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Graduate Theses and Dissertations Graduate School

March 2018

Design, Synthesis, Application of BiodegradablePolymersMussie GideUniversity of South Florida, mussie@mail.usf.edu

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Scholar Commons CitationGide, Mussie, "Design, Synthesis, Application of Biodegradable Polymers" (2018). Graduate Theses and Dissertations.https://scholarcommons.usf.edu/etd/7625

Design, Synthesis, Application of Biodegradable Polymers

by

Mussie Gide

A thesis submitted in partial fulfillment

of the requirements for the degree of

Master of Science

Department of Chemistry

College of Arts and Sciences

University of South Florida

Major Professor: Jianfeng Cai, Ph.D.

James Leahy, Ph.D.

Shengqian Ma, Ph.D.

Chen Yu, Ph.D.

Date of Approval:

March 20, 2018

Keywords: Antimicrobial polymers, Dendrimers, Amphiphilic, Unimolecular micelles, Host defense

Peptide

Copyright © 2018, Mussie Gide

DEDICATION

To my parents, for their enormous support. To Selam Hagos for her amazing heart. To my

brother Samuel Gide and my uncle George Ghide for their everlasting encouragement. To Efrem

Moconnen and Sami Abdelwahab, for their indispensable friendship. To everyone who was ever

there for me when I needed them.

ACKNOWLEDGEMENTS

I would like to thank my research advisor, Dr. Jianfeng Cai, for the support and guidance

he has given during my academic study at USF. I would like to thank him for his guidance, endless

support and patience. It is very rare to find an advisor who always have time for their students to

listen to their little problems, I feel I am fortunate and I thank my advisor for always finding time

and encouraging to overcome the barriers during my research work.

I would like to thank my thesis committee members, Dr. James Leahy, Dr. Shengqian Ma,

Dr. Chen Yu, for their extended support, advice and encouragement in accomplishing my Master’s

degree at USF.

I want to express my honest thanks to every member in the Cai’s group. I enjoyed every

moment in my lab. Dr. Alekhya Nimmagadda mentored me to understand and apply all the basic

principles of polymer synthesis and all related staffs of my research. Especial thanks to Dr. Yan

Shi and Dr. Teng Peng for their motivations and help in all my research progress. I am thankful to

Tim, Annemarie and Sylvia for their efforts during my thesis writing. I also enjoyed time with

other lab members, Olapeju, Lulu, Ma Su, Fengyu, Dr. Peng Sang, Chunpu, Dr. Mengmeng, Dr.

Mi and Minghui, Songyi Xue, Cong Pan and Ruixuan Gao. I would specially thank my friend and

lab mate Sami for bearing brotherly advice and making every moment in the lab lively.

The time I spent with my room mates Selam, Sami and Efrem was cherished. Furthermore,

I would like to thank my family for their endless love and support. Finally, I would like to remark

this is thesis is dedicated to Selam Hagos and all my family and friends, who encouraged and

motivated me all the time during my study at USF.

i

TABLE OF CONTENTS

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

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

LIST OF ABBREVIATIONS ........................................................................................................ vi

ABSTRACT .................................................................................................................................... x

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

1.1 Introduction ........................................................................................................................... 1

1.2 Outline of the Thesis ............................................................................................................. 2

1.3 Reference .............................................................................................................................. 3

CHAPTER 2: PEPTIDE BASED POLYCARBONATE POLYMESRS WITH POTENT AND

SELECTIVE ANTIMICROBIAL ACTIVITY .............................................................................. 5

2.1 Introduction ........................................................................................................................... 5

2.2 General Overview of Polymers ............................................................................................. 6

2.3 Statement of purpose of our work ......................................................................................... 7

2.4 Experimental: Materials and Methods .................................................................................. 7

2.4.1 Synthesis of Monomer 1 (M1) (Figure 2.1) ................................................................... 7

2.4.2 Synthesis of Monomer 2 (M2) - (Figure 2.2) ................................................................ 8

2.4.3 Synthesis of Monomer 3 (M3) - (Figure 2.3) ................................................................ 9

2.4.4 Synthesis of Polycarbonate Copolymers ..................................................................... 10

2.4.4.1 Random copolymerization of M1 and M2 (Figure 2.4) ............................................ 10

2.4.4.2 Random copolymerization of M1 and M3 (Figure 2.6) ............................................ 12

2.4.5 Antimicrobial Assay .................................................................................................... 13

2.4.6 MTT Assay .................................................................................................................. 13

2.5 Results and Discussion ....................................................................................................... 14

2.6 Conclusion .......................................................................................................................... 18

2.7 References ...................................................................................................................... 19

CHAPTER 3: LIPIDATED DENDRIMERS AS POTENT AND BROAD SPECTRUM

ANTIBACTERIAL AGENTS ...................................................................................................... 22

ii

3.1 Introduction ......................................................................................................................... 22

3.2 Dendrimer Overview .......................................................................................................... 24

3.3 Result and Discussion ......................................................................................................... 25

3.3.1 Solid phase Synthesis of lipidated dendrimers: ........................................................... 25

3.3.2 Antimicrobial and Hemolytic activity ......................................................................... 25

3.3.2 Mechanism of Action ................................................................................................... 29

3.3.3 Bacterial Kinetic Study ................................................................................................ 30

3.3.4 Bacterial Biofilm Assay ............................................................................................... 31

3.4 Materials and methods ........................................................................................................ 32

3.4.1 Antimicrobial Assay .................................................................................................... 32

3.4.2 Hemolysis Assay .......................................................................................................... 33

3.4.3 Fluorescence Microscopy ............................................................................................ 33

3.4.4 Time kill study ............................................................................................................. 34

3.4.5 TEM Study ................................................................................................................... 34

3.4.6 Biofilm assay ............................................................................................................... 35

3.5 Conclusion .......................................................................................................................... 35

3.6 References ........................................................................................................................... 36

CHAPTER 4: UNIMOLECULAR NANOPARTICLES AS POTENT ANTIMICROBIAL

AGENTS ....................................................................................................................................... 44

4.1 Introduction ......................................................................................................................... 44

4.2 Overview of Unimolecular Micelle Hyperbranched Polymers .......................................... 46

4.3 Experimental Section .......................................................................................................... 47

4.3.1 Synthesis of Hyperbranched Polyester Polyacid (HBPP) (Figure4.1)......................... 47

4.3.2 Synthesis of hydroxyl terminated hyperbranched polyester (Figure 4.3) .................... 48

4.3.3 Synthesis of Hydrophilic monomer (M1) (Figure 4.5) ................................................ 49

4.3.4 Synthesis of Hydrophobic monomer (M2) (Figure 4.6) .............................................. 50

4.3.5 Synthesis of Unimolecular micelle hyperbranched polymers (Figure 4.7 & Figure 4.8)

............................................................................................................................................... 50

4.3.6 Antimicrobial Assay .................................................................................................... 52

4.4 Results and Discussion ....................................................................................................... 53

4.5 Conclusion .......................................................................................................................... 56

4.6 References ........................................................................................................................... 57

CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS ................................................ 61

iii

LIST OF TABLES

Table 2.1 Copolymers synthesized from M 1 and M 2 monomers .................................................... 12

