

OH

C7H

11O

6

191.0556 Da

C7H

15O

2

131.1072 Da

Molecular Formula = C14

H26

O8

146.0

130.1

O

OOH

OH

O

O

O

OH

C7H

11O

6

191.0556 Da

C11

H23

O2

187.1698 Da

56.2.

Appendices

145

Figure A7.3: MALDI TOF Spectrum of Dex100G8

Figure A7.4 MALDI TOF spectrum ofDex6G12

OO

OH

OH

O

O

OOH

C7H

11O

6

191.0556 Da

C15

H31

O2

243.2324 Da

242.2

O

OOH

OH

O

O

O

OH

C9H

17O

6

221.1025 Da

C11

H23

O2

187.1698 Da

C18

H35

O8

379.2332 Da

C7H

11O

6

191.0556 Da

~186

Appendices

146

Figure A7.5 MALDI TOF spectrum of Dex100G12


O

OOH

OH

O

O

O

OH

C7H

11O

6

191.0556 Da

C17

H35

O2

271.2637 Da

28.0

27.0

O

OOH

OH

O

O

O

OH

C7H

11O

6

191.0556 Da

C15

H31

O2

243.2324 Da

242.3

117.7

Appendices

147


Figure A7.8 MALDI TOF Spectrum of Dex6G16

O

OOH

OH

O

O

O

OH

C7H

11O

6

191.0556 Da

C19

H39

O2

299.295 Da 187.3

OO

OH

OH

O

O

OOH

C7H

11O

6

191.0556 Da

C17

H35

O2

271.2637 Da 56.0

Appendices

148


OO

OH

OH

O

O

OOH

C7H

11O

6

191.0556 Da

C19

H39

O2

299.295 Da

127.3

Appendices

149

APPENDIX 8

MALDI TOF spectra of nanoparticles

Figure A8.1 MALDI TOF spectrum of PECADex100G4

Figure A8.2: MALDI TOF spectrum of PECADex6G8

O

O

OHOO

O

O

OH

O

O

N

C14

H25

O8

321.1549 Da

C6H

7NO

2

125.0477 Da

130.2

OO

OH

O O

O

O

OHO

O

N

C18

H33

O8

377.2175 Da

C6H

7NO

2

125.0477 Da

125.1

??

Appendices

150



O

O

OHO

OO

O

OH

O

ON

C18

H33

O8

377.2175 Da

C6H

7NO

2

125.0477 Da

125.1

OO

OHO O

O

O

OH

O

O N

C22

H41

O8

433.2801 Da

C6H

7NO

2

125.0477 Da

125.1

125.1

Appendices

151



O

OOH

O

O

O

O

OH

O

O

N

C28

H55

O8

519.3897 Da

C6H

7NO

2

125.0477 Da

125.1

O

O

OHO

O

O

O

OH

O

O N

C28

H55

O8

519.3897 Da

C6H

8NO

2

126.0555 Da

C31

H58

NO8

572.4162 Da

C3H

5O

2

73.029 Da

Appendices

152

APPENDIX 9

Confocal microscopy image of PECA-Dex100G8 tagged with MIA in PBS

125.1

125.1

Appendices

153

APPENDIX 10

SEM image of PECA-Dex100G8 EGFP nanoparticles

Appendices

154

APPENDIX 11

Equations used to calculate the polymer mass composition from the results of elemental analysis

100*).....*()...()*...(

)]..*)4...(*[)..*...()...**..(%

ChainGlycerolAlkylofmassmolarDSdDexofmassmolarXPECAofmassmolar

massatomicHratiomgHDSdMassatomicHratiomDexHratiomPECAHXmassatomicHH

100*).....*()...()*...(

)]..*)2...(*[)..*...()...**..(%


massatomicCratiomgCDSdMassatomicCratiomDexCratiomPECACXmassatomicCC

100*).....*()...()*...(

)...**..(%


ratiomPECANXmassatomicNN

100*).....*()...()*...(

)*...(%


XPECAofmassmPECA

Appendices

155

APPENDIX 12

Calibration curves used for the determination of Curcumin

y = 159.56x + 0.019 R² = 0.9964

0

0.5

1

1.5

2

2.5

3

0 0.002 0.004 0.006 0.008 0.01 0.012 0.014 0.016 0.018

Ab

sorb

ance

@ 4

35

.54

nm

Concentration µg/µl

Calibration curve of Curcumin in DMSO

y = 97.601x + 6.5747 R² = 0.9997

0

10

20

30

40

50

60

0 0.1 0.2 0.3 0.4 0.5 0.6

Flu

ore

sce

nce

inte

nsi

ty

µg/mL

Curcumin calibration curve in PBS (0.1% Polysorbate 80)

Appendices

156

APPENDIX 13

SEM images of native dextran and Dex100G4

native dextran

Dex100G4

Appendices

157

APPENDIX 14

Calibration curves for Doxorubicin

y = 34296x + 10.862 R² = 0.9995

0

10

20

30

40

50

60

0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 0.0014

Flu

ore

sce

nce

inte

nsi

ty

concentration mg/mL

Calibration curve of Doxorubicin in DMSO

y = 9020.8x + 0.2966 R² = 0.9947

0

1

2

3

4

5

6

7

0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007

Flu

ore

sce

nce

inte

nsi

ty

Concentration mg/mL

Calibration curve of doxorubicin in PBS

Appendices

158

APPENDIX 15

Calibration curve for BSA

Appendices

159

APPENDIX 16

Calibration curve for Evans blue

Calibration curve of Evans blue

y = 0.0254x + 0.0088 R² = 0.9999

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 10 20 30 40 50 60 70 80

Ab

sorb

ance

Concentration µg/mL

Calibration curve of Evans blue in water