design, synthesis and applications of polymer biomaterials

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Graduate Theses and Dissertations Graduate School

2-24-2015

Design, Synthesis and Applications of PolymerBiomaterialsFrankie CostanzaUniversity of South Florida, [email protected]

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Design, Synthesis and Applications of Polymer Biomaterials

by

Frankie Costanza

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Department of Chemistry

College of Arts and Sciences

University of South Florida

Major Professor: Dr. Jianfeng Cai, Ph.D.

Kirpal Bisht, Ph.D.

Edward Turos, Ph.D.

Chuanhai Cao, Ph.D.

Date of Approval:

February 24th, 2015

Keywords: Antimicrobial polymers, drug delivery, amphiphilic, micelle

Copyright © 2015, Frankie Costanza

DEDICATION

To Michael and Victoria Costanza

ACKNOWLEDGMENTS

I would like to express my gratification to my parents Michael and Victoria Costanza

who have always been my beacon of light through my hardest of times over the past many years.

This Doctorate would not be possible without their love and support for me during this time of

my life. I would also like to express my gratification to my mentor, Dr. Jianfeng Cai, for

allowing me to work in his laboratory as well as providing me with guidance, support and

understanding as I have endured numerous personal issues over the years. Without his

compassion, I would have not accomplished this milestone in my life. It is through him that I can

see what a truly hard working mentor and researcher really is. He has accepted all members of

his group into his home, and into his family, thereby making everyone feel as though they belong

here. For these reasons I can say with great respect, that it has been an honor to work and learn in

your laboratory. I would also like to thank Dr. Kirpal Bisht, Dr. Edward Turos, and Dr. Chuanhai

Cao for their support and insights over the years as well as for the use of their laboratory

instruments. Next, I would like to thank the entire Cai group as my support system for the times

when research became the most frustrating. Without their professional help, I would not have

been able to conduct many experiments as efficiently. In particular I would like to thank Shruti

Padhee for the antibacterial assays and Yan Wang for the in vivo mouse studies. Lastly, I would

like to thank Zoya Khan for being the foundation of my social support system over the past 3

years. All of the mentioned above people have always stood by me and helped me achieve this

degree with their support and patience, and again, I would like to thank each and every one of

you.

F.C.

i

TABLE OF CONTENTS

LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iii

LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vi

CHAPTER 1: INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Antibacterial Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

1.2 Drug Delivery Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3 Outline of Dissertation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

1.4 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

CHAPTER 2: INVESTIGATION OF ANTIMICROBIAL PEG-POLY(AMINO ACID)s. . . . .10

2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

2.3 Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.5 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

CHAPTER 3: PEG-POLY (AMINO ACID)s – ENCAPSULATED

……………TANSHINONE IIA AS POTENTIAL THERAPEUTICS FOR

…………... THE TREATMENT OF HEPATOMA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28



3.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3.5 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

CHAPTER 4: PEG-POLY (AMINO ACID)S/MICRORNA COMPLEX

…………… NANOPARTICLES EFFECTIVELY ARREST THE GROWTH AND

…………….METASTASIS OF HEPATOMA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52



4.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.5 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS. . . . . . . . . . . . . . . . . . . . . . . . .73

ii

APPENDIX A: SUPPORTING INFORMATION FOR INVESTIGATION OF PEG-

……………. POLY(AMINO ACID)S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

A1. Synthesis of PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

A2. The Morphology of PEG-poly(amino acid)s in water. . . . . . . . . . . . . . . . . . . . . . . . 79

A3. Antimicrobial activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80

A4. Hemolytic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81

A5. Fluorescence microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

A6. SEM of bacteria after treatment with polymer P5. . . . . . . . . . . . . . . . . . . . . . . . . . . 82

A7. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83

iii

LIST OF TABLES

Table 2.1: The antimicrobial and hemolytic activities of PEG-poly(amino acid)s. . . . . . . . . . . .17

Table A1: Molecular weights of the polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

iv

LIST OF FIGURES

Figure 1.1: Difference between bacterial and mammalian cell membranes. . . . . . . . . . . . . . . . . . 3

Figure 1.2: Graphical representation of our drug delivery system used for miRNA-139. . . . . . . .6

Figure 2.1: Synthesis of PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Figure 2.2: The nanomorphology of the PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . .17

Figure 2.3: Fluorescence micrographs of B. subtilis treated with polymer. . . . . . . . . . . . . . . . . .21

Figure 2.4: SEM pictures of bacteria treated with polymer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

Figure 3.1: The structure of Tanshinone IIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

Figure 3.2: Synthesis and NMR of the PEG-poly (amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . .38

Figure 3.3: Nanomorphology of poly (amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39

Figure 3.4: In vitro cytotoxicity of nanoparticles in MHCC97-H hepatoma cells. . . . . . . . . . . . 40

Figure 3.5: Biodistribution of TanshinoneIIA NPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Figure 3.6: Tumor inhibitory effect of TSIIA nanoparticles in vivo. . . . . . . . . . . . . . . . . . . . . . .43

Figure 3.7: (A) Kaplan-Meier overall survival analysis and (B) Life extended rate of the

MHCC97-H hepatoma-bearing mice treated with TSIIA nanoparticles. . . . . . . . . . . . . . . . . . . .44

Figure 3.8: Representative histopathological sections of the tumors from tumor-bearing mice. .45

Figure 3.9: Image of hepatoma-bearing mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Figure 4.1: Synthesis and NMR of PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Figure 4.2: SEM pictures of prepared nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62

Figure 4.3: The tumor growth curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Figure 4.4: Survival time of hepatoma-bearing mice in different groups. . . . . . . . . . . . . . . . . . . 64

Figure 4.5: Image of orthotropic hepatoma-bearing mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66

v

Figure 4.6: The immunohistochemical staining of CD31 antigen. . . . . . . . . . . . . . . . . . . . . . . . .67

Figure 4.7: Wound closure healing assay in miR-139 nanoparticles. . . . . . . . . . . . . . . . . . . . . . .68

Figure A1a: Synthesis of N-Carboxyanhydrides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76

Figure A1b: Synthesis of PEG-OTs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76

Figure A1c: Synthesis of PEG-N3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Figure A1d: Synthesis of PEG-NH2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Figure A1e: Synthesis of Polymer P3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Figure A2: SEM image showing the morphology of PEG-poly(amino acid)s . . . . . . . . . . . . . . .79

Figure A3: NMR of Polymer P2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84

Figure A4: NMR of Polymer P3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85






Figure A10: NMR of Polymer P9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Figure A11: NMR of Polymer P10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

vi

ABSTRACT

The emergence of antibiotic resistant bacteria has prompted the research into novel kinds

of antibacterial small molecules and polymers. Nature has solved this issue with the use of

cationic antimicrobial peptides, which act as nonspecific antibiotics against invading species.

Herein, we have tried to mimic this general mechanism in a biocompatible and biodegradable

polymer micelle based on the polymerization of naturally occurring amino acids lysine and

phenylalanine linked to a PEG tether. This amphiphilic structure allows for the spontaneous

collapse into stable nanoparticles in solution, which contains a hydrophilic outer layer and a

hydrophobic core. Our polymers have shown activity against clinically relevant strains including

Methicillin Resistant S. epidermidis, B. subtilis, K. pneumoniae, and P. aeruginosa.

To further the application of our biopolymers, we have used them as drug delivery

vehicles as well. First, we have used an anionic analogue based on glutamic acid to encapsulate a

super hydrophobic drug Tanshinone IIA, and use it against a hepatoma bearing mouse model.

Second, we have used a cationic analogue to form a complex with miRNA-139 and use it against

a hepatoma bearing mouse model as well. In both cases, our PEG poly(amino acids)s have

shown promising efficacy in drastically reducing the tumor size compared to the control only.

Taken together, our results show that our nanoparticles have the potential to be versatile

biomaterials as antibacterials as well as drug delivery vehicles in vivo.

1

CHAPTER 1: INTRODUCTION

1.1 Antibacterial Polymers

Clinics today are facing an increasing problem of infections associated with drug

resistance to conventional antibiotics1. One of the most common infections which account for

more than half of all reported skin infections, and causes an estimated 20,000 deaths annually is

from methicillin-resistant staphylococcus aureus and a similar strain from methicillin-resistant

staphylococcus epidermidis2. This problem becomes even more pronounced during surgery

because bacteria can easily colonize the surface of living and non-living substrate including the

raw patient’s tissue, as well as surgical devices, synthetic implants, orthopedics, and catheters,

etc3. However, this problem is not limited to the clinic and also applies to drinking water, food

packaging and agriculture4-6. This brings about an inherent need to develop new antibacterials

that are broad spectrum by design, but more importantly, are intrinsically difficult for the

bacteria to develop resistance.

In designing a new broad spectrum anti-bacterial material, there is one general structural

feature that has been employed by many different researchers to again activity against gram

positive and gram negative bacteria. Generally speaking, this involves having a proper balance of

cationic charge, which draws the material close to the negatively charged bacterial membrane,

and hydrophobic residues, which interact with the phospholipid bilayer3,7,8. This theology is

based on the natural defense mechanisms of many living organisms which synthesize cationic

antimicrobial peptides (CAPs) when under attack. While each peptide is different and unique to

2

the particular organism, they all generally consist of about 12-50 residues, in which

approximately 50% of them are hydrophobic. While the mechanism of these CAPs is still under

some debate, it is generally believed that it does not interact with any receptor on the cell

surface9. Instead, CAPs (as well as broad spectrum antimicrobial polymers [AMPs]6) target the

natural differences between bacterial and mammalian cells. When a bacterial cell is approached

by an AMP or CAP, hydrophobic interactions prevail and may sink the AMP or CAP into the

bacterial membrane, and thus places a torsional strain on the membrane bound phospholipids,

which can further lead to membrane disruption and subsequent cell lysis9.

Eukaryotic mammalian cells (including human erythrocytes) contain up to 25%

cholesterol9 in their lipid bilayers and are predominantly composed of zwitterionic (overall

neutral) phospholipids such as phosphatidylcholine, phosphatidyethanolamine, and

sphingomyelin10. Furthermore, most of the lipids in mammalian cells that do have a net negative

charge are actually located on the inner leaflet of the cell instead of the outer leaflet (Figure 1.1).

Bacteria on the other hand, have a structurally different outer membrane. First, they lack

cholesterol in their membranes, which is thought to act as a stabilization factor for mammalian

cells11. This alone can make bacterial membranes easier to disrupt compared to mammalian ones.

Second, the outer leaflet of the bacterial cell are generally composed of negatively charged

phospholipids such as phosphatidylglycine, cardiolipin and phosphatidylserine10. More

specifically in gram positive cells, some negative charge is thought to arise from lipoteichoic

acids (which links the outer thick peptidoglycan layer to the inner cell membrane) and its

associated negatively charged phosphate groups linked via phosphodiester bonds. In contrast,

gram-negative cells have only a thin peptidoglycan layer, but contain a second outer cell

membrane on top of this with the outer leaflet being composed mostly of lipopolysaccharides

3

that are highly anionic7. The outer membrane of gram negative cells do not contain teichoic

acids, but they do contain phosphate groups in a similar environment as the phosphates in

teichoic acids12. Discounting surface layer antigens, it can be said that the outer membranes of

gram positive and gram negative bacteria carry an overall net negative charge, which is greater

than that of mammalian cells which are relatively neutral. Therefore, a more electrostatically

favorable interaction exists between CAPs and AMPs with bacteria as compared to mammalian

cells.

Figure 1.1: Difference between bacterial and mammalian cell membranes.

4

In applying this theology to designing a functional biomaterial, we have developed

amphiphilic nanoparticles based on naturally occurring amino acids linked to a mono methyl

poly ethylene glycol (mPEG) tether. PEG is an FDA approved water soluble polymer for in vivo

use due to its excellent biocompatibility and enzyme degradability13. The use of PEG here is

highly advantageous in that it is highly hydrophilic and helps the ensuing polymer collapse into a

micelle as it has a hydrophobic core. Equally as important, PEG has non-adhesive antifouling

properties14, which prevent protein and cell adhesion to the nanoparticle. This prevents the

aggregation of debris on the outside of the particle which would hinder its activity due to the

particle being blocked from interacting with bacterial membranes. This most accepted reason for

this property is due to a layer of tightly bound water molecules that surrounds the PEG surface,

which effectively prevents protein adhesion15.

To ensure the polymer is fully biocompatible, the bulk of the material is made from poly

amino acids. Briefly, positively charged, negatively charged, and hydrophobic natural L-amino

acids (lysine, glutamic acid, and phenylalanine respectively) were cyclized via triphosgene into

N-carboxyanhydride (NCA) monomers and purified by recrystallization. Then, mPEG-NH2 was

used as the initiator for the polymerization and various equivalents of the amino acid monomers

were added to develop a library of polymers. To lessen the hydrophobicity of the core, we have

used a hybrid core that is composed of a random set of charged residues to hydrophobic residues.

In theory, by having the charged segments (which have favorable electrostatic interactions to

approach the bacterial membrane) next to the hydrophobic moieties, this allows the hydrophobic

phenylalanine rings to move closer to the bacterial membrane and increase the membrane

depolarization leading to cell lysis. Taken together, our compounds show broad spectrum activity

5

against both gram positive and gram negative strains and form nanoparticles in the sub 200nm

range.