Table 2.2 Antibacterial activity of polycarbonates ..................................................................................... 17

Table 3.1 The Antibacterial and Hemolytic Activity of Lipidated Dendrimers ......................................... 27

Table 4.1 Unimolecular micelle hyperbranched polymers ............................................................52

Table 4.2 The Antibacterial Activity of Unimolecular micelle hyperbranched polymers ............55

iv

LIST OF FIGURES

Figure 2.1 Synthesis of the Monomer 1 ....................................................................................8

Figure 2.2 Synthesis of Monomer 2 ..........................................................................................9

Figure 2.3 Synthesis of the Monomer 3 ..................................................................................10

Figure 2.4 Synthesis of Random Amphiphilic Polycarbonates ...............................................11

Figure 2.5 1HNMR of Polymer MG-P7 .................................................................................. 11

Figure 2.6 Synthesis of MG-P9 amphiphilic polycarbonate ...................................................13

Figure 2.7 IC50 of Polymer MG-P9 against HepG-2 and HEK-293T cell lines ..................... 18

Figure 2.8 IC50 of Polymer MG-P7 against HepG-2 and HEK-293T cell lines ..................... 18

Figure 3.1 Solid phase Synthesis of D-A dendrimers .............................................................26

Figure 3.2 Solid phase Synthesis of D-B dendrimers .............................................................28

Figure 3.3 Fluorescence micrographs of MRSA and E. Coli that were treated or not treated with

25 μg/mL of D-A-2 for 2 h ....................................................................................29

Figure 3.4 TEM micrographs of MRSA and E. coli treated with 10 µg/mL of D-A-2.……...30

Figure 3.5 Time kill study of D-A-2 against MRSA E. coli.....................................................31

Figure 3.6 Inhibition of biofilms of MRSA and E. coli by D-A-2 .........................................32

Figure 4.1 Synthesis of HBPP .................................................................................................48

Figure 4.2 1H NMR of HBPP ..................................................................................................48

Figure 4.3 Synthesis of hydroxyl terminated hyperbranched polyester ..........................................49

Figure 4.4 1H NMR of hydroxyl terminated hyperbranched polyester ...................................49

Figure 4.5 Monomer 1 (M1) synthesis ....................................................................................50

v

Figure 4.6 Monomer 2 (M2) synthesis ....................................................................................50

Figure 4.7 Synthesis of unimolecular nanoparticles with random chains of polycarbonates…51

Figure 4.8 Synthesis of unimolecular nanoparticles with random chains of polycarbonates...52

vi

LIST OF ABBREVIATIONS

WHO World Health Organization

HDPs Host-defense peptides

MRSA Methicillin-Resistant Staphylococcus Aureus

TEM Transmission Electron Microscopy

LPS Lipopolysaccharides

DCM Dichloro methane

TEA Triethylamine

HCl Hydrochloric Acid

TFA Trifluoracetic acid

NMR Nuclear Magnetic Resonance

MIC Minimum inhibitory concentration

CFU Colony forming unit

MRSE Methicillin-resistant Staphylococcus epidermidis

VREF Vancomycin-resistant E. faecalis

vii

PBS Phosphate Buffered Saline

DAPI 4, 6-Diamidino-2-phenylindole dihydrochloride

PI Propidium iodide

PAMAM Polyamidoamine

HOBT 1-hydroxybenzotriazole monohydrate

DIC Diisopropylcarbodiimide

Boc Tert-Butyloxycarbonyl

Fmoc Fluorenylmethyloxycarbonyl chloride

DMF Dimethylformamide

HPLC High-performance liquid chromatography

MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight

TIS Triisopropylsilane

NMP N-Methyl-2-Pyrrolidone

E.Coli Escherichia Coli

P. aeruginosa Pseudomonas aeruginosa

FM Florescence Microscopy

TSB Tryptic soy broth

viii

OD Optical Density

DIPEA Diisopropylethylamine

TFE Tetrafluoroethylene

MeOH Methanol

MTT (3-(4,5-Dimethylthiazol -2-yl)-2,5-Diphenyl tetrazolium Bromide)

PA Pseudomonas aeruginosa

NaHCO3 Sodium bicarbonate

THF Tetrahydrofuran

Na2SO4 Sodium sulfate

E. faecalis Enterococcus faecalis

M Molarity

PEG Polyethylene glycol

Dde Dichlorodiphenyldichloroethylene

MDC 5-methyl-2-oxo-1,3-dioxane-5-carbonyl chloride

TU 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2- thiourea catalyst

DBU 1,8-diazabicyclo [5.4.0] undec-7- ene

ix

MWCO Molecular Weight Cut-Off's

CH3CN Acetonitrile

DIC Diisopropylcarbodiimide

ROP Ring opening Polymerization

SnCl2

HepG-2

Stannous chloride

liver hepatocellular cells

HEK-293T Human embryonic kidney Cells

C. diff UK6 Clostridium difficile

HBPP Hyperbranched Polyester Poly acid

x

ABSTRACT

Bacterial infections have posed a serious threat to the public health due to the significant

rise of the infections caused by antibiotic resistant bacteria. There has been considerable interest

in the development of antimicrobial agents which mimic the natural HDPs, and among them

biodegradable polymers are newly discovered drug candidates with ease of synthesis and low

manufacture cost compared to synthetic host defense peptides. Herein, we present the synthesis of

biocompatible and biodegradable polymers including polycarbonate polymers, unimolecular

micelle hyperbranched polymers and dendrimers that mimic the antibacterial mechanism of HDPs

by compromising bacterial cell membranes. The developed amphiphilic polycarbonates are highly

selective to Gram-positive bacteria, including multidrug resistant pathogens and the unimolecular

micelle hyperbranched polymers showed promising broad-spectrum activity. However, lipidated

amphiphilic dendrimers with low molecular weight display potent and selective antimicrobial

activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant

strains. In addition to antibacterial activity against planktonic bacteria, these dendrimers were also

shown to inhibit bacterial biofilms effectively. These class of polymers may lead to a useful

generation of antibiotic agents with practical applications.

1

CHAPTER 1: INTRODUCTION

1.1 Introduction

Antibiotics are agents that can be produced by microorganisms and have the capacity to

inhibit the growth of similar microorganisms. In the history of medicine, antimicrobials are one of

the most powerful forms of chemotherapy. With the discovery of new antibiotics, the death rate

caused by infectious diseases was decreased from 797 per hundred thousand in 1900 to 36 per

hundred thousand in 1980.1 However, although the number of discovered antibacterial medications

continues to grow, bacterial resistance to many of these medications become a threat to the current

prevention and treatment of infections. Indeed, within the past twenty years, bacterial resistance

has been considered the greatest challenge to the antibiotic era; 30% of all deaths in America was

due to tuberculosis, pneumonia and gastrointestinal infections caused by resistant bacteria.2

Antibiotic resistance can occur via three general mechanisms.3 First, bacterial destruction

to the antibiotics by specific enzymes for instance some bacteria produce beta lactamase enzyme

which degrades beta lactam drugs. Another approach to drug resistance is when bacteria undergo

mutational changes in their structural and functional makeup by acquiring new genes from other

strains. This results in changes in the bacterial receptor conformation which leads to less

susceptibility of the bacteria towards the drug. An example of this type of bacterial resistance is

mostly shown by Vancomycin-resistant Enterococcus. The third major course of bacterial

resistance is through overexpression of several efflux pumps which leads to banishing of the drugs

from the bacterial cell which results in a decrease in concentration of the drug to a level which is

2

below the toxic threshold. An example of this mechanism is shown by gram-positive and gram-

negative bacteria but mainly by Pseudomonas bacterial strains.2,3,4

One of the promising ongoing research topics to address the challenges created by the

resistant bacteria is to mimic host defense peptides (HDP) by unnatural peptidomimetics including

β-peptides,5 peptoids,6 oligoureas,7 AApeptides.8 Small peptidomimetics can mimic the function

and mechanism of natural HDPs and have shown promising potent, broad spectrum antimicrobial

activity. HDPs, however, are generally not cost effective, as it is difficult to scale up the reaction

to produce in extensive quantities.9 Recently, polymers such as methacrylates,10 norborenene,11

amidoamines,12 polystyrenes, polycarbonates13, Dendrimers14 and hyperbranched unimolecular

nanoparticles15 are getting more attention as novel anti-infective candidates.

Polymers are macromolecular compounds composed of repeating units of monomers

which are linked to each other with a chemical bond. Polymers are currently becoming main

interest of the biomedical era.16 With the current innovations in polymer chemistry, polymers are

functionalized in various architectural designs, including dendrimers and unimolecular micelles.

Dendrimers are highly branched globular structures consisting of a central core from which

identical fragments are built up to make star-like macromolecular structures.17

1.2 Outline of the Thesis

In this thesis, we discuss the design, synthesis, and antibacterial application of biodegradable

polymers.

In chapter 2, we studied the design and antibacterial activity of amphiphilic peptide-based

polycarbonate polymers.

In chapter 3, we discuss the design, solid phase synthesis and the antibacterial activity of

lipidated dendrimers

3

In chapter 4, we describe the design, development and antibacterial activity of unimolecular

micelle nanoparticle.

In chapter 5, we recapitulate the research findings and conclude the future directions of our

research study.

1.3 Reference

1. Walsh & Wright. Introduction: Antibiotic Resistance. Chem. Rev. 105, 391–394 (2005).

2. Wright, G. D. Bacterial resistance to antibiotics: Enzymatic degradation and

modification. Mech. Antimicrob. Resist. Oppor. New Target. Ther. 57, 1451–1470 (2005).

3. Fair, R. J. & Tor, Y. Antibiotics and Bacterial Resistance in the 21st Century. Perspect.

Med. Chem. 6, PMC.S14459 (2014).

4. Guilhelmelli, F. et al. Antibiotic development challenges: the various mechanisms of

action of antimicrobial peptides and of bacterial resistance. Front. Microbiol. 4, (2013).

5. Karlsson, A. J., Pomerantz, W. C., Weisblum, B., Gellman, S. H. & Palecek, S. P.

Antifungal Activity from 14-Helical β-Peptides. J. Am. Chem. Soc. 128, 12630–12631 (2006).

6. Patch, J. A. & Barron, A. E. Helical Peptoid Mimics of Magainin-2 Amide. J. Am. Chem.

Soc. 125, 12092–12093 (2003).

7. Violette, A. et al. Mimicking Helical Antibacterial Peptides with Nonpeptidic Folding

Oligomers. Chem. Biol. 13, 531–538 (2006).

8. Li, Y. et al. Helical Antimicrobial Sulfono-γ-AApeptides. J. Med. Chem. 58, 4802–4811

(2015).

4

9. Costanza, F. et al. Investigation of antimicrobial PEG-poly(amino acid)s. RSC Adv 4,

2089–2095 (2014).

10. Chen, Y. et al. Amphipathic antibacterial agents using cationic methacrylic polymers

with natural rosin as pendant group. RSC Adv. 2, 10275–10282 (2012).

11. AL-Badri, Z. M. et al. Investigating the Effect of Increasing Charge Density on the

Hemolytic Activity of Synthetic Antimicrobial Polymers. Biomacromolecules 9, 2805–2810

(2008).

12. Calabretta, M. K., Kumar, A., McDermott, A. M. & Cai, C. Antibacterial Activities of

Poly(amidoamine) Dendrimers Terminated with Amino and Poly(ethylene glycol) Groups.

Biomacromolecules 8, 1807–1811 (2007).

13. Engler, A. C. et al. Polycarbonate-Based Brush Polymers with Detachable Disulfide-

Linked Side Chains. ACS Macro Lett. 2, 332–336 (2013).

14. Chen, C. Z. & Cooper, S. L. Recent Advances in Antimicrobial Dendrimers. Adv. Mater.

12, 843–846 (2000).

15. Zhou, Y., Huang, W., Liu, J., Zhu, X. & Yan, D. Self-Assembly of Hyperbranched

Polymers and Its Biomedical Applications. Adv. Mater. 22, 4567–4590 (2010).

16. Paul, D. R. & Robeson, L. M. Polymer nanotechnology: Nanocomposites. Polymer 49,

3187–3204 (2008).

17. Tülü, M. & Ertürk, A. S. Dendrimers as Antibacterial Agents. Search Antibact. Agents

(2012). doi:10.5772/46051

5

CHAPTER 2: PEPTIDE BASED POLYCARBONATE POLYMERS WITH POTENT AND

SELECTIVE ANTIMICROBIAL ACTIVITY

2.1 Introduction

Natural antimicrobial peptides (HDPs) are one of the first lines of defense within our body

when bacteria or other microbes attack the body. They have broad-spectrum antimicrobial

activities.1 HDPs are found in many species, including humans, animals, plants, and invertebrates.

The mechanism of action for these peptides are not receptor-based interaction, but through direct

action on the bacteria’s membranes.2 The slowly progressing development of replacements for the

current ineffective antibiotic treatment results in a major risk to public health through multidrug

resistant bacteria. Different studies have revealed that bacteria can develop resistance to drugs in

numerous ways including effluxing the drug from inside the cell through membrane bounded

pumping proteins, undergoing mutational changes within the main receptors of the drug, and

developing enzymes which can selectively degrade the antibiotics.3 Presently, to face the

challenges caused by the multi-drug resistant bacteria various novel antimicrobial agents have

been developed. Compounds having cationic groups have arisen as auspicious candidates to

replace the current antibacterial drugs which already faced resistance. This is due to the short

amphiphilic positively charged peptides have the tendency to mimic HDPs and are selective

towards bacteria. Presently researchers are mainly working on synthetic peptides, polymers and

lipids as potential cationic compounds.4 Herein we report the synthesis and antimicrobial

6

application of peptide based polycarbonate polymers, in continuation to projects previously done

in our lab.5

2.2 General Overview of Polymers.

Polymers are large macromolecules consisting of repeating units, named monomers, linked

together through a covalent bond. Polymers can be obtained from natural sources called natural

polymers or artificially synthesized like that of synthetic polymers. Polymers have diverse

structural arrangements, ranging from linear to branched and cross-linked arrangement. Linear

polymers have long and straight chained arrangements like polyethylene and polyvinyl chloride.