1.2 Drug Delivery Polymers

Due to our polymers exhibiting excellent water solubility as well has having a defined

secondary nanoparticle structure with a hydrophobic core; we anticipated that they would also

make suitable drug delivery vehicles for hydrophobic drugs. As a proof of concept, we have used

a super hydrophobic drug, Tanshinone IIA, to be delivered by our PEG poly amino(acid)s in a

live mouse model. Tanshinone IIA is a small molecule isolated from salvia miltiorrhiza and

exhibits broad spectrum anti-tumor activity16-18. While its direct mechanism of action is

unknown, it is believed to have multiple intra- and extra- cellular targets19. For a molecule such

as Tanshinone which is highly insoluble in water and thus, unable to be administered in vivo, a

drug delivery vehicle must be used. We have used a glutamic acid analogue of our polymers for

this purpose. Its negative charge prevents it from aggregation with many human serum proteins,

many of which are also negatively charged, and our hybrid core is hydrophobic enough to ensure

that Tanshinone IIA is encapsulated and able to be administered in vivo. We have used a hybrid

core instead of a purely hydrophobic core because it is hydrophobic enough to drive the collapse

of the micelle and encapsulate the drug, but also contains less electrostatic interactions with the

drug which will aid in its release to occur more rapidly. Our results show that compared to

Tanshinone IIA alone, our encapsulated Tanshinone IIA has reduced the hepatoma tumor

volume by approximately 70%, which allows it to be a suitable carrier for this drug.

To further the clinical application of our polymers, we have also used a lysine analogue

of our nanoparticle to encapsulate and deliver a small interfering RNA, known as miRNA-139,

to be administered in a live mouse model as well. MicroRNAs are small non-coding RNA

6

molecules that regulate post-transcriptional gene expression. Since they can bind to any specific

mRNA sequence, they have potential for the regulation of virtually any gene, and have already

been implicated in many biological functions such as cell proliferation, metastasis,

differentiation and apoptosis20,21. For our delivery system of miRNA-139, we have used a lysine

analogue of our polymers due to its cationic nature, which has electrostatically favorable

interactions with the anionic charged miRNA. Again, we have used a hybrid core so as to limit

hydrophobic interactions, as well as facilitate the release of the miRNA due to the less tightly

packed hydrophobic core. Compared to miRNA-139 alone, our encapsulated miRNA-139 has

shown the ability to stunt the growth of a hepatoma tumor by approximately 75% in a live mouse

model (Figure 1.2). Taken together, these results show that our PEG poly amino(acid)s show

great versatility in delivering novel therapeutic agents, as well as inhibiting the proliferation of a

broad spectrum of bacterial strains.

Figure 1.2. Graphical representation of our drug delivery system used for miRNA-139 in

vivo.

7

1.3 Outline of the dissertation

In this dissertation, we discuss the design, synthesis, and applications of mPEG poly

amino (acid)s as well as testing of the compound in a live mouse model.

In chapter 2, we outline the synthetic design behind our positively charged polymer

library for use as broad spectrum antibacterial agents against both Gram positive and Gram

negative strains. The in vitro testing is also described.

In chapter 3, we describe the drug delivery application of a negatively charged polymer

for the delivery of a super hydrophobic drug, Tanshinone IIA. Its in vivo application is discussed

and data for its tumor suppression in a live mouse model is presented.

In chapter 4, we describe the drug delivery application of a positively charged polymer

for the delivery of miRNA-139. The rational of the design, techniques used, and data for the in

vivo tumor suppression of our delivery system is presented.

In chapter 5, we summarize the major findings of this dissertation and then conclude with

the future directions of this research.

1.4 References

(1) Alekshun, M. N.; Levy, S. B.: Molecular Mechanisms of Antibacterial Multidrug

Resistance. Cell 2007, 128, 1037-1050.

(2) Taubes, G.: The Bacteria Fight Back. Science 2008, 321, 356-361.

(3) Li, Y.; Fukushima, K.; Coady, D. J.; Engler, A. C.; Liu, S.; Huang, Y.; Cho, J. S.;

Guo, Y.; Miller, L. S.; Tan, J. P. K.; Ee, P. L. R.; Fan, W.; Yang, Y. Y.; Hedrick, J. L.: Broad-

Spectrum Antimicrobial and Biofilm-Disrupting Hydrogels: Stereocomplex-Driven

Supramolecular Assemblies. Angewandte Chemie 2013, 125, 702-706.

(4) Kang, D. H.; Gupta, S.; Rosen, C.; Fritz, V.; Singh, A.; Chander, Y.; Murray, H.;

Rohwer, C.: Antibiotic Uptake by Vegetable Crops from Manure-Applied Soils. Journal of

Agricultural and Food Chemistry 2013, 61, 9992-10001.

8

(5) Page, K.; Wilson, M.; Parkin, I. P.: Antimicrobial surfaces and their potential in

reducing the role of the inanimate environment in the incidence of hospital-acquired infections.

Journal of Materials Chemistry 2009, 19, 3819-3831.

(6) Kenawy, E.-R.; Worley, S. D.; Broughton, R.: The Chemistry and Applications of

Antimicrobial Polymers: A State-of-the-Art Review. Biomacromolecules 2007, 8, 1359-1384.

(7) Li, P.; Poon, Y. F.; Li, W.; Zhu, H.-Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.;

Lamrani, M.; Beuerman, R. W.; Kang, E.-T.; Mu, Y.; Li, C. M.; Chang, M. W.; Jan Leong, S. S.;

Chan-Park, M. B.: A polycationic antimicrobial and biocompatible hydrogel with microbe

membrane suctioning ability. Nat Mater 2011, 10, 149-156.

(8) Zhou, C.; Qi, X.; Li, P.; Chen, W. N.; Mouad, L.; Chang, M. W.; Leong, S. S. J.;

Chan-Park, M. B.: High Potency and Broad-Spectrum Antimicrobial Peptides Synthesized via

Ring-Opening Polymerization of α-Aminoacid-N-carboxyanhydrides. Biomacromolecules 2009,

11, 60-67.

(9) Glukhov, E.; Stark, M.; Burrows, L. L.; Deber, C. M.: Basis for Selectivity of

Cationic Antimicrobial Peptides for Bacterial Versus Mammalian Membranes. Journal of

Biological Chemistry 2005, 280, 33960-33967.

(10) Salick, D. A.; Kretsinger, J. K.; Pochan, D. J.; Schneider, J. P.: Inherent

Antibacterial Activity of a Peptide-Based β-Hairpin Hydrogel. Journal of the American

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(11) Matsuzaki, K.: Why and How are Peptide–Lipid Interactions Utilized for Self-

Defense? Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta (BBA) -

Biomembranes 1999, 1462, 1-10.

(12) Jiang, W.; Saxena, A.; Song, B.; Ward, B. B.; Beveridge, T. J.; Myneni, S. C. B.:

Elucidation of Functional Groups on Gram-Positive and Gram-Negative Bacterial Surfaces

Using Infrared Spectroscopy. Langmuir 2004, 20, 11433-11442.

(13) Brandl, F. P.; Seitz, A. K.; Teßmar, J. K. V.; Blunk, T.; Göpferich, A. M.:

Enzymatically degradable poly(ethylene glycol) based hydrogels for adipose tissue engineering.

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S.: Antibacterial and Antifouling Polymer Brushes Incorporating Antimicrobial Peptide.

Bioconjugate Chemistry 2008, 20, 71-77.

(15) Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S.: Protein Adsorption on

Oligo(ethylene glycol)-Terminated Alkanethiolate Self-Assembled Monolayers: The Molecular

Basis for Nonfouling Behavior. The Journal of Physical Chemistry B 2005, 109, 2934-2941.

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Underlying Treatment Mechanism of Psoriasis. Evidence-Based Complementary and Alternative

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S. Y.; Chang, C. S.: The selective inhibitory effect of a synthetic tanshinone derivative on

prostate cancer cells. Prostate 2012, 72, 803-816.

(18) Yan, M. Y.; Chien, S. Y.; Kuo, S. J.; Chen, D. R.; Su, C. C.: Tanshinone IIA

inhibits BT-20 human breast cancer cell proliferation through increasing caspase 12, GADD153

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2008, 9, 102-114.

10

CHAPTER 2: INVESTIGATION OF ANTIMICROBIAL PEG-POLY(AMINO ACID)S

Note to reader

This chapter has been previously published and has been reproduced by permission of The

Royal Society of Chemistry. Original citation:

Costanza, F.; Padhee, S.; Wu, H.; Wang, Y.; Revenis, J.; Cao, C.; Li, Q.; Cai, J.:

Investigation of antimicrobial PEG-poly(amino acid)s. RSC Advances 2014, 4, 2089-2095.

2.1 Introduction

The extensive use of antibiotics in recent decades for medical and agricultural purposes

has elicited the widely known issue of bacterial resistance.1 As such, the development of new

antimicrobial agents with novel mechanisms has been a constant area of research. This includes

the need for new antibacterial materials to prevent contamination in food packaging, storage,

medical supplies, and drinking water,2-4 in addition to drug discovery. One approach to

circumvent resistance could be the development of antimicrobial peptides (AMPs). 1,5,6 Cationic

AMPs exist in most organisms and are the first line of natural defense against infections.

Although they possess different secondary structures or sometimes even random structures in

solution, they are believed to adopt an amphipathic conformation when interacting with

negatively charged bacterial membranes, and subsequently disrupt the function of bacterial

membranes. 5 As this biophysical interaction lacks defined bacterial targets, this mechanism of

action is not expected to develop resistance as easily as traditional antibiotics. Based on such

11

recognition, many classes of AMP mimetics have been developed. 7-14 These small

peptidomimetics are capable of mimicking the function and mechanism of AMPs, and are shown

to have potent and broad spectrum antimicrobial activity. However, their step-by-step

preparation is tedious, and they are generally not economical to produce in substantial quantities,

limiting the potential of widespread use as affordable biomaterials in the future.

Recently, there have been some advances in polymer antimicrobial agents such as

polymers based on methacrylates, 15,16 norborenes, 17,18 amidoamines,19 and others. 3,20-23 These

polymers contain both hydrophobic components and cationic components, which are critical for

the disruption of bacterial membranes. Many of them have also been shown to kill both Gram-

positive and Gram-negative bacteria, including clinically relevant pathogens. In addition, they

are generally prepared through a one-pot polymerization, allowing the generation of products in

large amounts. As such, there is promising potential for these synthetic materials to be used as

antimicrobial agents to treat bacterial infections. However, the development of biodegradable

antimicrobial nanoparticles is very rare. Poly(amino acid)s,23 synthesized by ring-opening

polymerization, show attractive features for further exploration. They have been widely used for

drug delivery and other biomedical applications24-27 as they are completely biodegradable.

However, their antimicrobial development is rare. 23 In this study, we designed, synthesized and

studied the antimicrobial activity of biodegradable polymer nanoparticles - PEGylated

amphiphilic poly (amino acids). The PEG is introduced to the polymers because PEG

(polyethylene glycol) is essentially non-toxic and biocompatible.7,28-30 Meanwhile, PEGylation

promotes the formation of a corona which comprises the outer shell of the nanoparticles.

Additionally, PEG is widely available, inexpensive, highly water soluble and easily modifiable

by size and end group functionality. It has also been used in the development of peptide

12

nanoparticles with a hydrophilic corona which would enhance the stability of the micelle and

increase circulation time.31-33 Another attractive aspect of using PEG in a block type

antimicrobial nanoparticle is that it has non-adhesive antifouling properties which can prevent

protein and cell adhesion.29 This is a key feature of the material as any cellular debris that can

potentially adhere to the outer surface would likely inhibit its activity. Another report shows that

PEG does not reduce activity to bacteria, while also may potentially decrease toxicity to human

cells.19 As such, we also expected that introduction of a PEG segment to poly(amino acid)s will

promote the formation of nanoparticles, which may facilitate bacterial membrane disruption, and

therefore possess improved antimicrobial activity.

2.2 Materials and Methods

(All polymers were prepared by a similar method). 0.4 g of PEG-NH2 was dissolved in

10 ml of anhydrous dioxane and purged with N2. Separately 0.49 g (20 eq) of Z-lysine NCA was

dissolved in 5 ml anhydrous dioxane, passed through a 2 micron filter to remove any insoluble

decomposed NCA, thoroughly purged with N2 and then added via syringe. The reaction was then

allowed to proceed for 3 days under an active N2 atmosphere. Then 10 equivalents of Z-lysine

NCA and 15 equivalents of phenylalanine NCA were dissolved in 5 ml anhydrous dioxane,

passed through a 2 micron filter, purged, and added via syringe. Polymerization was allowed to

continue for another 3 days. The clear solution was then precipitated into ether and the product

was collected via filtration. The polymer was then dissolved in 10 ml of TFA and to this

solution, 10 equiv. of HBr in AcOH (33% v/v conc.) was added and stirred for 4 hours. The

product was then precipitated into ether and collected via filtration. Finally, the compounds were

13

dissolved in DMSO (dimethyl sulfoxide) and purified via dialysis (MWCO 3,500) for 3 days.

The water was changed daily and the final product was achieved after freeze dying.

The bacterial strains used for testing the efficacy of polymers were collected from the

American Type Culture Collection (ATCC) and were multi-drug resistant S. epidermidis

(RP62A), B. subtilis (BR151), K. pneumoniae (ATCC 13383) and multi-drug resistant P.

aeruginosa (ATCC 27853). The antimicrobial activities of the polymers developed were

determined in sterile 96-well plates by the 2-fold serial-dilution method. Bacterial cells were

grown overnight at 37 °C in 5 mL medium after which a bacterial suspension of approximately

106 CFU/mL in Luria broth or trypticase soy was prepared ensuring that the bacterial cells were

in the mid-logarithmic phase. Aliquots of 50 µL bacterial suspension were added to 50 µL of

medium containing different concentrations of polymers for a total volume of 100µL in each

well. The 96-well plates were incubated at 37 ºC for about 20 h. The Biotek microplate reader

was used to measure the optical density (OD) at a wavelength of 600 nm after about 20 h. The

experiments were carried out as three independent biological replicates, each in duplicate. The

lowest concentration at which complete inhibition of bacterial growth is observed is defined as

the minimum inhibitory concentration (MIC).