Branched polymers contain some branches on the linear backbone, furthermore cross-linked

polymers are formed from conjugated and covalently bonded linear polymers.6 Generally

polymers are synthesized by a process called polymerization, where several types of

polymerization methods are applied today. In a polymerization reaction, the monomers used

should have a functional group that enables bond formation. Homo-polymerization and

copolymerization are the two most common practiced polymerization methods. In homo-

polymerization, one type of monomer is linked together, while in copolymerization two or more

distinct types of monomers are used.

Based on the sequential arrangement of the monomers several types of copolymers can be

synthesized including block copolymers, graft copolymers and random copolymers.7 Natural and

synthetic polymers are a necessity in our daily life. Growing biomedical applications of polymers

continue to be discovered and researched from time to time. It has been more than half a century

that drugs conjugated with polymers have been applied in biomedical fields for therapeutic

applications. As part of the therapeutic applications of polymers, polycarbonates are developed,

and their application is being studied. The mechanism of action is similar to the antimicrobial HDP

7

through disrupting the microbial membrane by the interaction of the cationic hydrophilic unit with

the negatively charged membranes.8,9

2.3 Statement of purpose of our work

In our lab, previous group members designed and synthesized amphiphilic polycarbonate

polymers which have primary amino groups. According to their findings, the polymers exhibited

potent antimicrobial activity and selectivity against gram-positive bacteria, including multidrug

resistant strains. Encouraged by the promising results obtained, we have planned certain

modifications in the composition of the monomers that may lead to the development of new

polymers which could potentially display broad spectrum antibacterial activity. Phenylalanine,

lysine and 4-Bromo benzoic acid were used as components of the backbone of 5-methyl-2-oxo-

1,3-dioxane-5-carbonyl chloride (MDC) and polymerization of the monomers was done

following the ring opening polymerization mechanism (ROP) as reported by Yang and Hedrick

et al.10 Lysine derived carbonate monomer was designated to afford the hydrophilic feature of

the polymer as it has a free primary amino groups, and both phenylalanine and 4- Bromo benzoic

acid derived monomers will offer hydrophobic characteristics to the polymer.11,10

2.4 Experimental: Materials and Methods

2.4.1 Synthesis of Monomer 1 (M1) (Figure 2.1)

To synthesize Monomer 1, First Di-tert-butyl (6-((2-hydroxyethyl)amino)-6-oxohexane-

1,5-diyl) (S)-dicarbamate was prepared by dissolving 1 equiv N2,N6-bis(tert-butoxycarbonyl)-L-

lysine and 1.5 equiv ethanolamine in Dimethylformamide (DMF), to this solution 1.5 equiv 1-

ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 1.5 equiv

Hydroxybenzotriazole (HOBT) coupling agents were added with a 1.5 equiv N,N-

Diisopropylethylamine (DIPEA) base. The mixture stirred for 6 h and after the reaction was

8

completed, work up was done using ethyl acetate and the organic layer was washed with 1N HCl

(100 mL×3) and brine (50 mL×1). Then product was dried with sodium sulfate and the solvent

was removed with a rotavapor and it was purified using flash chromatography and we got 80%

yield. As part of the monomer constituent, 5-methyl-2-oxo-1,3-dioxane-5-carbonyl chloride

(MDC) was also synthesized following the procedures given by Yang and Hedrick.10 Lastly

Monomer 1 was synthesized by dissolving 1.5 equiv MDC and 1 equiv di-tert-butyl (6-((2-

hydroxyethyl)amino)-6-oxohexane-1,5-diyl)(S)-dicarbamate in 30 mL Dichloromethane (DCM)

in a 100 mL round bottom flask, to which 1.5 equiv triethylamine (TEA) base was added. The

reaction was left for 4 h in an ice bath and then the product was extracted by DCM and was washed

with 1 N HCl (100 mL×3), brine (50 mL×1) and then dried over sodium sulfate. The solvent was

removed in vacuo to give oily product, which was further purified by flash chromatography (ethyl

acetate: hexane 1:1) to give the final white sticky solid product.

Figure 2.1 Synthesis of the Monomer 1.

2.4.2 Synthesis of Monomer 2 (M2) - (Figure 2.2)

To synthesize Monomer 2, first tert-butyl (S)-(1-((2-hydroxyethyl) amino)-1-oxo-3-

phenylpropan-2-yl) carbamate was prepared by dissolving 1 equiv tert-butoxycarbonyl-L-

phenylalanine and 1.5 equiv Ethanolamine in DMF with coupling agents 1.5 equiv EDC, 1.5 equiv

HOBT and 1.5 equiv DIPEA base. The reaction was left for 6 h, after the reaction was completed

9

work up was done by ethyl acetate and the product was washed with 1N HCl (100 mL×3) and

Brine (50 mL×1). The organic solvent was then dried with sodium sulfate and removed by

rotavapor to get white solid product. From that Monomer 2, was prepared by the adding the above

product (1 equiv tert-butyl (S)-(1-((2-hydroxyethyl)amino)-1-oxo-3-phenylpropan-2-yl)

carbamate) with 1.5 equiv MDC which is synthesized according to Yang and Hedrick,10 and the

mixture was dissolved in 30 mL DCM with 1.5equiv TEA in a 100 mL round bottom flask. The

reaction was left for 4 h in an ice bath and then the product was extracted by DCM and was washed

with 1 N HCl (100 mL×3), brine (50 mL×1) and then dried over sodium sulfate. The solvent was

removed by rotavapor to give solid product, which was further purified by flash chromatography.

Figure 2.2 Synthesis of the Monomer 2.

2.4.3 Synthesis of Monomer 3 (M3) - (Figure 2.3)

First 4-bromo-N-(2-hydroxyethyl) benzamide was prepared by dissolving 1 equiv 4-

bromobenzoic acid and 1.5 equiv Ethanolamine in DMF with coupling agents, 1.5 equiv EDC, 1.5

equiv HOBT and 1.5 equiv DIPEA base. The reaction was left for 6 h, after the reaction was completed

work up was done by ethyl acetate and the product was washed with 1N HCl (100 mL×3) and Brine

(50 mL×1). Then the organic solvent was dried with sodium sulfate and vacuo to give a yellowish

solid product. From that Monomer 3 was synthesized by dissolving 1equiv 4-bromo-N-(2-

hydroxyethyl) benzamide, 1.5 equiv MDC and 1.5 equiv TEA in 30 mL DCM in a 100mL round

10

bottom flask. The reaction was completed left for 4 h and then the product was extracted by DCM and

was washed with 1 N HCl (100 mL×3), brine (50 mL×1) and then dried over sodium sulfate. The

solvent was removed in vacuo to give solid product, which was further purified by flash

chromatography.

Figure 2.3 Synthesis of the Monomer 3.

2.4.4 Synthesis of Polycarbonate Copolymers

2.4.4.1 Random copolymerization of M1 and M2 (Figure 2.4)

Batches of polymers were synthesized from M1 and M2 monomers (Table 2.1). All polymers

were synthesized via ring opening polymerization (ROP) method. The detailed synthesis of random

copolymer MG-P7 will be given as an example and all further polymers will follow the reaction steps

and conditions. First 1 equiv initiator benzyl alcohol was dissolved in 10 mL of DCM in a round

bottom flask after purging with nitrogen gas. Then the two monomers, 30 equiv M1 (hydrophilic

monomer) and 20 equiv M2 (hydrophobic monomer) were added to the flask together, to this mixture

1 equiv (3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea catalyst (TU) and 1 equiv 1,8-

diazabicyclo [5.4.0] undec-7-ene (DBU) base were added. The reaction was left stirring for 6 h, and

then 1.3 equiv benzoic acid was added to quench the reaction. The polymers were then purified by

dialysis against methanol and distilled water for 3 days using dialysis tubing MWCO 3500 with

methanol and water being replaced twice a day. The polymer solvent was then vacuumed and then the

polymers were treated with 15 ml of a 1:1 TFA:DCM mixture for 3 h to deprotect the Boc protecting

11

group in order to obtain the free amine functional groups.12 Again, the polymers were purified in a

dialyzing tube against methanol and water for 4 days and then freeze dried to get the final product and

the product was characterized by 1HNMR (Figure 2.5).13 As such, a series of polycarbonate polymers

containing varying numbers of hydrophobic and hydrophilic groups were prepared in the same manner

(Table 2.1).

Figure 2.4 Synthesis of Random Amphiphilic Polycarbonates.

Figure 2.5 1HNMR of Polymer MG-P7

12

Table 2.1 Copolymers synthesized from M 1 and M 2 monomers.

Compound

Type of Co-

Polymer

Hydrophilic Units

M1

Hydrophobic units:

M2

Ideal Molecular

Weight

MG-P1 Random 10 30 13932.96

MG-P2 Random 15 25 13837.95

MG-P3 Random 15 30 15589.81

MG-P4 Random 20 25 15494.80

MG-P5 Random 20 40 20750.36

MG-P6 Random 30 10 13552.92

MG-P7 Random 30 20 17056.63

MG-P8 Random 30 30 20560.34

MG-P9 Random 20 20 14459.48

2.4.4.2 Random copolymerization of M1 and M3 (Figure 2.6)

Polymer MG-P9 was prepared in similar fashion as above procedure, but the hydrophobic

monomer M3 was used instead of M2.

13

Figure 2.6 Synthesis of MG-P9 amphiphilic polycarbonate.

2.4.5 Antimicrobial Assay

Minimum Inhibitory Concentration (MIC) of the polycarbonate polymers was determined

using a broth micro dilution method in 96-well plates against Methicillin-resistant S. aureus (MRSA,

ATCC 33591), Clostridium difficile (C. diff UK6) and Gram-Negative bacteria E. coli (ATCC 25922).

Bacterial cells were grown overnight at 37 °C in 4 mL TSB. Approximately 106 CFU/mL bacterial

suspension in TSB was prepared. Aliquots of 50 µL bacterial suspension were added to 50 µL of the

polycarbonate polymers to prepare serial diluted concentrations (50 to 0.25 µg/mL) in each well. The

plates were then incubated at 37 °C for 20h. Optical density (OD) at 600 nm was measured after 18h

using Biotek microplate reader and the MIC was determined accordingly.14

2.4.6 MTT Assay

The cytotoxicity of the polymers was determined by a cell viability assay called MTT assay.

MTT is a tetrazolium salt which is very soluble in water and turns to an insoluble purple formazan

through the cleavage of the tetrazolium ring by the dehydrogenase enzyme once it is inserted into the

cell. Formazan will be accumulated in the cell as it becomes impermeable through the cell

membrane.15,16 MTT is expressed in terms of IC50 ,which is defined as the concentration of a drug that

inhibits the growth of 50% of the viable body cells. In this assay, an MTT (3-[4,5-dimethylthiazol-2-

yl]-2,5-diphenyltetrazolium bromide) reagent was used. To determine the IC50 of the synthesized

polymers, first 100μL of HepG-2 (human liver carcinoma cells) and HEK-293T (Human embryonic

14

kidney cells) cells with a consistent density (5,000-10,000 cells per well) were added to the well plates

and incubated overnight. And different polymer concentrations ranging from 128 μg/mL to 0.125

μg/mL were prepared and added to the cell culture medium and incubated for 16-48h. MTT solution

was prepared by dissolving MTT in PBS, and then 10μl of the MTT stock solution was added to each

well and allowed to incubate for 4 h at 37 °C. Solubilizing of formazan was done by carefully removing

the media from each well without disturbing the cells and 100μl of DMSO was added to each well

with continuous mixing by pipetting up and down. After incubating the mixture at 37°C for 15

minutes, absorbance was measured at 540 nm immediately.17

2.5 Results and Discussion

Recently, the synthesis of polycarbonate polymers from monomers with primary amino groups

using benzyl alcohol initiators through ring-opening polymerization (ROP) of MDC was reported as

an effective way of synthesizing polycarbonate polymers in our lab.12,10 These polymers, containing

primary amino groups with appropriate hydrophobic group showed optimal amphiphilicity and were

found to be potent and highly selective antimicrobial polymers, but they were only active against

gram-negative bacteria.12 To extend this work, we sought to make a change in the previously used

monomers by some other peptide scaffolds as peptides are potential biological molecules that can be

modified and mimic the antimicrobial mechanism of natural host defense peptides. Studies revealed

that cationic peptide Epsilon-poly-L-lysine has antimicrobial activity and is used as a food

preservative.18 Considering the application of lysine as an antibacterial residue, we designed our

monomer where lysine could be a main chain and linked to MDC backbone by ethanolamine to

produce hydrophilic M1 monomer. Hydrophobic groups are necessary for a polymer to produce

antimicrobial effect, so we selected another peptide with hydrophobic characteristics to be

incorporated in to the MDC backbone to make M2 monomer. Phenylalanine was used to synthesize

15

the hydrophobic monomer as it has been proven for its antibacterial applications mixed with cationic

peptides.19 Furthermore, to see the effect of non-peptide hydrophobic residue, 4-bromobenzoic acid

was used in synthesizing another hydrophobic monomer which is M3. This monomer was selected

because many antimicrobial analogs with halogen group in their structures like chlorine, fluorine and

bromine have been discovered as potent antibacterial residues and we decided to take advantage of

those groups.20

The synthesis of the polymers was direct and straight forward. Copolymerization was done

through ROP following the published protocols given by Yang and Hedrick et al.10 Compared to

diblock copolymers random copolymers have proven potent antimicrobial activity according to the

previous study done in our lab.12 As Random core structure ease the interaction of the polymer with

the membrane of the bacteria via the cationic and hydrophobic interactions,21we decided to focus our

synthesis mainly on random copolymers.

Amphiphilicity is the most crucial factor to determine antimicrobial potency of a polymer. To

see the effect, we synthesized nine polymers by intermixing M1 and M2 monomers as well as M1 and

M3 monomers (Table 2.1). The synthetic strategy employed to make random copolymers is shown in

Fig 2.4 and Fig 2.6. To explore our proposal, the polymers were tested against clinically relevant

threatening strains of Gram-positive bacteria Methicillin-resistant Staphylococcus aureus (MRSA,

ATCC 33591), and a Gram-negative bacteria Escherichia coli (E. coli, ATCC 25922) for their

antimicrobial activity (Table 2.2).