Freshly drawn human red blood cells (hRBC’s) were used for the assay. The blood

sample was washed with PBS buffer several times and centrifuged at 700g for 10 min until a

clear supernatant was observed. The hRBC’s were re-suspended in 1X PBS to get a 5% v/v

suspension which was used to perform the assay. 50 µL of different polymers solutions were

added to sterile 96-well plates. Then 50 µL of 5% v/v hRBC solution was added to make up a

total volume of 100 µL in each well. The 0% hemolysis point and 100% hemolysis point were

determined in 1 X PBS and 0.2% Triton-X-100 respectively. The 96 well plate was incubated at

14

37 °C for 1h and centrifuged at 3500 rpm for 10 min. The supernatant (30 µL) was then diluted

with 100 µL of 1XPBS and hemoglobin was detected by measuring the optical density at 360nm

by Biotek microtiter plate reader (Type: Synergy HT).

% hemolysis = (Abs sample -Abs PBS )/(Abs Triton

–Abs PBS ) x 100

A double staining method with DAPI (4’,6-diamidino-2-phenylindole

dihydrochloride,Sigma,>98%) and PI (propidium iodide, Sigma) as fluorophores was used to

visualize and differentiate the viable from the dead B. subtilis cells. DAPI being a double

stranded DNA binding dye, stains all bacterial cells irrespective of their viability. Ethidium

derivatives such as propidium iodide (PI) is capable of passing through only damaged cell

membranes and intercalates with the nucleic acids of injured and dead cells to form a bright red

fluorescent complex. The bacterial cells were first stained with PI and then with DAPI (2-(4-

Amidinophenyl)-6-indolecarbamidine dihydrochloride). The bacterial cells were grown until

they reached mid-logarithmic phase and then ~ 2x10 3 cells were incubated with 100 µg/mL

polymer P5 for 4 h. Then the cells were pelleted by centrifugation at 3000 g for 15 min in an

Eppendorf microcentrifuge. The supernatant was decanted and the cells were washed with 1X

PBS several times and then incubated with PI (5 µg/mL) in the dark for 15 min at 0 oC. The

excessive PI was removed by washing the cells several times with 1X PBS several times. Lastly,

the cells were incubated with DAPI (10 µg/mL in water) for 15 min in the dark at 0oC. Finally,

the excessive DAPI solution was removed by washing it with 1X PBS. The controls were

performed following the exact same procedure for bacteria without P5. The bacteria were then

examined by using the Zeiss Axio Imager Z1 optical microscope with an oil-immersion objective

(100X). 7,8

15

MRSE and B.subtilis were grown to an exponential phase and approximately about 2 x

106 cells were incubated with the polymer for about 20 h. The cells were then harvested by

centrifugation (3000 g) for 15 min. After pelleting the cells, they were washed three times with

DI water. The cells were then fixed with 2.5% (w/v) glutaraldeyhyde in nanopure water for about

30 min, followed by extensive wash with DI water to get rid of any excess glutaralhedyde.

Graded ethanol series (30%, 50%, 70%, 95% and 100%, 5 min each) were then used to

dehydrate the cells. Following the dehydration of cells, hexamethyldisilazane was added for

about 2 minutes. Then, about 2 µL of sample was added to a silicon wafer followed by Au/Pd

coating, and = the samples were observed at 25KV with a HITACHI S-800 scanning electron

microscope.

2.3 Results and Discussions

To test our hypothesis, we synthesized a series of PEGylated poly(amino acid)s

containing both hydrophobic groups and cationic groups (Figure 2.1) via ring-opening

polymerization of N-carboxyanhydrides (NCAs). 34 NCAs can easily be synthesized and attained

in large quantities from natural amino acids at low cost, which makes them attractive in

designing new materials. 35 The synthetic procedure was adapted from a previously published

protocol. 35 Briefly, Phe-NCA and Z-Lys-NCA (Figure 2.1) were prepared through the treatment

of amino acids phenylalanine or Z-Lysine with triphosgene, respectively. Next, CH3O-PEG-NH2

was used as the initiator to react with NCAs to generate poly(amino acid)s via the ring-opening

polymerization. In the synthesis of two polymers (P2 and P3), to ensure the formation of cationic

shell of nanoparticles, Z-Lysine-NCA was first added into the flask containing the initiator and

reacted for three days at the room temperature. Subsequently, Z-Lysine-NCA and Phe-NCA

were then added in one batch to form a hybrid segment, which forms the core structure of

16

nanoparticles in solution. Such a random core structure was expected to facilitate the interaction

with the bacterial membranes through both charge and hydrophobic forces. 23 The random core

was also designed to limit the packing density of the phenylalanine rings, which allows for a

more overall amphipathic material. As such, a series of polymers containing varying numbers of

hydrophobic and hydrophilic groups were prepared (Table 2.1). To test if the charge is critical to

the polymers’ antimicrobial activity, one negatively charged polymer was synthesized using both

Glu-NCA and Phe-NCA.

ONH Dioxane

N2, r.t., 3d

O

HN

NH

O

NHO

O

1. TFAHBr / AcOH

c equiv. Z-Lys NCA+ d equiv. Phe NCA

a

H

b

b equiv Z-Lys NCA

DioxaneN2, r.t., 3d

2. Dialysis O

HN

NH

O

NH2

ab

HNO

O

O

R

NCA

H

a

HN

NH2

O

NH

Od

Hc

Figure 2.1 Synthesis of PEG-poly(amino acid)s.

The structures of these polymers was characterized by the 1H NMR (Appendix A3-A11),

and their nanomorphology was evaluated by scanning electron microscopy. As expected, most of

these poly(amino acid)s form nanoparticles in water with sizes ranging from 50 to 200 nm

(Figure A2). The zoomed-in pictures (Figure 2.2b and 2.2c) clearly show that these nanoparticles

17

contain a hydrophobic core and a cationic shell including a PEG corona. As expected, polymer

P9 and P10 do not form nanoparticles due to the lack of the hydrophobic components. These

polymers were tested against a range of Gram-positive and Gram-negative bacteria. Their

antimicrobial activity is shown in Table 2.1.

Figure 2.2 The nanomorphology of the PEG-poly(amino acid)s P5. a-c, SEM pictures. a is a

zoomed-out view, while both b and c are the zoomed-in views. d is the schematic illustration of

PEG-poly(amino acid)s core-shell structure.

18

Table 2.1. The antimicrobial and hemolytic activities of PEG-poly(amino acid)s. The

microbial organisms used are K. pneumoniae (ATCC 13383), and P. aeruginosa (ATCC 27853),

methicillin-resistant S. epidermidis (RP62A), B. subtilis (BR151). The minimum inhibitory

concentration (MIC) is the lowest concentration that completely inhibits growth after 24 h. HC50

is the concentration causing 50% hemolysis. The antimicrobial peptide Magainin II was included

as a control.

O

HN

NH

O

NH2

ab

HN

NH2

O

NH

Od

Hc O

HN

NH

Oab

HN

O

NH

Od

Hc

O

O

HO

HO

P2-P10 P1

#

MR

SE

(µM

)

B.

Subtili

s

(µM)

K.

Pneum-

oniae

(µM)

P.

Aerug-

inosa

(µM)

mPE

G

(mw

)

Lysine

Content

b [(c

Phenyl-

alanine

Content

d)]

%

hydroph

obic

content

hemolytic

activity

HC50(µM)

MW

(Theor

etical)

Aver

age

size

of

nano

parti

cles

(nm)

P1 >100 >100 >100 >100 5000

Glu 20

(10 15) 33 - 10880

70-

80

P2 55

2.8-

5.5

27.5 -

55.3 >100 5000 10 (10 10) 33 9 9030

80-

90

P3

9-

22.6 4.5-9 45.3 >100 5000 20 (10 15) 33 25 11045

40-

50

P4

6.5-

13

1.6-

3.2 65 >100 5000 (10 10) 50 10 7750

60-

80

P5

5.3-

10.5

5.3-

10.5

26.3-

52.6 21-52.6 2000 (10 10) 50 6 4750

200-

250

P6

9.1-

18.2

18.2-

45.5

45.5-

91.6

45.5-

91.6 2000 (10 15) 60 5 5485

80-

100

P7

6.7-

13.3

3.4-

6.7 >100

33.3-

83.6 2000 (20 20) 50 3 7500

80-

100

P8

1.4-

2.8

5.7-

11.4 >100 28-56 2000 (30 20) 40 3 8780

100-

110

P9

5.5-

11 5.5-11 >100 >100 2000 20 0 0 >50 4560

P10

6.5-

12.8

6.5-

12.8 63.6-128 >100 2000 15 0 0 >50 3920

Mag

ainin

II11 30 16 40 40 2478

19

The negatively charged PEG-poly(amino acid)s P1, which contains glutamate residues,

did not show any antimicrobial activity under the tested conditions. This is consistent with the

hypothesis that negatively charged bacterial membranes interact weakly with molecules bearing

negative charge due to electrostatic repulsion. 36 The outer membrane of bacteria has a net

positive charge and thus the lack of activity of P1 is expected. The impact of PEGylation on the

hemolytic activity can be illustrated by P4, which contains a longer PEG corona than P5, but has

less hemolytic activity. It suggests that PEGylation does decrease the hemolytic activity of

polymers compared to the non-PEGylated analogues, although the impact is not significant.23

However, a long PEG corona may prevent the interaction of the polymer with the bacterial

membrane and thus less reactive against bacterial membranes. This is evidenced by the fact that

P4 is not as broad-spectrum active as P5. It must be noted that hemolytic activity is more tightly

related to the hydrophobic content of the polymers. Polymers P9 and P10 do not contain any

hydrophobic content, and they do not show discernible hemolytic activity even up to 50 µM.

Also, they are not active against Gram-negative bacterial membranes, indicating that the

existence of hydrophobic segments, and thus the formation of nanoparticles are important for the

disruption of membranes of Gram-negative bacteria. However, they are highly selective against

Gram-positive bacteria, including clinically-related multi-drug resistant S. epidermidis,

suggesting their potential application as novel antimicrobial biomaterials. This is consistent with

other research and is likely due to the inability of the material to penetrate the sturdy outer

membrane of Gram negative bacteria 37. Increasing hydrophobic contents lead to the most potent

antimicrobial polymers P5 and P6, which are active against all the tested bacteria. In fact, both

P5 and P6 have comparable antimicrobial activity to the antimicrobial peptide magainin II

against the tested strains. Our results also suggest that the selectivity between antimicrobial

20

activity and hemolytic activity can be tuned. P2 and P3 have similar hydrophobic content and

antimicrobial activity; however, P3 is less hemolytic than P2. It is possible that the cationic shell

(outer layer) of P3 is thicker than P2, which prevents the hydrophobic contents from non-

specifically interacting with the membrane due to increased hindrance. These effects are not

evident in the other synthesized polymers, in which the hydrophobic Phe-NCA and cationic Lys-

NCA were added simultaneously to produce hybridized core structures, and as such, their

selectivity is low. Therefore, we speculate the addition of a cationic shell (b portion), such as a

poly-lysine segment, into polymer P5 could allow for a more selective polymer to be obtained

with higher antimicrobial activity but less hemolytic activity.

We also examined the impact of particle size and molecular weight of poly(amino acid)s

on the antimicrobial activity. Although molecular weights of poly(amino acid)s do not have an

obvious impact on the antimicrobial activity and hemolytic activity, it seems that the size of

nanoparticles does affect these activities in some way. The smaller sized particles tend to have

lower toxicity to blood cells. For instance, P3 has the smallest particle size and it has the least

hemolytic activity. A similar trend is observed for other polymers, except for P5. We

hypothesize that the larger the particle size, the less specific interaction it will have with red

blood cells, thereby allowing the compound to be more hemolytic.

The ability of the polymers to damage bacterial membranes was first evaluated by

fluorescence microscopy using a double staining approach. In this assay, B. subtilis was co-

stained by DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide). DAPI can pass

through the intact bacteria membranes irrelevant of bacterial viability; 8,10,11,36 however, PI can

only stain bacteria by intercalating nucleic acids in their nucleus after the membranes have been

disrupted. Figure 2.3 shows B. subtilis with intact cell membranes do not show fluorescence

21

after the incubation with PI. However, after 2 h treatment of B. subtilis with the most potent

polymer P5, a strong red fluorescence was observed inside the bacterium, indicating PI has

permeated into the cell’s nucleus. This demonstrates that the membranes of B. subtilis have been

disrupted.

Figure 2.3. Fluorescence micrographs of B. subtilis treated with 100 µg/ml polymer P5 for 2 h.

a1, control, no treatment, DAPI stained; a2, control, no treatment, PI stained; a3, control, no

treatment, the merged view. b1, P5 treatment, DAPI stained; b2, P5 treatment, PI stained; b3, P5

treatment, the merged view.

The ability of the polymer P5 to compromise the integrity of bacterial membranes was

further assessed by SEM (Figure 2.4). After treatment of B. subtilis with P5, the membranes of

B. subtilis are completely disrupted (Figure 2.4b). The same disruption is also observed after the

22

treatment of MRSE with P5. Figure 2.4d shows MRSE after incubation with P5 in which the

membranes of MRSE were damaged, leading to the aggregation of debris due to loss of

membrane zeta potential. 7-9 It is important to note that these polymers do not have any defined

targets and are interacting non-specifically, which would allow for some MRSE to survive once

they become shielded from nearby debris, as shown in figure 2.4d. Figure 2.4e clearly shows that

one core-shell nanoparticle from P5 is approaching the surface of a MRSE cell, while the other

nanoparticle is already merging into the membrane of the bacteria. These results support that the

polymer P5 kills bacteria via membrane disruption. Meanwhile, it is also possible that multiple

polymer nanoparticles interact with the membrane of the same bacteria cell simultaneously to

cause bacterial cell lysis.

Figure 2.4 SEM pictures. a, B. subtilis, no treatment; b, B. subtilis, treatment with 100 µg/mL

polymer P5. c, MRSE, no treatment; d, MRSE, treatment with 100 µg/mL polymer P5, a zoomed-

out review; e, MRSE, treatment with 100 µg/mL polymer P5, a zoomed-in view.

23

2.4 Conclusions

In summary, we have successfully synthesized a series of PEGylated poly (amino acids)

via ring-opening polymerization of NCAs. The reaction is run in one pot, and large quantities

can be produced easily. Our results show that these biodegradable nanoparticles have broad-

spectrum activity against both Gram-positive and Gram-negative bacteria. PEGylation can not

only promote the formation of nanoparticles, but also potentially decrease hemolytic activity to a

certain extent, even though both hemolytic activity and broad-spectrum activity are primarily

determined by the hydrophobic content. The selectivity of the polymers can be tuned by varying

both the ratio and numbers of hydrophobic and cationic groups. Both fluorescence microscopy

and SEM suggest that these PEGylated poly(amino acid)s kill bacteria by disrupting the integrity

of their membranes. Surprisingly, the polymers containing only PEG and lysine segments, show

remarkable selectivity against Gram-positive bacteria, and do not display any hemolytic activity

under our experimental conditions. It may suggest that these polymers can be not only used to

develop antimicrobial biomaterials to prevent contamination in food packaging, storage and

medical supplies, but also used as potential therapeutics to treat gram positive bacterial

infections. Furthermore, our core-shell PEG-poly(amino acid)s nanoparticles are also capable of

encapsulating drug molecules, making it possible to load small molecular antibiotics and to kill

bacteria more effectively and synergistically via membrane disruption as well as targeted

inhibition of bacterial functions. Such dual-functional antimicrobial nanoparticles are currently

being developed in our lab.

24

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28

CHAPTER 3: PEG-POLY (AMINO ACID)s – ENCAPSULATED TANSHINONE IIA AS

POTENTIAL THERAPEUTICS FOR THE TREATMENT OF HEPATOMA

Note to reader

This chapter has been previously published and has been reproduced by permission of

The Royal Society of Chemistry. Original citation:

Wang, Y*.; Costanza, F.*; Wu, H.; Song, D.; Cai, J.; Li, Q.: PEG-poly(amino acid)s-

encapsulated tanshinone IIA as potential therapeutics for the treatment of hepatoma. Journal

of Materials Chemistry B 2014, 2, 3115-3122.

3.1 Introduction

There have been many developments of drug delivery systems using amino acid

polymeric scaffolds including poly(sodium N-acryloyl-L-valinate-co-alkylacrylamide)1 as

carriers of nonsteroidal anti inflammatories, poly (L-lysine)-block-poly(L-phenylalanine)2, poly-

L-amino acid (homopolymer)3 as carriers for DNA delivery or doxorubicin as a model

encapsulated drug, and poly(L-lactide-β-γ-benzyl glutamate)4, among many others. However,

there are limitations to some of these such as inadequate water solubility and lack of a defined

amphiphilic water soluble structure in aqueous medium. This reduces circulation time as the

material is rapidly cleared by the kidneys during circulation. For those without a proper peptide

backbone, the materials are not biodegradable, which is very undesirable1.

There have been other kinds of drug delivery systems unrelated to peptides. One

intriguing example involves the emulsification of oleic acid as a surfactant in chloroform with

29

polystyrene allyl alcohol followed by a second emulsification with poly vinyl alcohol. A DNA

plasmid (hydrophilic model) or doxorubicin (hydrophobic model) nanocapsule is achieved after

evaporation of the organic solvent5. While this approach is certainly unique, its polymer

backbone is not ideal for a biological system. Other interesting delivery systems utilize

derivatives of chitin (one of the most abundant polysaccharides) to form nanoparticles from its

derivative chitosan, and have shown a model encapsulation of doxorubicin as well6,7. This

approach is valid as chitosan is non-toxic, biocompatible and very abundant, but there are issues

with water solubility at physiological pH levels.

Another class of drug delivery systems that have been gaining traction over the past

decade are those of liposomes, which are essentially made of phospholipid spheres in solution.

They have been used for aptamer delivery and labeled with FTIC8, and have also been used as a

delivery of the model drug doxorubicin,9 among many others10. However, a major limitation of

liposomes is their lack of stability as they are made up of small molecules and therefore, their

critical micelle concentration is higher than that of polymer based nanoparticles. Upon dilution in

a physiological system, they have a greater capacity to fall apart and the controlled release of the

drug is lost. One way of circumventing this issue is by attaching on PEG to prolong their

circulation time and in vivo stability upon delivering doxorubicin11.

While many groups have reported a novel kind of drug delivery system based on various

approaches already mentioned, often times only a model drug is used for encapsulation and for

proof of principle. Here, we have used our delivery system to encapsulate Tanshinone IIA and

test it in a live mouse model. Tanshinone IIA (Figure 3.1) is a lipo-soluble small molecule

isolated from salvia miltiorrhiza, and displays broad-spectrum anti-tumor activity.12-17 It is

believed to have multiple intra- and extra-cellular targets18 and therefore is less probable to

30

develop drug-resistance in tumor cells. Some known targets include inhibition of human

esterases and carboxylesterases19, inhibition of hypoxia inducible factor-120, dilation of coronary

blood vessels via activating potassium channels21, and induced maspin expression22 among many

more undiscovered targets. However, Tanshinone IIA (TSIIA) has some intrinsic drawbacks

such as poor water solubility and a short half-life.23 This has prompted the ongoing research to

develop new delivery systems to increase the solubility of TSIIA and to improve its anti-tumor

efficacy. 17,18,23 In order to advance the application and efficacy of TSIIA as a novel anti-cancer

therapeutic, the exploration of new polymer delivery systems are needed. As such, herein we

report the design and synthesis of amphiphilic PEG-poly(amino acid)s (PPAAs), which were

used as a novel nanocarrier to encapsulate and deliver TSIIA. The method of delivery is that of

passive diffusion out of the polymer and into circulation as the drug is noncovalently attached.

Many other drug delivery systems utilize polymer drug conjugates in which the drug is

covalently attached to the nanoparticle. Unless there is a clear method of release, this should be

avoided as it does decrease the activity of the drug by making it harder to reach its target24.

However, this also does limit some potential toxic effects the drug may have as well.

O

O

O

Figure 3.1 The structure of Tanshinone IIA.

31

Compared to other polymer delivery systems, poly (amino acid)s have many attractive

features. The synthesis of poly (amino acid)s is very straightforward, which can be achieved

through the ring-opening polymerization of N-carboxyanhydrides (NCAs).25 NCAs can be

obtained in one step upon treatment of side chain protected natural amino acids with phosgene or

triphosgene followed by crystallization as a primary purification method. Since the subsequent

polymerization is living, the monomers can be added batch by batch, which therefore allows for

easy synthesis of poly (amino acid)s with multiple blocks. As there are 20 natural amino acids,

the potential is limitless to obtain poly (amino acid)s that contain a diverse array of functional

groups with tunable charges. Additionally, they are biocompatible and completely

biodegradable26, and have low toxicities, which further augment their application for drug

delivery. 27-29 Furthermore, poly (amino acid)s can be easily modified with other functional

groups. For example, PEG can be attached to the terminus of poly (amino acid)s, which helps to

prevent self-aggregation and prolongs its half-life, etc.30 Therefore, we expect that PEG-

poly(amino acid)s will be an excellent carrier for the delivery of TSIIA and will improve its anti-

tumor efficacy.

3.2 Materials and Methods

Amino acids were purchased from Purebulk and Chemimpex. Other chemicals were

purchased either from Fisher Scientific or Sigma-Aldrich, and used directly without further

purification. Tanshinone IIA (98.4%) was purchased from Xian Guanyu Pharmaceutical Co,.

Ltd, China. MHCC97-H hepatoma cells, and BALB/c male athymic nude mice from Shanghai

Cancer Institute; and CCK-8 Cell Proliferation Assay Kit was purchased from Cayman

Chemical. D-Luciferin potassium salt was purchased from Synchem-Pharma Co., Ltd.

32

BALB/c male athymic nude mice (5 weeks old, body weight 14.8 ± 0.9 g) were

purchased from the Department of Experimental Animals of Shanghai Fudan University. All

animals were housed at 22°C on a 12-h light/dark cycle. All experiments were performed

according to the recommendations of the local animal protection legislation for conducting

animal studies approved by the Ethics Committee of Shanghai University of Traditional Chinese

Medicine (SYXK 2005-0008).

CH3O-PEG5000-NH2 (0.4g, 0.08 mmol) was dissolved in 10 ml of anhydrous dioxane

and purged with N2. Separately 20 equiv. (0.42g, 1.6 mmol) of Glu (OBz)-NCA was dissolved in

5 ml anhydrous dioxane, passed through a 2 μM filter to remove any decomposed NCA,

thoroughly purged with N2 and then added via syringe. The reaction was then allowed to proceed

for 3 days under an active N2 atmosphere. Then, 10 equiv. (0.21g, 0.8 mmol) of Glu (OBz)-NCA

and 15 equiv. of Phe-NCA (0.229g, 1.2 mmol) were dissolved in 5 ml anhydrous dioxane, passed

through a 2 μM filter, purged, and added via syringe. Polymerization was allowed to continue for

another 3 days. The clear solution was then precipitated into ether and the product was collected

via filtration.

The polymer was dissolved in 10 ml of TFA and to this solution, 10 equiv. of HBr in

AcOH (33% v/v conc.) was added and stirred for 4 hours. The product was then precipitated into

ether and collected via filtration.11,12

The polymer was dissolved in minimal DMSO and added to dialysis tubing (MWCO =

3,500) followed by immersion in water. Dialysis was carried out for 3 days, replacing the water

daily. Any precipitate was then filtered out, and the clear filtrates were lyophilized to afford the

final products.

33

5 mg TSIIA and 45 mg PEG-poly (amino acid)s were dissolved in 700 μL DMSO,

followed by drop-wise addition of 30 mL 30% tert-butanol/water. The resulting solution was

stirred for 3h at room temperature, and then lyophilized to produce a solid puffy cake. The solid

was re-dissolved in 30% tert-butanol/water, stirred for 3h and lyophilized. The resulting solid

was again dissolved in pure water, stirred for 3h, and the lyophilization provided the desired

TSIIA-loading poly (amino acid)s nanoparticles.

The cytotoxicity of nanoparticles against MHCC97-H hepatoma cells was determined

using CCK-8 cell viability assay19. Briefly, MHCC97-H hepatoma cells (1×104/well) were

seeded in 96 well and incubated overnight. The cells were then incubated for 24 h and 48h at

37°C/5% CO2 using different concentrations (5, 10, 20μg/mL)of TSIIA, (50, 100,

200μg/mL)PEG-poly(amino acid)s nanoparticles, and (50, 100, 200μg/mL; containing 5, 10,

20μg/mL TSIIA)PEG-poly (amino acid)s-encapsulated TSIIA nanoparticles. 10 µl cell counting

kit-8 (CCK-8; Dojindo Molecular Techinologies, Inc, Japan) was added to each well and the

plates were incubated for an additional 1h at 37°C/5%. Finally, the cell viability was measured as

the absorbance at 450 nm (A450) using a microplate reader (Bio-Rad, Model 680, Hercules, CA,

USA). All experiments were performed in triplicate.

The following study consisted of tissue distribution, targeted imaging and therapeutic

evaluation was carried out when the tumor grew to approximately 0.1 cm3. Four to six week old

Male BALB/c nude mice were subcutaneously injected with 2x106 human liver cancer cells

MHCC97-H. After 7-10 days when implanted tumors reached 1-1.2 cm in diameter, the mice

were sacrificed under sterilized condition, the xenografic tumors were dissected out, minced into

1mm3 pieces, and were then implanted into the right flanks of nude mice. When these tumors

reached 1 cm in diameter, they were minced to 1-mm3 chunks to establish the model of the

34

orthotopic liver cancer. The procedure was first done by making a 1-cm incision along the lower

edge of the left ribcage, which exposes the left lobe of liver. Then, a space was generated

between the liver and its capsule using forceps, into which tumor chunks were inserted.

Hemostatic measures were applied during surgery and no active bleeding was detected before

closure.

To evaluate TSIIA biodistribution, the six HCC-bearing mice (three mice per group)

were injected with TSIIA and TSIIA NPs at doses equivalent to 1 mg TSIIA per kg in 0.2 ml of

PBS via the caudal vein. The heart, liver, spleen, lung, kidney, colon, and tumors were collected

24 h post-injection. Biodistribution was calculated as the percentage of injected dose (ID) per

gram of tissue (%ID/g). Values are expressed as mean ± standard deviation (n = 3).