As shown in Table 2.2, Polymers MG-P1:MG-P8 showed similar antimicrobial activity

against MRSA at 3µg/mL except for MG-P4 which does not show any activity. Polymer MG-P4 had

almost equal ratios of hydrophilic and hydrophobic residues 20 equiv and 25 equiv respectively. The

remaining seven polymers had unequal ratios of the residue and showed moderate antibacterial activity

16

against MRSA. The obtained data reveals that the cationic group in the polymer is vital for the

interaction of the polymers with the negatively charged membrane of the bacterial cell. Though this

aids in the activity of cell death, the component of hydrophobicity is necessary for the eradication of

bacterial cells.22 Furthermore, in order to study more about the importance of hydrophobic residue in

a polymer we synthesized polymer MG-P9 from M1 and M3 monomers where M3 is non-peptide

analog with bromophenyl group in its structure. We hypothesized the activity of the polymers will be

enhanced, as halogen encompassing analogs have already been proven to have potent antibacterial

activities.22 To synthesize polymer MG-P9, the same type of hydrophilic monomer M1 was used and

a new hydrophobic monomer M3 was used instead of M2. MG-P9 had 20 equiv hydrophobic and 20

equiv hydrophilic residues. As expected, the obtained MIC result was encouraging, and it was 0.75 µg

/mL. This value clearly reveals the presence of halogens in the hydrophobic residue increases the

hydrophobicity and ensures potent antimicrobial activity. Unfortunately, the desired activity against

gram negative bacteria was not obtained.

To further confirm the antimicrobial application of the polymers, we tested the polymers

against other gram-positive C. diff UK6 bacteria and the result obtained by all polymers was

encouraging. As shown in Table 2.2, polymers MG-P1, MG-P4, MG-P7 and MG-P9 were found to

be the most potent polymers against C. diff bacteria as their MIC result is below 1µg /mL. Polymer

MG-P4, which showed no activity against MRSA, displayed potent activity towards C. diff UK6 at

0.25 µg /mL. Similarly, polymer MG-P9 showed potent activity of 0.25 µg /mL, as we expected. From

the MIC results of all the polymers and especially that of polymer MG-P9, we can suggest that the

presence of halogens, especially bromine, has a significant role in the antimicrobial potency.

17

Table 2.2 Antibacterial activity of polycarbonate Polymers.

Compound

Hydrophilic

units

Hydrophobic

units

MIC- Gram Positive bacteria

(µg/mL)

MTT

(IC50) (µg/mL)

MRSA C.diff UK6 HepG-2 HEK-293T

MG-P1 10 30 6-3 0.5 54.74 49.01

MG-P2 15 25 6-3 4 - -

MG-P3 15 30 6-3 4 - -

MG-P4 20 25 NA 0.25 47.13 74.19

MG-P5 20 40 6-3 2 - -

MG-P6 30 10 6-3 1 65.98 75.15

MG-P7 30 20 < 3 0.5 129.8 126.1

MG-P8 30 30 6-3 2 - -

MG-P9 20 20 0.75 0.25 86.26 82.5

The cytotoxic activity of the polymers was also analyzed by an MTT assay against HepG-

2 (human liver carcinoma cells) and HEK-293T (Human embryonic kidney cells). The obtained

results reveal that the most potent polymers, (MG-P1, MG-P4, MG-P6, MG-P7 and MG-P9)

which had MIC less than 1μg/mL against C. diff UK6, showed better selectivity. Notably, Polymer

MG-P9 had IC50 of 86.26 μg/mL and 82.5 μg/mL against HepG-2 and HEK-293T respectively

which is > 300-fold of selectivity for C. diff UK6 (Figure 2.7). Similarly, polymer MG-P7 had an

IC50 of 129.8 μg/mL and 126.1 μg/mL against HepG-2 and HEK-293T respectively (Figure 2.8)

which is > 300-fold of selectivity for C. diff UK6. The results from the MTT assay confirmed that

18

the polymers were highly selective towards bacteria and we decided to do further animal study for

polymers MG-P9 and MG-P7 to research their cytotoxic property in animals.

Figure 2.7 IC50 of Polymer MG-P9 against HepG-2 and HEK-293T cell lines.

Figure 2.8. IC50 of Polymer MG-P7 against HepG-2 and HEK-293T cell lines.

2.6 Conclusion

To summarize, we have developed a series of polycarbonate antimicrobial polymers based on

ring opening polymerization method. These polymers display good potency against multidrug-

resistant Gram-positive bacteria. Both peptide and nonpeptide analogues were used as hydrophilic

and hydrophobic monomers to synthesize the polycarbonate polymers. The intermixed polymer from

lysine derived hydrophilic monomer and nonpeptide bromophenyl derived hydrophobic monomer

19

showed the most potent activity towards gram – positive bacteria and the polymer from peptide derived

lysine and phenyl alanine monomers showed the highest selectivity. Two polymers are being selected

for further in vivo study and the study is currently ongoing.

2.7 References

1. Arnt, L., Nüsslein, K. & Tew, G. N. Nonhemolytic abiogenic polymers as antimicrobial

peptide mimics. J. Polym. Sci. Part Polym. Chem. 42, 3860–3864 (2004).

2. Lienkamp, K. et al. Antimicrobial Polymers Prepared by ROMP with Unprecedented

Selectivity: A Molecular Construction Kit Approach. J. Am. Chem. Soc. 130, 9836–9843 (2008).

3. Wright, G. D. Bacterial resistance to antibiotics: Enzymatic degradation and modification.

Mech. Antimicrob. Resist. Oppor. New Target. Ther. 57, 1451–1470 (2005).

4. Carmona-Ribeiro, A. M. & de Melo Carrasco, L. D. Cationic Antimicrobial Polymers and

Their Assemblies. Int. J. Mol. Sci. 14, 9906–9946 (2013).

5. Li, Y. et al. Helical Antimicrobial Sulfono-γ-AApeptides. J. Med. Chem. 58, 4802–4811

(2015).

6. Paul, D. R. & Robeson, L. M. Polymer nanotechnology: Nanocomposites. Polymer 49,

3187–3204 (2008).

7. Young, R. J. & Lovell, P. A. Introduction to Polymers, Third Edition. (CRC Press, 2011).

8. Santos, M. R. E. et al. Recent Developments in Antimicrobial Polymers: A Review.

Materials 9, (2016).

9. Lee, J. & Lee, D. G. Antimicrobial Peptides (AMPs) with Dual Mechanisms: Membrane

Disruption and Apoptosis. J. Microbiol. Biotechnol. 25, 759–764 (2015).

20

10. Chin, W. et al. Biodegradable Broad-Spectrum Antimicrobial Polycarbonates:

Investigating the Role of Chemical Structure on Activity and Selectivity. Macromolecules 46,

8797–8807 (2013).

11. Nimmagadda, A. et al. Polycarbonates with Potent and Selective Antimicrobial Activity

toward Gram-Positive Bacteria. Biomacromolecules 18, 87–95 (2017).