The forty-eight mice were classified into 4 groups on the basis of the solutions they were

administered: control group (normal saline, 13.5 mL/kg), blank nanoparticles group (blank NPs,

10mg/kg), TSIIA solution group (TSIIA, 1 mg/kg), and PEG-poly (amino acid)s-encapsulated

TSIIA nanoparticles group (TSIIA NPs, 10mg/kg, containing TSIIA 1mg/kg), by injection

through the vena caudalis at 1vic/2day. Fourteen days after the injection, six mice in each group

were randomly evaluated for survival time. Life extended rate (L) was calculated as follows:

L=(STtest-STcontrol)/STcontrol×100%

where STcontrol and STtest are the average survival time (days) for the mice administered with

normal saline and with tested agents respectively. The toxicities of the NPs were evaluated by

monitoring changes in body weight. The remaining six mice in each group were killed, and the

tumors were removed for examination. The longest (a) and the longest (b) vertical dimensions of

the tumor were measured. The size (V) of the tumor was calculated using the following equation:

35

Representative formalin-fixed, paraffin-embedded tissue blocks from each tumor were analyzed

by conventional hematoxylin-erosin (HE) staining. The degree of necrosis in each tumor was

visually graded as follows: +, no necrosis present or slight necrosis in fragments; ++, mid-range

necrosis, absence of nuclei from many cells with or without massive cytoplasmic damage; and

+++, severe necrosis, total loss of cytoplasm of the cancer cells.

The in vivo optical imaging in MHCC97-H hepatoma-bearing mice was evaluated using

CRI Maestro TM In vivo Fluorescence Imaging System from Cambridge Research &

Instrumentation (Massachusetts, USA). The twenty-four mice were classified into 4 groups:

control group (normal saline, 13.5 mL/kg), blank nanoparticles group (blank NPs, 10mg/kg),

TSIIA solution group (TSIIA, 1 mg/kg), and PEG-poly (amino acid)s-encapsulated TSIIA

nanoparticles group (TSIIA NPs, 10mg/kg, containing TSIIA 1mg/kg), by injection through the

vena caudalis at 1vic/2day. Prior to in vivo imaging, the mice were anesthetized with

phenobarbital sodium. D-luciferin solution (150 mg/kg) was injected i.p 5 min. before imaging.

Exposure times of the bioluminescence imaging ranged from 10 to 30s. Fluorescence images

were obtained at an excitation wavelength of 490 nm and emission wavelength of 535 nm.

Exposure times ranged from 1 to 2 min. and the fluorescence images were captured using CRI

Maestro TM In vivo Fluorescence Imaging System in vivo optical imaging.

3.3 Results and Discussion

To test our hypothesis, we synthesized a PPAA containing both hydrophilic and

hydrophobic groups (Figure 2) via ring-opening polymerization of N-carboxyanhydrides (NCAs).

2

2abV =

36

28 In an aqueous solution, the PEG-poly (amino acid)s are expected to form amphiphilic micelles

containing a hydrophobic core and a hydrophilic corona. In our attempt, we prepared PPAAs

containing glutamate and phenylalanine residues. In brief, Glu(OBz)-NCA and Phe-NCA were

synthesized by reacting amino acids phenylalanine or H-Glu(OBz)-OH with triphosgene (Figure

3.2a). 28 Then, CH3O-PEG5000-NH2 was used to initiate the ring-opening polymerization of the

NCAs (Figure 3.2b) to produce the desired poly (amino acid)s. The core-shell nanostructures were

achieved by first adding 20 equiv. Glu(OBz)-NCA and allowing the reaction to proceed for three

days at room temperature. Subsequently, 10 equiv. Glu(OBz)-NCA and 15 equiv. Phe-NCA were

added simultaneously in order to form an anionic-hydrophobic hybrid core in solution. This hybrid

core design is different from other previously reported PEG-poly(amino acid)s di-block

copolymers, in which the core contains only exclusively hydrophobic groups such as Phe. 28 We

anticipated the hybrid core to be advantageous as it is hydrophobic enough to encapsulate TSIIA,

yet hydrophilic enough (due to the presence of anionic charges) to aid in its water solubility. This

type of core can minimize the probability of aggregation, enhance the targeted delivery of TSIIA,

and aid in its release as it is more weakly bounded to the core compared to previously reported

literature31. The sequence of the poly (amino acid)s (poly dispersity Index: 1.05 by gel permeation

chromatography) was also confirmed by NMR (Figure 3.2c).

The PPAAs were then used to encapsulate TSIIA by adopting a previously reported

protocol. 32,33 Briefly, TSIIA and PEG-poly (amino acid)s were dissolved in DMSO, followed by

drop-wise addition of 30% tert-butanol/water and shaken overnight.. The solution was lyophilized

and re-dissolved in 30% tert-butanol/water and again shaken overnight. This solution was again

freeze dried and re-dissolved in water to be shaken overnight. Lastly, the solution was lyophilized

to produce the final TSIIA-loaded nanoparticles.

37

Figure 3.2. a, synthesis of Phe-NCA and H-Glu(OBz)-NCA; b, synthesis of the PEG-poly (amino

acid)s. c, 1H-NMR (800 MHz, DMSO-d6) of the PEG-poly (amino acid)s. Amino acid residues

shown in the bracket were added in one batch.

The nanomorphology of these NPs was confirmed by SEM. As expected, irrelevant of

encapsulation of TSIIA, poly (amino acid)s all form nanoparticles in water with sizes ranging from

50 to 100 nm (Figure 3.3). Interestingly, poly (amino acid)s containing TSIIA show a smaller size

38

distribution, probably due to the strong hydrophobic interactions between TSIIA and Phe residues

in the polymer, which leads to a tighter core in the nanoparticles.

Figure 3.3 Nanomorphology of poly (amino acid)s. a, SEM image of poly (amino acid)s; b, SEM

image of TSIIA-loading poly (amino acid)s. c, Size distribution of poly (amino acid)s obtained by

dynamic light scattering.

Cytotoxicity results showed that PEG-poly (amino acid)s-encapsulated TSIIA

nanoparticles effectively increase the cytotoxicity for MHCC97-H hepatoma cells as compared

with TSIIA alone (Figure 3.4). At 24 h post-incubation, the cell viability of MHCC97-H

hepatoma cells was higher than that of 48 h post-incubation. The MHCC97-H hepatoma cells

treated with increasing doses of PEG-poly(amino acid)s nanoparticles showed 94% to 98%

39

viability, respectively. This finding suggests that the blank PEG-poly(amino acid)s nanoparticles

were non-toxic at each of the tested concentrations. Specifically, the cytotoxicity of PEG-poly

(amino acid)s-encapsulated TSIIA nanoparticles was significantly improved over time, revealing

that the PEG-poly (amino acid)s-encapsulated TSIIA nanoparticles can enhance the cytotoxicity

of TSIIA for MHCC97-H hepatoma cells. This higher cytotoxicity is very beneficial for

antitumor therapy in vivo.

Figure 3.4. In vitro cytotoxicity of nanoparticles in MHCC97-H hepatoma cells. (A) 24 h-

treatment, and (B) 48 h-treatment. *p＜0.05 when compared with TSIIA; **p＜0.01 when

compared with TSIIA.

Biodistribution of free TSIIA and PEG-poly (amino acid)s-encapsulated TSIIA

nanoparticles in HCC-bearing mice in various tissues and organs is shown in Figure 3.5. After 24

h of injection, greater concentrations of free TSIIA were found in the heart, lung and kidney than

TSIIA NPs. In tissues, TSIIA concentration 24h post-TSIIA NPs injection followed the order of

tumor > liver > kidney > lung > spleen > colon > heart, whereas tumor accumulation of free

40

TSIIA was markedly lower. For TSIIA NPs, around 16.3% ID/g (injected dose / gram) was

found in tumor at 24 h post-injection whereas for free TSIIA found was around 1.2%ID/g. This

suggests that the TSIIA NPs does show mild selectivity for the tumor over other organ systems,

but the TSIIA itself is being widely distributed. Overall, there were significant differences in the

uptake of free TSIIA and TSIIA NPs by different tissues and organs. Therefore, with TSIIA

loaded NPs, a greater amount of the drug reached the tumor site than would have if injected

without a delivery system.

Figure 3.5. Biodistribution of TanshinoneIIA NPs. Data are shown as the percentage of injected

dose per gram of organ weight (%ID/g). **P < 0.01 compared to the other groups. Data is

presented as mean ± SD (n = 3).

**

41

To assess the potential of poly (amino acid)s with encapsulated TSIIA as a novel anti-

cancer agent, its in vivo therapeutic efficacy using a hepatoma-bearing mouse model was first

evaluated, as TSIIA was previously reported to have significant activity against hepatoma. 17,34-36

First, the ability of encapsulated TSIIA to prevent the growth of hepatic tumor was evaluated.

After administration of the polymer nanoparticles, the tumor volume was measured at

predetermined intervals so as to calculate the inhibition rate. As illustrated in Figure 3.6A,

compared to the control group in which only saline was administered, poly (amino acid)s alone

(denoted as blank NPs hereafter) did not stop tumor growth. Consistent with previous findings,

TSIIA is an effective anti-tumor agent, which suppressed the growth of the hepatic tumor by 40%.

However, as seen after 4 weeks of treatment, encapsulated TSIIA (denoted as TSIIA NPs) was a

superior therapeutic agent, as it successfully arrested the growth of the hepatic tumor by 75%.

Compared to TSIIA alone, the anti-tumor efficacy of TSIIA NPs inhibited another 50% tumor

growth. The results demonstrate that the delivery of TSIIA NPs by PPAAs is an effective approach

to treat hepatoma, suggesting the potential of further development of PPAAs for anti-cancer drug

delivery.

With regard to toxic side effects, the body weight of the animals showed no significant

difference between the control and treated groups (Figure 3.6B), but the difference of tumor size

is conspicuous, suggesting that the PPAAs effectively enhanced antitumor efficiency with minimal

toxicity. However, the slight decrease in body weight in the TSIIA only group shows that without

the drug delivery platform, the drug itself is likely quite toxic.

42

Figure 3.6 Tumor inhibitory effect of TSIIA nanoparticles in vivo. (A) The tumor growth curves

(mm3), (B) body weights (g). Data is represented as the mean ± standard deviation (n = 6).

The effect on the survival time of mice with hepatoma was also determined. As shown in

Figure 3.7A, all the mice eventually died of hepatoma. Among the four test groups, TSIIA NPs

exhibited the most promising results to improve the overall survival rate, with the longest survival

time of 37.50±1.87 days (P<0.01), compared with the saline control group that had the shortest

survival time of 24.83±2.56 days. Interestingly, TSIIA alone did not substantially improve the

survival time, which may be due to the insolubility and the short half-life. Blank NPs as well only

showed a very marginal effect on the mouse survival. The results indicate that among the four

groups, TSIIA NPs show the most potent anticancer activity, which further augments its potential

as an anti-cancer nanomedicine (Figure 3.7B). Meanwhile, the results further support that PPAAs

are excellent nanocarriers for therapeutic development. The TSIIA NPs have improved survival

time by more than double compared to the TSIIA alone proving that a drug delivery vehicle is

required for proper delivery and activity.

43

Figure 3.7. (A) Kaplan-Meier overall survival analysis and (B) Life extended rate of the

MHCC97-H hepatoma-bearing mice treated with TSIIA nanoparticles. **p＜0.01vs the other

groups. Data is represented as the mean ± standard deviation (n = 6)

In addition, the degree of tumor putrescence in each group was also examined by electron

microscope with conventional HE staining as illustrated in Figure 3.8. The necrosis area in the

TSIIA NPs group was significantly greater than that of the other groups, which further

demonstrated the powerful capability of preventing the growth of the tumor. The TanshinoneIIA

group also displayed a certain extent of necrosis, which might be due to its high concentration in

solution and its intrinsic anti-tumor efficacy and cytotoxic effects.

44

Figure 3.8. Representative histopathological sections of the tumors from tumor-bearing mice (HE

Stained, magnification×200). (A) minimal necrosis after treatment with normal saline; (B)

minimal necrosis after treatment with blank nanoparticles; (C) mid-range necrosis after treatment

with free Tanshinone IIA; (D) severe necrosis after treatment with TSIIA nanoparticles.

Subcutaneous xenografting has been a widely used model to evaluate the tumor growth in

vivo. In our current report, we established an orthotopic murine liver cancer model by directly

implanting human hepatic cancer cell line MHCC97-H-Luc into the mouse liver. This model has

shown the ability to not only retain the morphology and biological characteristics of primary

human liver cancer, but also to mimic its microenvironment with the cancer cells capable of

metastasizing.37 To continuously observe and record in vivo tumor growth, we used a luciferase

labeled human hepatic cancer cell line MHCC97-H-Luc for this model. Twenty days after

implantation, when orthotopic tumors grew to an average size of 1 mm3, tumor-bearing animals

were randomly allocated into 4 different treatment/injection groups: saline control, blank NPs,

TSIIA, TSIIA NPs. Tumor size was determined on day 3, 6, 18, 24 and post-treatment. As shown

45

in Figure 3.9, on day 3, no significant difference of tumor size was observed among any

experimental group. On day 14, we found significantly smaller tumors in the mice treated with

TSIIA NPs compared to the controls (P<0.01). On day 24, tumors in the control groups grew to

considerable size and had detectable metastasis, while tumors in TSIIA treatment group were

smaller in spite of having some metastasis. In contrast, tumors were much smaller in the TSIIA-

nanoparticle group (P<0.01), and more interestingly, exhibited the smallest metastatic sites. The

results suggest that TSIIA NPs also inhibit tumor metastasis, as well as growth.

Figure 3.9. Image of hepatoma-bearing mice. The color bars (from blue to red) represent the

change of fluorescence intensity from low to high. Data is represented as the mean ± standard

deviation (n = 6)

46

3.4 Conclusion

In conclusion, we report the design and synthesis of PEG-poly (amino acid)s copolymer

nanoparticles. The nanoparticles encapsulated Tanshinone IIA and enhanced its antitumor

activity in vivo using a hepatoma-bearing mouse model. The increase in anti-tumor efficacy is

not due to the polymer nanoparticles themselves; instead, it comes from Tanshinone IIA after

encapsulated delivery. Our results suggest that PEG-poly (amino acid)s encapsulated Tanshinone

IIA are a potential novel nanomedicine for the treatment of hepatoma, and meanwhile, PEG-poly

(amino acid)s are an excellent class of nanocarriers for further development and delivery of

hydrophobic anti-cancer therapeutics.

3.5 References

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expression of TCTP, MCL-1 and Bcl-xL. Molecular Medicine Reports 2013, 7, 1045-1049.

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(18) Xu, S. W.; Liu, P. Q.: Tanshinone II-A: new perspectives for old remedies. Expert

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(19) Hatfield, M. J.; Tsurkan, L. G.; Hyatt, J. L.; Edwards, C. C.; Lemoff, A.; Jeffries,

C.; Yan, B.; Potter, P. M.: Modulation of Esterified Drug Metabolism by Tanshinones from

Salvia miltiorrhiza (“Danshen”). Journal of Natural Products 2013, 76, 36-44.

(20) Dat, N. T.; Jin, X.; Lee, J.-H.; Lee, D.; Hong, Y.-S.; Lee, K.; Kim, Y. H.; Lee, J.

J.: Abietane Diterpenes from Salvia miltiorrhiza Inhibit the Activation of Hypoxia-Inducible

Factor-1. Journal of Natural Products 2007, 70, 1093-1097.

(21) Wu, Y.-B.; Ni, Z.-Y.; Shi, Q.-W.; Dong, M.; Kiyota, H.; Gu, Y.-C.; Cong, B.:

Constituents from Salvia Species and Their Biological Activities. Chemical Reviews 2012, 112,

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(22) Liu, W.; Zhou, J.; Geng, G.; Shi, Q.; Sauriol, F.; Wu, J. H.: Antiandrogenic,

Maspin Induction, and Antiprostate Cancer Activities of Tanshinone IIA and Its Novel

Derivatives with Modification in Ring A. Journal of Medicinal Chemistry 2011, 55, 971-975.

(23) Zhang, W. L.; He, H. L.; Liu, J. P.; Wang, J.; Zhang, S. Y.; Zhang, S. S.; Wu, Z.

M.: Pharmacokinetics and atherosclerotic lesions targeting effects of tanshinone IIA discoidal

and spherical biomimetic high density lipoproteins. Biomaterials 2013, 34, 306-319.

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of Materials 2012, 24, 840-853.



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(26) Hamley, I. W.: PEG–Peptide Conjugates. Biomacromolecules 2014, 15, 1543-

1559.

(27) Matsumura, Y.: Poly (amino acid) micelle nanocarriers in preclinical and clinical

studies. Advanced drug delivery reviews 2008, 60, 899-914.

(28) Koo, A. N.; Lee, H. J.; Kim, S. E.; Chang, J. H.; Park, C.; Kim, C.; Park, J. H.;

Lee, S. C.: Disulfide-cross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-

mediated intracellular drug delivery. Chemical communications (Cambridge, England) 2008,

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acid)s nanogels as efficient intracellular drug delivery platforms. Polymer Chemistry 2011, 2,

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49

(30) Whitehead, K. A.; Langer, R.; Anderson, D. G.: Knocking down barriers:

advances in siRNA delivery. Nature Reviews Drug Discovery 2009, 8, 129-138.

(31) Lalatsa, A.; Schatzlein, A. G.; Mazza, M.; Thi, B. H. L.; Uchegbu, I. F.:

Amphiphilic poly(l-amino acids) - New materials for drug delivery. Journal of Controlled

Release 2012, 161, 523-536.

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one-step drug-loading procedure for water-soluble amphiphilic nanocarriers. Pharmaceutical

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Polymeric Nanoparticles. Pharmaceutical research 2009, 26, 1025-1058.

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Tanshinone IIA, Isolated from Salvia miltiorrhiza Bunge, Exerts Anticancer Activity Through

Inducing Apoptosis in Hepatoma Cells. American Journal of Chinese Medicine 2013, 41, 197-

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Development of intravenous lipid emulsion of tanshinone IIA and evaluation of its anti-

hepatoma activity in vitro. International Journal of Pharmaceutics 2012, 424, 76-88.

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50

CHAPTER 4: PEG-POLY (AMINO ACID)S/MICRORNA COMPLEX

NANOPARTICLES EFFECTIVELY ARREST THE GROWTH AND METASTASIS OF

HEPATOMA

Note to reader

This chapter has been submitted for publication and is currently in the peer review

process.

4.1 Introduction

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-

transcriptional gene expression.1,2 They function by specifically binding to their mRNA targets,

inhibiting protein translation, or destroying target mRNAs.3,4 MiRNAs have been found to play

key roles in various biological functions, including cell proliferation, metastasis, differentiation,

and apoptosis.5,6 The aberrant expression of miRNA is associated with a variety of diseases

including many types of cancers.7,8 Specifically, miRNAs can act as tumor suppressors or

oncogenes, which makes them promising targets for anti-cancer therapeutic development.9 In

contrast to small molecules, which generally act by hindering certain receptors, enzymes, ion

channels and metabolic pathways, interfering RNA can regulate a specific gene transcript

anywhere in the body10. This allows enormous specification and diversification when targeting

any kind of illness. Utilizing these natural processes of RNA interference, miRNAs show

considerable promise as they are quickly becoming a next generation of therapeutics for diseases

51

such as cancer11. Furthermore, since miRNAs are very small compared to a full size RNA, they

can easily be synthesized which allow them to become economically feasible for industrial

demands.

Despite the potential, there are significant challenges for the development of miRNA-

based therapeutics. One problem is the delivery of miRNAs, as they are not cell permeable and

are highly negatively charged.12-14 In addition, they also face obstacles such as short circulation

time, aggregation with serum proteins, and rapid enzymatic degradation.15,16 This elicits a need

to develop new kinds of nontoxic and biocompatible delivery systems that can further its clinical

application. In theory, a suitable carrier should protect the miRNA from enzymatic degradation,

so that it is transported it to the target of interest still intact. This should be followed by

internalization and subsequent release inside the cell so that it can intersect the mRNA at the

desired location.

As such, nanostructures such as polymeric micelles and liposomes have been widely used

as nanocarriers for targeted delivery of miRNAs to specific cells or tissues.15-18 These non-viral

transport mediums are less likely to provoke an immune response and are much less expensive to

produce compared to their viral counterparts. In order for successful delivery, the miRNA must

first form a stable complex with, and become shielded by the delivery system so as to prevent it

from degradation in the body. Both of these conditions are met by using a PEG tether as a

driving force for the collapsing of a stable nanoparticle as well as forming a protective shield

before reaching the target. Next, the nanocarrier must be internalized via endocytosis, which is

typically accomplished nonspecifically19. Finally, the cargo or miRNA must be released from the

nanocarrier before being degraded. Due to the less hydrophobic nature of our hybrid core, we

52

anticipate that our miRNA should be sufficiently released faster than endosomal enzymes can

degrade the miRNA.

To date, the most widely used delivery systems for miRNA are cationic lipid based

nanoparticles, many of which also use PEG as a shield and stabilizer20,21. More specifically,

liposomes have even shown the ability to directly target the hepatocytes for miRNA delivery10.

For this general purpose however, liposomes have many intrinsic drawbacks such as the

premature release of the cargo, rapid clearance from the blood and uptake by macrophages.22

Other popular approaches are cationic polymeric based particles such as poly(2-

(dimethylamino)ethyl methacrylate23 which has been used to form a complex with DNA. Here,

PEG was also used as a hydrator as well as to ameliorate the electrostatic interactions so as to

allow decomplexation inside the cell more readily. The polymeric nature of the nanoparticle

allows for greater stability compared to a lipid based liposome counterpart. However, it is

important to note that both liposome and polymeric based nanoparticles are produced from self-

assembly in solution, and as so, they are not exceptionally stable compared to a much more

robust delivery system such as an inorganic gold nanoparticle24. However, particles such as these

are inherently more toxic and less biodegradable and so; researches must fine tune their delivery

system to have a well-balanced profile of toxicity, stability, and biodegradability.

Among the different nanocarriers developed for drug delivery, poly (amino acid)s have

many unique advantages. Poly (amino acid)s are polypeptides that can be synthesized

conveniently using the ring-opening polymerization of N-carboxyanhydrides (NCAs).25 Since

NCAs can be easily synthesized on a large scale from natural amino acids, poly (amino acid)s

can be tuned straightforwardly with diverse functional groups and charges. Due to their intrinsic

low toxicity and full biodegradability, they have been widely used for drug delivery.26,27,28

53

Furthermore, the addition of a PEG segment to polymers has been shown to mitigate the problem

of non-specific interactions between cationic nanoparticle/RNA complexes and negatively

charged serum proteins due to its antifouling properties29, prevent self-aggregation, and evade

the immune system.15 However, to the best of our knowledge, the use of PEGylated poly (amino

acid)s for the delivery of miRNAs is not yet reported. Herein, we report the development of

PEG-poly (amino acid)s as a novel carrier for the delivery of miRNA-139. Low expression of

miR-139 has been linked to a few cancers including hepatocellular carcinoma, colorectal cancer,

leukemia, glioblastoma, etc.30-34 Up regulation of miRNA-139 has been shown to suppress tumor

growth, migration and metastasis by targeting the 3’-untranslated region of some oncogenic

mRNAs such as Rho-kinase 2, 32 insulin-like growth factor receptor 31 and MCl-1 mRNA.34 As

such, systemic delivery of miRNA-139 is expected to lead to novel anti-cancer therapeutics. In

this report, we show that the positively charged poly (amino acid)s with a PEG tail can form

defined nanoparticles in aqueous solution. Such nanoparticles can be used to form a complex

with the negatively charged miRNA-139. These PEG-poly (amino acid)s/miRNA-139 complex

nanoparticles (denoted as miR-139 NPs hereafter for simplification) effectively suppress the

growth of a hepatoma tumor in MHCC97-H hepatoma-bearing mice by significantly reducing

85% of the tumor size in four weeks, while subsequently also doubling their lifespan. In addition,

these nanoparticles have also been shown to prevent hepatic tumor cell migration and metastasis.

Our results suggest that PEG-poly (amino acid)s are promising non-viral gene vectors for the

delivery of miRNAs for the treatment of cancers.

54

4.2 MATERIALS AND METHODS

Amino acids used for the synthesis of poly (amino acid)s were purchased from purebulk

inc. The miRNA mimics and miRNA control were purchased from Biomics Biotechnologies

(Nantong, China). MHCC97-H hepatoma cells, and BALB/c male athymic nude mice from

Shanghai Cancer Institute; and CCK-8 Cell Proliferation Assay Kit was purchased from Cayman

Chemical. D-Luciferin potassium salt was purchased from Synchem-Pharma Co., Ltd.

CH3O-PEG2000-NH2 was dissolved in 10 ml of anhydrous dioxane and purged with N2.

Separately 20 eq of Z-lys-NCA was dissolved in 5 ml anhydrous dioxane, passed through a 2 µM

filter to remove any decomposed NCA, thoroughly purged with N2 and then added via syringe.

The reaction was then allowed to proceed for 3 days under an active N2 atmosphere. Then 10

equiv. of Z-Lys-NCA and 15 equiv. of Phe-NCA were dissolved in 5 ml anhydrous dioxane,

passed through a 2 μM filter, purged, and added via syringe. Polymerization was allowed to

continue for another 3 days. The clear solution was then precipitated into ether and the product

was collected via filtration.

The polymer was dissolved in 10 ml of TFA and to this solution, 10 equiv. of HBr in

AcOH (33% v/v conc.) was added and stirred for 4 hours. Product was then precipitated into

ether and collected via filtration.

The polymer was dissolved in minimal DMSO and added to dialysis tubing (MWCO =

3,500) followed by immersion in water. Dialysis was carried out for 3 days and the water was

replaced daily. Any precipitate was then filtered out, and the clear filtrates were lyophilized to

afford the final products.

55

To form the PEG-poly(amino acid)s/miR nanoparticle complex, 1 mg miRNA-139 and

15 mg PEG-poly (amino acid)s were dissolved in 200 μL DMSO, followed by drop-wise

addition of 10 mL water. The resulting solution was stirred for 3h at room temperature, and the

lyophilization provided the desired product.

The following targeted study consisting of tissue distribution, targeted imaging and

therapeutic evaluation was carried out when the tumor grew to approximately 0.1 cm3. Male

BALB/c nude mice, 4-6 weeks old, were subcutaneously injected with 2x106 human liver cancer

cells MHCC97-H. After 7 -10 days, when implanted tumors reached 1-1.2 cm in diameter, the

mice were sacrificed under sterilized condition, the xenographic tumors were dissected out,

minced into 1mm3 pieces, which were then implanted into the right flanks of nude mice. When

these tumors reached 1 cm in diameter, they were minced to 1-mm3 chunks for establishing the

orthotopic liver cancer model. The procedure was first done by making a 1-cm incision along

the lower edge of left ribcage, to expose the left lobe of liver. Then, a space was generated

between the liver and its capsule using forceps, into which tumor chunks were inserted.

Hemostatic measures were applied during surgery and no active bleeding was detected before

closing abdominal incision. No fasting was applied to the animals.

The mice were classified into 4 groups on the basis of the solutions they were

administered: control group(normal saline, 13.5 mL/kg), blank NPs(10mg/kg) group, miR-

control NPs(10mg/kg) group, miR-139 NPs (10mg/kg) group, by injection through the vena

caudalis at 1vic/2day. 7 days after the injection, six mice in each group were chosen randomly

for evaluation of survival time. Survival rate (SR) was calculated as follows:

56

where STcontrol and STtest are the average survival time (days) for the mice administered with

normal saline and with test agent respectively. The toxicities of the NPs were evaluated by

monitoring changes in body weight.