12. Nimmagadda, A. et al. Polycarbonates with Potent and Selective Antimicrobial Activity

toward Gram-Positive Bacteria. Biomacromolecules 18, 87–95 (2017).

13. Costanza, F. et al. Investigation of antimicrobial PEG-poly(amino acid)s. RSC Adv 4,

2089–2095 (2014).

14. Teng, P. et al. Small Antimicrobial Agents Based on Acylated Reduced Amide Scaffold.

J. Med. Chem. 59, 7877–7887 (2016).

15. Fotakis, G. & Timbrell, J. A. In vitro cytotoxicity assays: Comparison of LDH, neutral red,

MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol.

Lett. 160, 171–177 (2006).

16. Al-Sheddi, E. S. et al. Novel All Trans-Retinoic Acid Derivatives: Cytotoxicity, Inhibition

of Cell Cycle Progression and Induction of Apoptosis in Human Cancer Cell Lines. Molecules 20,

8181–8197 (2015).

17. Vybrant® MTT Cell Proliferation Assay Kit. Available at:

https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/mtt-assay-

protocol/vybrant-mtt-cell-proliferation-assay-kit.html. (Accessed: 6th February 2018)

21

18. Hyldgaard, M. et al. The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine.

Appl. Environ. Microbiol. 80, 7758–7770 (2014).

19. Vaara, M. & Porro, M. Group of peptides that act synergistically with hydrophobic

antibiotics against gram-negative enteric bacteria. Antimicrob. Agents Chemother. 40, 1801–1805

(1996).

20. Garrison, A. T. et al. Structure–Activity Relationships of a Diverse Class of Halogenated

Phenazines That Targets Persistent, Antibiotic-Tolerant Bacterial Biofilms and Mycobacterium

tuberculosis. J. Med. Chem. 59, 3808–3825 (2016).

21. Zhou, C. et al. High Potency and Broad-Spectrum Antimicrobial Peptides Synthesized via

Ring-Opening Polymerization of α-Aminoacid- N -carboxyanhydrides. Biomacromolecules 11,

60–67 (2010).

22. Garrison, A. T. et al. Structure–Activity Relationships of a Diverse Class of Halogenated

Phenazines That Targets Persistent, Antibiotic-Tolerant Bacterial Biofilms and Mycobacterium

tuberculosis. J. Med. Chem. 59, 3808–3825 (2016).

22

CHAPTER 3: LIPIDATED DENDRIMERS AS POTENT AND BROAD SPECTRUM

ANTIBACTERIAL AGENTS

3.1 Introduction

The resistance developed by bacteria against conventional antibiotics have contributed to

the sharp rise in illness and deaths caused by bacterial infections that were once curable.1

Conventional antibiotics are generally small molecules that exert their activity by targeting specific

cellular nucleic acids and proteins, or cell wall enzymes of bacteria. Bacteria are likely to develop

mutations rapidly on the targets upon prolonged antibiotic treatment, leading to the development

of drug resistant bacterial strains.1 In order to combat the emerging resistance, research efforts

have been focused on developing host-defense peptides (HDPs) as bacteria are believed to have

less probability to develop resistance to HDPs due to their distinct antimicrobial mechanisms.2

It is known that HDPs are naturally occurring peptides that are rich in cationic and

hydrophobic residues. Despite the diversity in three-dimensional structures, upon association with

the bacterial membranes, HDPs generally obtain globally amphipathic structures which are critical

for membrane action on bacteria.3 The interaction occurs considerably selectively for bacteria as

the outer leaflet of membranes of bacteria is predominantly rich in negatively charged

phospholipids.4 In addition, in Gram-positive bacteria, teichoic acids or lipo-teichoic acids are

frequently identified on the peptidoglycan layer, whereas lipopolysaccharides are common

components on the outer membranes of Gram-negative bacteria.5 These negative charges greatly

23

contribute to the initial attraction of the catatonically charged HDPs onto the bacterial membranes.

Subsequently, the hydrophobic patches of HDPs help to penetrate the bacterial membranes through

hydrophobic interactions with phospholipids. In contrast, the outer surface of mammalian cell

membranes is largely zwitterionic as they are dominated by neutral phospholipids such as

cholesterol, phosphatidylcholine, and sphingomyelin, whereas their negatively charged

phospholipids are essentially located in the inner leaflet of membranes. As such, HDPs have less

probability to interact with mammalian cells compared with bacteria, which is believed to account

for at least a significant part of the selectivity of HDPs.6 Since membrane action lacks specific

molecular targets, it is generally believed that HDPs are less prone to development of antibiotic

resistance.7

Owing to the abovementioned advantages, HDPs have received notable attention as a new

generation of antimicrobial agents combating antibiotic resistance. However, there are noticeable

limitations associated with HDPs, including low-to-moderate activity, potential high cost of

manufacturing, susceptibility to proteolytic degradation etc.8 It is conceivable that the

antimicrobial agents which can mimic the mechanism of HDPs but with enhanced selectivity and

antimicrobial activity will be one viable strategy for antibiotic development to combat resistance.

To date, unnatural peptidomimetics such as β-peptides,9 peptoids,10a-10b oligoureas,11

AApeptides,12a-12f have been developed to mimic HDPs and target a wide range of bacterial strains.

These peptidomimetics are generally more active and more metabolically stable than natural

HDPs, however, they still suffer from potentially high manufacturing cost and difficulty in scale-

up.13 Another alternative approach is to develop cationic antimicrobial polymers including poly

(α-amino acid)s,14 nylon-3 polymers,15 polyacrylates,16,17 polycarbonates,18 and dendrimers such

as PAMAM,19 poly(propylene imine),20 etc. Herein we are exploring to design and synthesize

24

efficient and cost effective lipidated poly lysine dendrimer which has biomedical applications

especially as antibacterial agent.

3.2 Dendrimer Overview

Dendrimers are highly branched globular structures consisting of a central core from which

identical fragments are built up to make star-like macromolecular structures.21 Compared with

linear polymers, dendrimers are normally in the nano size scale and have narrow polydispersity,

uniform nanomorphology, and tunable surface entities.22 The terminal groups of the arms of the

dendrimer determine its solubility and reactivity,21 as well as capability for further modification.