The remaining mice in each group were killed, and the tumors were removed for

examination. The longest (a) and the longest (b) vertical dimensions of the tumor were

measured. The size (V) of the tumor was calculated using the following equation:

The in vivo optical imaging of 4 groups in MHCC97-H hepatoma-bearing mice were

evaluated using CRI Maestro TM In vivo Fluorescence Imaging System from Cambridge

Research & Instrumentation (Massachusetts, USA).

Prior to in vivo imaging, the mice were anesthetized with Phenobarbital sodium. D-

luciferin solution (150 mg/kg) was injected i.p 5 min before imaging. Exposure times of

bioluminescence imaging ranged from 10 to 30 s. Fluorescence imaging was obtained with an

excitation wavelength of 490 nm and emission wavelength of 535 nm. Exposure times ranged

from 1 to 2 min. and the fluorescence images were captured using CRI Maestro TM In vivo

Fluorescence Imaging System In vivo optical imaging

Tissue sections of 5-μm in thickness were cut from formalin fixed and paraffin embedded

tissue blocks. After deparaffinization and rehydration, endogenous peroxidases were quenched in

0.3% H202 diluted in methanol. Tissue sections were then incubated overnight at room

temperature (RT) with mouse anti human CD31 antibody(1:400; Dako, Glostrup, Denmark).

100STtest STcontrol

SRSTcontrol

−= ×

2

2abV =

57

Immunodetection was performed incubating the slide with a goat anti-mouse antibody and

horseradish peroxidase (HRP)-streptavidin complex (both Dako) for 30 min at RT. Staining was

visualized using 0.05% 3,3’-diaminobenzidine containing 0.0038% H202. Representative

micrographs were taken under an Olympus BX-51TF microscope equipped with a DP23-3-5

camera. The CD31 positive microvessels in the tumor-bearing area were quantified by

computerized image analysis. Four representative areas in tumor for CD31 staining were selected

and photographed at 200 × magnification.

For cell motility assay, 8 × 105 cells were seeded onto 60-mm dishes and grown for 24 h.

A linear wound was made by scraping a pipette tip across the confluent cell monolayer. Cells

were rinsed with PBS and grown in 1640 supplemented with 10% FBS for additional 48 h. The

cell motility in terms of wound closure was measured by photographing at three random fields at

the time of wounding (time 0) and at 12, 24 and 48 h after wounding. 38

4.3 Results and Discussion

To test our hypothesis, we synthesized a PEGylated poly (amino acid)s containing both

cationic and hydrophobic groups (Figure 4.1) via ring-opening polymerization of N-

carboxyanhydrides (NCAs). 25 The synthetic procedure was adapted from a previously published

protocol.27 Briefly, Z-Lys-NCA and Phe-NCA were first synthesized through the treatment of

amino acids phenylalanine or Z-Lysine with triphosgene, respectively (Figure 4.1a). Next, CH3O-

PEG2000-NH2 was used as the initiator to react with both NCAs to produce poly (amino acid)s

via the ring-opening polymerization (Figure 4.1b). In order to generate defined core-shell

nanoparticles, 20 equiv. Z-Lysine-NCA was first added into the flask containing CH3O-PEG-NH2

and the reaction was allowed to proceed for three days at the room temperature. Subsequently, 10

58

equiv. Z-Lysine-NCA and 15 equiv. Phe-NCA were then added in one batch to form the cationic-

hydrophobic hybrid core of nanoparticles in solution. We hypothesized that such a hybrid core

would be superior to a pure hydrophobic core consisting of phenylalanines because the core is still

positively charged, which increases the solubility as well as the electrostatic attraction with

negatively charged miRNAs, but is still hydrophobic enough to cause the self-assembly into

nanostructures. The NMR of the poly (amino acid)s is shown in Figure 1c.

ONH Dioxane

N2, r.t., 3d

O

HN

NH

O

NHO

O

1. TFAHBr / AcOH

10 equiv. Z-Lys-NCA+ 15 equiv. Phe-NCA

45

H

20

20 equiv Z-Lys-NCA

DioxaneN2, r.t., 3d

2. Dialysis

O

HN

NH

O

NH2

4520

HNO

O

O

R

Z-Lys-NCA

HN

NH

O

NH2

H

15

H2N OH

O

R

R =

NH

O

O

or

Phe-NCA

a

b

45

Triphosgene

10

H

O

59

Figure 4.1. a, synthesis of Phe-NCA and Z-Lys-NCA; b, synthesis of PEG-poly (amino acid)s. c, 1H-NMR (800 MHz, DMSO-d6) of the PEG-poly (amino acid)s. Amino acid residues shown in

the bracket were added in one batch, and the residues were randomly mixed.

The PEG-poly (amino acid)s/miRNA-139 complex nanoparticles (miRNA-139 NPs) were

then prepared by adapting a previously reported protocol. 35,36 Briefly, 1 mg miRNA-139 and 15

mg PEG-poly (amino acid)s were dissolved in DMSO, followed by slow addition of water. The

nanoparticles (NPs) were obtained after lyophilization. Meanwhile, miR-67 (miR-control), a

miRNA that has a minimum sequence similarity to miRNAs in human and mouse, 37 was included

as a negative control.

The nanomorphology of these NPs was confirmed by SEM. The PEG-poly (amino acid)s

and their complexes with miRNAs form nanoparticles with sizes ranging from 50 to 200 nm

(Figure 4.2). PEG-poly (amino acid)s nanoparticles themselves tend to aggregate together (Figure

4.2a); however, after forming complexes with miRNAs, the nanoparticles are more defined and

60

segregated (Figure 4.2b and 4.2c). It is possible that the presence of miRNAs close to surface of

nanoparticles lead to electrostatic repulsion among nanoparticles.

Figure 4.2. SEM pictures of prepared nanoparticles. a, PEG-poly(amino acid)s (denoted as blank

NPs thereafter); b, miR-139 NPs; c, miR-control NPs. Bar = 50 nm.

To evaluate the in vivo anti-tumor effect of miR-139 NPs, we tested these particles in a

mouse xenograftic model of hepatoma cell line MHCC97-H. After four weeks, in comparison to

the control groups (only saline, miR-139, blank NPs, or miR-control NPs), the miR-139 NPs

treatment showed the most significant inhibition of the tumor growth, with treated tumors being

1/5 the average size of the ones in the control groups (P＜0.05) (Figure 4.3). Our result further

indicated that the repressive effect on the tumor growth by miR-139 NPs was very specific, since

blank NPs or scramble miR-control NPs did not exhibit any inhibitory effect on tumors.

Although as anticipated, miR-139 alone did exhibit the ability to block the tumor growth, and the

efficacy is much less than that of miR-139 NPs. These results clearly demonstrate that the novel

miR-139 NPs are effective anti-tumor agents. It also indicates that PEG-poly(amino acid)s are

very effective nanocarriers for the delivery and the in vivo efficacy of miRNAs.

61

Figure 4.3. The tumor growth curves (mm3). Data is represented as the mean ± standard

deviation (n = 6).

The effect on the survival time of mice with hepatoma was also determined (Figure 4.4).

Although all the mice eventually died of hepatoma, administration of miR-139 NPs had greatly

improved the life-span of mice bearing hepatoma. As seen in Figure 4.4, among the tested

agents, the miR-139 NPs group showed that it had the most promising prospect to improve the

survival condition, coupled with the longest survival time of 50.33 ± 3.08 days (P<0.01),

compared with the saline control group with the shortest survival time of 25.50 ± 1.87 days.

Neither NPs alone nor miR-control NPs had virtually any effect in the improvement of the

survival time. Although as expected, miR-139 displayed certain antitumor efficacy, but it is far

inferior to that of miR-139 NPs. Overall, the results demonstrate that the complexation of PEG-

poly (amino acid)s with miRNAs greatly enhances the therapeutic efficacy of miRNAs.

62

Figure 4.4 Survival time (day) of hepatoma-bearing mice in different groups. Data is

represented as the mean ± standard deviation (n =10).

Subcutaneous xenograft has been a widely used in vivo tumor growth model of cancer

cells, but in many ways, has failed to retain and recapitulate characteristics of the primary

tumors. In our current report, we established an orthotopic murine liver cancer model by directly

implanting human hepatic cancer cell line, MHCC97-H, into a mouse liver. Not only does this

model retains the morphology and biological characteristics of a human liver cancer, but it also

mimics the microenvironment with which the cancer cells are capable of metastasizing in. To

continuously observe and record in vivo tumor growth, we used a luciferase labeled MHCC97-H

human hepatic cancer cell line and renamed it “MHCC97-H-Luc”. Twenty days after

implantation, when orthotopic liver tumors grew to an average size of 1 mm3, tumor-bearing

animals were randomly allocated into 5 different treatment/injection groups similar to Figure 4.4.

Tumor size was determined on day 7, 14, 21, and 28 post-treatment. As shown in Figure 4.5, on

63

day 7, no significant difference of tumor size was observed among mice, treated vs. controls. At

day 14 and beyond, significantly smaller tumors were observed in miR-139-NPs treated mice

compared to the controls (P<0.01). On day 28, tumors in saline, blank NPs and miR-control NPs

group grew to such a considerable size that they nearly took over the whole livers, and showed

detectable metastasis. Mice in miR-139 had smaller tumor sizes, however, the difference was

marginal compared to other control groups. In contrast, tumors were much smaller in miR-139

NPs treatment group (P<0.01), and more interestingly, no metastatic sites were observed.

Figure 4.5. Image of orthotropic hepatoma-bearing mice. The color bars (from blue to red)

represent the change of fluorescence intensity from low to high.

Since the loss of miR-139 was closely associated with aggressive pathologic features such

as metastasis in primary human hepatoma cases, we hypothesized that miR-139 NPs could

64

impede the migratory and invasive capabilities of hepatoma cells. As such, we also evaluated the

effect of miR-139 NPs on tumor angiogenesis. First, by immunohistochemistry staining, we

showed that the expression of CD31, a blood vessel endothelial cell marker, was present in the

control xenograft tumors, whereas its expression was weak or negative in the tumor treated with

miR-139 NPs (Figure 4.6). Semi-quantitative analysis of the microvessel area revealed that the

average MVD (microvessel density) value in miR-139 NPs treated tumor was significantly lower

than that of the other control groups. (p< 0.01).

Figure 4.6. The immunohistochemical staining of CD31 antigen showed that there were

micro-vessels around tumor cells (the brown parts). A, control group (saline, 13.5 mL/kg); B,

blank NPs (10mg/kg) group; C, miR-control NPs (10mg/kg) group; D, miR-139

(0.5mg/kg)group; E, miR-139 NPs (10mg/kg)group. Quantitative analysis was performed by

counting the number of tumor foci in 10 randomly selected high-power fields under microscope

(×200).

Next, we assessed the anti-metastasis effect of miR-139 NPs on MHCC97-H cells in vitro

by a wound healing assay. As shown in Figure 4.7, miR-139 NPs remarkably attenuated the

migratory ability of MHCC97-H hepatoma cells, as indicated by the decrease in the numbers of

65

migrated cells, compared to the controls. These results are consistent with the previous findings

in that miR-139 NPs played a significant role in hepatocarcinoma cell migration. 32

Figure 4.7. Wound closure was delayed in miR-139 nanoparticles transfected cells in

12h,24h and 36h time points in MHCC97-H cells

4.4 Conclusion

In conclusion, we designed and synthesized PEGylated poly (amino acid) nanoparticles,

for the purpose of using them as nanocarriers to form a miR-139 NP complex. Although the use

of nanoparticles for microRNA delivery is common, the employment of PEG-poly (amino acid)s

for such a purpose is rare. We believed that PEG-poly (amino acid)s would be versatile carriers

66

because they are amendable to further modification on their functional groups. Meanwhile, the

presence of a PEG tail is expected to minimize side reactions and to increase the bioavailability

of microRNAs. Indeed, miR-139 complex nanoparticles effectively suppress tumor growth in a

traditional xenograftic model, as well as in a newly established orthotopic liver cancer model of

human hepatoma cell line MHCC97-H, and displays significant inhibitory impacts on cancer cell

migration and metastasis. Our results suggest that PEG-poly (amino acid)s are potent non-viral

gene vectors for the delivery of therapeutic miRNAs for the treatment of cancers.

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70

CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS

In this dissertation, we have explored mPEG poly amino (acid)s as broad spectrum

antibacterial polymers as well as versatile drug delivery polymers. In this chapter, we want to

summarize our results and provide some perspective for future directions.

We have developed polymer micelles based on the ring opening polymerization of N-

carboxy anhydrides by mPEG-NH2. By using natural L-amino acids, our material is fully

biodegradable and biocompatible which makes it very suitable to be used in vivo. While here we

have only used three representative amino acids based on the cationic side chain of lysine,

anionic side chain of glutamic acid, and hydrophobic side chain of phenylalanine, in theory, any

natural amino acid can be used as long as the side chain is protected during the polymerization.

With 20 natural amino acids, and hundreds more unnatural amino acids being used in research,

this leads the way to develop new types of polymers with enormous diversity. Furthermore,

instead of using PEG as an initiator, any other polymer containing a nucleophilic primary amine

group can be used as an initiator to encourage even more diversity. Together, this allows one to

attach a different tether, use different equivalents of monomers to control the polymer size, and

use different amino acids which provide the polymer with different kinds of functionalization to

be used in post polymerization modification.

Our first application with our polymer micelles utilizes the differences in electrostatics

between bacterial membranes and eukaryotic membranes. Due to bacterial membranes having an

overall more net negative charge on their outer membrane compared to eukaryotic membranes

71

which are overall more neutral, this has allowed us to develop cationic polymer micelles that

selectively interact, and subsequently disrupt bacterial membranes more readily. The extent of

hemolysis against erythrocytes can be tuned by lowering the amount of hydrophobic content in

the polymers, but this also lessens its membrane disrupting ability. Also, our polymers allow for

the further functionalization to increase the antibacterial activity due to its reactive amine groups

that are still present. One could imagine attaching specific antibiotics or membrane targeting

ligands to the polymer in an effort to synergistically kill the microbes with greater activity. As

per our other two applications, antibacterial drugs could also be encapsulated by our polymers

for a more systemic effect.

Next, we have shown the ability of a negatively charged polymer to encapsulate a super

hydrophobic drug Tanshinone IIA and be delivered to a live liver cancer bearing mouse.

Polymeric micelles such as ours are gaining traction in the field of drug delivery due to their

small size, relatively narrow distribution of particles, ability to encapsulate hydrophobic drugs,

and their ability to prolong the circulation time of the drug. There were no additional side effects

noticed from our delivery method, and we observed a marked decrease in tumor size which

exemplifies our delivery method as safe and effective. Furthermore, the nanoparticles formed are

all less than 200 nanometers, which is thought of being the maximum size that a particle can be

in order to cross the blood brain barrier should one try and use this delivery system to target

brain chemistry. While unnecessary in this application, one could imagine loading a different

kind of novel therapeutic into our polymer with the end goal of delivering the cargo through the

blood brain barrier.

Lastly, we have used our system to show a proof of concept in delivering a negatively

charged miRNA with our cationic based polymer. Again, no significant side effects were

72

observed and the tumor size was drastically reduced, which shows the versatility in our

nanoparticles to deliver various kinds of cargo. However, the major limitation of our design is

that it lacks targeting efficiency and the cargo is not being delivered directly at the site of

interest. This drawback has spawned the latest research in our lab in which we are currently

testing our nanoparticles with Tanshinone IIA, but this time with FTIC as a fluorescent labeling

polymer, as well as with RGD as a targeting peptide. This is made possible by the many

available functional groups that the polymer still possesses. At the time of this writing, the

results of our site specific targeting and labeling have not yet arrived, and therefore are not

present in this dissertation. However, we do expect that with targeting peptides such as RGD, our

efficiency to deliver site specific cargo will increase.

Finally, we would like to conclude by stating that our research has further validated the

use of mPEG poly amino (acid)s in being able to exhibit broad spectrum antibacterial activity in

addition to acting as a versatile drug delivery nanoparticle. We anticipate that in research to

come, this type of methodology will become more site specific as in our targeting approach, and

that new types of functionalities will allow this class of materials to achieve its full potential in

becoming a new generation of safe and effective polymer biomaterials.

73

APPENDIX A

Supporting info for: Investigation of antimicrobial PEG-poly(amino acid)s

A1. Synthesis of PEG-poly(amino acid)s

Figure A1a. Synthesis of N-carboxyanhydrides (NCAs):1

HNO

O

O

R

H2N

O

OH

R

R =

HN

O

O

O

O

Typically, to a round bottom flask, 5 g of the amino acid was suspended in approximately 130 mL

of anhydrous THF and heated to 60°C. Separately, 0.34 equivalents of triphosgene was dissolved

in 20 mL of anhydrous THF and added dropwise to the solution under an active N2 atmosphere

(final concentration ~0.15 M). After the solution turned clear in ~4 h, any insoluble precipitates

were filtered off. The solution was concentrated under vacuum and crystallized from THF/hexane.

Three to four subsequent recrystallizations were performed until pure white compounds were

obtained.

74

A1b. Synthesis of PEG-OTs:

OO

H

a

OO

a

S

O

O

a = 45 or 113TsCl

DCM

Typically, to a solution of 10 g of mPEG (mw = 2000 or 5000) in 50 mL of DCM, 0.416

ml of TEA was added and stirred for 10 min. Then 3.8 g of p-toluensulfonyl chloride (10 eq.)

dissolved in DCM was added and stirred for 20 h at room temperature. The solution was washed

with citric acid to neutralize TEA and then dried over NaSO4. The solvent was removed and

product was precipitated into ether.

A1c. Synthesis of PEG-N3:

OO

a

S

O

O

ON3

aa = 45 or 113

NaN3

DMF

5 g of mPEG-OTs was dissolved in 40 ml of DMF and 1.89 g (30 eq.) of NaN3 was

added. The reaction was heated to 90°C for 20 h. Then 200 ml of brine was added and extracted

into DCM. The extracts were concentrated and then precipitated into ether.

A1d. Synthesis PEG-NH2:

Excess water/brine was used to move all salts into the water layer along with DMF. The

DCM extracts were dried over NaSO4, concentrated under reduced pressure and precipitated into

ether.

75

ON3

a

ONH

H

a

a = 45 or 113

THF

LAH

2 g of PEG-N3 was dissolved in 50 ml of THF (minimal) and 200 mg of LAH (2 eq.) was

added at 0 °C. The reaction was allowed to reach room temperature and was stirred overnight.

Then 5 eq of water was added from a 4M NaOH solution and stirred until all the LiOH and Al(OH)3

salts have precipitated out as a white solid. The salts were filtered off, and the solvent was removed

to be re-dissolved in DCM. It was then washed with water/brine to remove any excess salts. DCM

layers were collected, dried over NaSO4, and precipitated into ether.

ONH Dioxane

N2, r.t., 3d

O

HN

NH

O

NHO

O

1. TFAHBr / AcOH

c equiv. Z-Lys NCA+ d equiv. Phe NCA

a

H

b

b equiv Z-Lys NCA

DioxaneN2, r.t., 3d

2. Dialysis O

HN

NH

O

NH2

ab

HNO

O

O

R

NCA

H

a

HN

NH2

O

NH

Od

Hc

76

A1e. Synthesis of polymer P3

(The other polymers were prepared by a similar method). 0.4 g of PEG-NH2 was dissolved

in 10 ml of anhydrous dioxane and purged with N2. Separately 0.49 g (20 eq) of Z-lysine NCA

was dissolved in 5 ml anhydrous dioxane, passed through a 2 micron filter to remove any

decomposed NCA, thoroughly purged with N2 and then added via syringe. The reaction was then

allowed to proceed for 3 days under an active N2 atmosphere. Then 10 equivalents of Z-Lysine

NCA and 15 equivalents of Phenylalanine NCA were dissolved in 5 ml anhydrous dioxane, passed

through a 2 micron filter, purged, and added via syringe. Polymerization was allowed to continue

for another 3 days. The clear solution was then precipitated into ether and the product was collected

via filtration.

A1f. Removal of Z protecting groups:

Polymers were dissolved in 10 ml of TFA and to this solution, 10 equivalents of HBr in

AcOH (33% v/v conc.) was added and stirred for 4 hours. Product was then precipitated into

ether and collected via filtration.

A1g. Purification

The polymers were dissolved in minimal DMSO and added to dialysis tubing (MWCO =

3,500) followed by immersion in water. Dialysis was carried out for 3 days replacing the water

daily. Any precipitate was then filtered out, and the clear filtrates were lyophilized to afford the

final products.

77

Table A1. Molecular Weights of the polymers

Polymer # P1 P2 P3 P4 P5 P6 P7 P8 P9 P10

MW (calculated) 10880 9030 11045 7750 4750 5485 7500 8780 4560 3920

A2. The morphology of PEG-poly(amino acid)s in water.

Figure A2. SEM image showing the morphology of PEG-poly(amino acid)s

A3. Antimicrobial activity

The bacterial strains used for testing the efficacy of polymers were multi-drug resistant S.

epidermidis (RP62A), B. subtilis (BR151), K. pnuemoniae (ATCC 13383) and multi-drug resistant

P. aeruginosa (ATCC 27853). The antimicrobial activities of the polymers developed were

78

determined in sterile 96-well plates by serial dilution method. Bacterial cells were grown overnight

at 37 °C in 5 mL medium after which a bacterial suspension of approximately 106 CFU/mL in

Luria broth or trypticase soy was prepared ensuring that the bacterial cells were in the mid-

logarithmic phase. Aliquots of 50 µL bacterial suspension were added to 50 µL of medium

containing different concentrations of polymers for a total volume of 100µL in each well. The 96-

well plates were incubated at 37 0C for about 20 h. The Biotek microplate reader was used to

measure the optical density (OD) at a wavelength of 600 nm after about 20 h. The experiments

were carried out as three independent biological replicates, each in duplicate. The lowest

concentration at which complete inhibition of bacterial growth is observed is defined as the

minimum inhibitory concentration (MIC).

A4. Hemolytic activity

Freshly drawn human red blood cells (hRBC’s) were used for the assay. The blood sample

was washed with PBS buffer several times and centrifuged at 700g for 10 min until a clear

supernatant was observed. The hRBC’s were re-suspended in 1X PBS to get a 5% v/v suspension

which was used to perform the assay. 50 µL of different polymers solutions were added to sterile

96-well plates. Then 50 µL of 5% v/v hRBC solution was added to make up a total volume of 100

µL in each well. The 0% hemolysis point and 100% hemolysis point were determined in 1 X PBS

and 0.2% Triton-X-100 respectively. The 96 well plate was incubated at 37 °C for 1h and

centrifuged at 3500 rpm for 10 min. The supernatant (30 µL) was then diluted with 100 µL of

1XPBS and hemoglobin was detected by measuring the optical density at 360nm by Biotek

microtiter plate reader (Type: Synergy HT).

% hemolysis = (Abs sample -Abs PBS )/(Abs Triton

–Abs PBS ) x 100

79

A5. Fluorescence microscopy

A double staining method with DAPI (4’,6-diamidino-2-phenylindole dihydrochloride,

Sigma, >98%) and PI (propidium iodide, Sigma) as fluorophores was used to visualize and

differentiate the viable from the dead B. subtilis cells. DAPI being a double stranded DNA binding

dye, stains all bacterial cells irrespective of their viability. Ethidium derivatives such as propidium

iodide (PI) is capable of passing through only damaged cell membranes and intercalates with the

nucleic acids of injured and dead cells to form a bright red fluorescent complex. The bacterial cells

were first stained with PI and then with DAPI. The bacterial cells were grown until they reached

mid-logarithmic phase and then ~ 2x10 3 cells were incubated with 100 µg/mL polymer P5 for 4

h. Then the cells were pelleted by centrifugation at 3000 g for 15 min in an Eppendorf

microcentrifuge. The supernatant was decanted and the cells were washed with 1X PBS several

times and then incubated with PI (5 µg/mL) in the dark for 15 min at 0 oC .The excessive PI was

removed by washing the cells several times with 1X PBS several times. Lastly, the cells were

incubated with DAPI (10 µg/mL in water) for 15 min in the dark at 0oC. Then finally the excessive

DAPI solution was removed by washing it with 1X PBS. The controls were performed following

the exact same procedure for bacteria without P5. The bacteria were then examined by using the

Zeiss Axio Imager Z1optical microscope with an oil-immersion objective (100X). 2,3

A6. SEM of bacteria after treatment with polymer P5.

MRSE and B.subtilis were grown to an exponential phase and approximately about 2 x 106

cells were incubated with polymer P5 for about 20 h. The cells were then harvested by

centrifugation (3000 g) for 15 min. After pelleting the cells, the cells were washed three times with

80

DI water. The cells were then fixed with 2.5% (w/v) glutaraldeyhyde in nanopure water for about

30 min, followed by extensive wash with DI water to get rid of any excess glutaralhedyde. Graded

ethanol series (30%, 50%, 70%, 95% and 100%, 5 min each) was then used to dehydrate the cells.

Following the dehydration of cells hexamethyldisilazane was added for about 2 min. Then about

2 µL of sample was added to a silicon wafer followed by Au/Pd coating, and then the samples

were observed at 25KV with a HITACHI S-800 scanning electron microscope.

A7. References



mediated intracellular drug delivery. Chemical communications (Cambridge, England) 2008,

6570-2.

(2) Niu, Y.; Padhee, S.; Wu, H.; Bai, G.; Harrington, L.; Burda, W. N.; Shaw, L. N.;

Cao, C.; Cai, J.: Identification of gamma-AApeptides with potent and broad-spectrum

antimicrobial activity. Chemical communications (Cambridge, England) 2011, 47, 12197-12199.

(3) Niu, Y.; Padhee, S.; Wu, H.; Bai, G.; Qiao, Q.; Hu, Y.; Harrington, L.; Burda, W.

N.; Shaw, L. N.; Cao, C.; Cai, J.: Lipo-gamma-AApeptides as a New Class of Potent and Broad-

Spectrum Antimicrobial Agents. J Med Chem 2012, 55, 4003-4009.

81

O

HN

NH

HN

NH2

O

O

O

NH2

a b cd

NH2

A

B

B

B

CC

CD

E

E

F

G

G

Figure A3: NMR of Polymer P2

1H-NMR (DMSO-d6, 800 MHz)

82

O

HN

NH

HN

NH2

O

O

O

NH2

a b cd

NH2

A

B

B

B

CC

CD

E

E

F

G

G



83

O

NH

HN

NH2

O

O

NH2

a cd

A

B

B

CC

E

F

G

D



84

O

NH

HN

NH2

O

O

NH2

a cd

A

B

B

CC

E

F

G

D



85

O

NH

HN

NH2

O

O

NH2

a cd

A

B

B

CC

E

F

G

D



86

O

NH

HN

NH2

O

O

NH2

a cd

A

B

B

CC

E

F

G

D



87

O

NH

HN

NH2

O

O

NH2

a cd

A

B

B

CC

E

F

G

D



88

Oa

HN

O

NH2

NH2b

B

CD

E

G



89

Oa

HN

O

NH2

NH2b

B

CD

E

G



design, synthesis and applications of polymer biomaterials

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