Dendrimers have attracted significant interest in potential application in the biomedical and

materials sciences.23 For instance, they have been widely studied in targeted drug delivery systems

for the treatment of cancer,24 as antimicrobial agents,25 enzyme catalysis and surface engineering

techniques.26

Indeed, different attempts have been made to synthesize dendrimer compounds by

introducing functional groups pertaining to antimicrobial activity, e. g., poly(amidoamine)

dendrimers with quaternary ammonium salts.27 Lysine dendrimers were synthesized by coupling

with other peptides and confer activity against bacteria.28 Herein, we are presenting a new class of

lipidated dendrimers that encompass lysine amino acids to present a multicharged cationic surface

and bear a hydrophobic domain which is composed of different lengths of lipid tails. We also

evaluated the dendrimers for their antibacterial activity and mechanism of action. Intriguingly,

these dendrimers showed potent and broad-spectrum activity against both Gram-positive and

Gram-negative bacteria, including multidrug-resistant strains, in addition, the simple design of the

dendrimers allows easy scale-up and optimization using the solid phase peptide synthesis.29

25

3.3 Result and Discussion

3.3.1 Solid phase Synthesis of lipidated dendrimers

Lipidated dendrimers with one lipid tail were initially synthesized on Rink amide resin

following the standard Fmoc chemistry protocol used for the solid phase peptide synthesis. Briefly,

20% piperidine in DMF was used to remove the Fmoc protecting group in every coupling cycle

which was followed by the coupling of 2 equiv of the desired amino acid, 4 equiv of HOBT (1-

hydroxybenzotriazole monohydrate) and DIC (diisopropylcarbodiimide) in DMF for 4 h.30 The

coupling reaction time was prolonged to 6-8h upon increasing the number of generations of the

dendrimers. In order to introduce hydrophobic lipid tails, initially 4-Methyltrityl-Lys(Fmoc)-OH

was attached onto the rink amide resin. Then the 4-Methyltrityl protecting group was selectively

removed under 2% TFA. The lipid tail was attached, followed by coupling of the Fmoc-

Lys(Fmoc)-OH monomer (Scheme 1) until the desired dendrimers were obtained. The lipidated

dendrimers were cleaved from the solid support and purified by HPLC and then tested for their

antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria (Table 2.1),

including three clinically threatening Gram-positive bacterial strains, methicillin-resistant S.

epidermidis (MRSE, RP62A), vancomycin-resistant E. faecalis (VREF, ATCC 700802), and

methicillin-resistant S. aureus (MRSA, ATCC 33591), and two Gram-negative bacterial strains,

E. coli (ATCC 25922), P. aeruginosa (ATCC 27853).

3.3.2 Antimicrobial and Hemolytic activity

As shown in Figure 3.1 and Table 3.1, the dendrimer D-A-1 which has only two positively

charged amino groups and one C16 tail already exhibited good antibacterial activity against Gram-

positive bacterial strains, with MICs of 3 µg/mL against MRSA and Enterococcus faecalis and 1.5

µg/mL against MRSE bacterial strains, respectively. Encouraged by the positive results, we

26

synthesized D-A-2 which bears twice the cationic groups and the same hydrophobic tail as D-A-

1. To our delight, the dendrimer exhibited potent and broad-spectrum activity against both Gram-

positive and Gram-negative bacterial strains with activity of 0.75 µg/mL against Gram-positive

strains and 3 µg/mL against Gram-negative strains. It is exciting that this dendrimer is highly

selective for bacteria because it only has very limited hemolytic activity (Table 3.1).

Figure 3.1 Solid phase Synthesis of D-A dendrimers.

To further understand the structure function relationship of this type of dendrimers in terms

of cationic charges, D-A-3 and D-A-4 were synthesized by increasing the terminal cationic amine

groups to 8 and 16, whereas the C16 hydrophobic tail was retained. Interestingly, bearing double

cationic charges, the activity of D-A-3 slightly reduced against all bacterial strains when compared

to D-A-2. D-A-4, containing 16 cationic charges, was found to be inactive against both Gram-

positive and Gram-negative bacterial strains. The results suggested that cationic charges by

themselves are not solely responsible for antimicrobial activity; optimized cationic charges and

27

hydrophobicity are necessary for both antibacterial activity and selectivity. Bearing balanced

cationic groups and hydrophobic tail, we hypothesized D-A-2 was able to selectively bind to

negatively charged bacterial membranes and insert its lipid tail into bacterial lipid layers. However,

with more cationic amino groups on the dendrimers, the hydrophobic tail was shielded in their

core, reducing its hydrophobic interactions with bacterial lipids, leading weaker or even abolished

capability to disrupt bacterial membranes.

Table 3. 1 The Antibacterial and Hemolytic Activity of Lipidated Dendrimers.

Next, we set out to evaluate the impact of hydrophobicity of the dendrimers on the

antimicrobial activity by synthesizing a series of dendrimers (D-A-2a to D-A-2d) which have the

same cationic charges as the most active dendrimer D-A-2 but varying hydrophobicity due to the

change in the length of the lipid tail. As shown in Figure 3.1 and Table 3.1, D-A-2a, D-A-2b, D-

A-2c, D-A-2d had a C14, C12, C10, C8 lipid tail, respectively. Interestingly, compared to D-A-2,

the dendrimers D-A-2a, D-A-2b exhibited reduced activity against Gram-positive bacteria,

whereas they lost activity against Gram-negative bacteria at the tested conditions. Furthermore,

Dendrimer

MW

Number

of positive

charges

Length

of

lipid tail

Gram-positive

bacteria (µg/mL)

Gram-negative

bacteria (µg/mL)

Hemolysis

(HC50 µg/mL)

Selectivity

indexof MRSA

(HC50/ MIC)

MRSA MRSE VREF E. coli P. A

D-A-1 511.80 2 16 3.0 1.5 3.0 >25 >25 125 42

D-A-2 768.15 4 16 0.75 0.75 0.75 3.0 3.0 >250 >333

D-A-3 1280.85 8 16 1.5 1.5 1.5 6.0 6.0 >250 >166

D-A-4 2306.25 16 16 >25 >25 >25 >25 >25 - -

D-A-2a 740.09 4 14 1.5 1.5 1.5 >25 >25 125 83

D-A-2b 712.06 4 12 3.0 1.5 3.0 >25 >25 125 42

D-A-2c 683.98 4 10 >25 >25 >25 >25 >25 - -

D-A-2d 655.93 4 8 >25 >25 >25 >25 >25 - -

D-B-1 878.39 2

Two C16

tails

>25 >25 >25 >25 >25 - -

D-B-2 1134.74 4 >25 >25 >25 >25 >25 - -

D-B-3 1647.44 8 >25 >25 >25 >25 >25 - -

28

D-A-2c and D-A-2d dendrimers completely abolished their activity (Table 3.1) toward both

Gram-positive and Gram-negative strains. This is consistent to previous findings,31,32 that C16

lipid tail is necessary to penetrate bacterial membranes. Ones with short lipid tails have less

probability to interact and lack capability to penetrate bacterial membranes.

Figure 3.2: Solid phase Synthesis of a series of D-B dendrimers.

To further evaluate the effect of hydrophobic lipid tails on the activity, we next synthesized

dendrimers containing two C16 lipid tails based on the most potent compound D-A-2. In order to

synthesize this type of dendrimer, after removal of the 4-Methyltrityl protecting group, Dde-

Lys(Dde)-OH monomer was coupled on to the first monomer on the solid support (Figure 3.2),

followed by the deprotection of the Dde group.33,34 Then to each of the two unprotected amines,

one C16 tail was attached. Their antimicrobial activity was also examined and shown in Table 3.1.

Interestingly, those sequences, D-B-1, D-B-2 to D-B-3, did not show any activity towards bacteria

29

under the tested conditions. Indeed, the results are consistent to our previous findings,35 and we

hypothesized that those dendrimers may self-assemble into micelles due to strong hydrophobic

interaction among each other, which deteriorates their ability to penetrate bacterial membranes.

3.3.2 Mechanism of Action

The antimicrobial activity of these dendrimers suggested that both cationic and

hydrophobic groups are required to be present in balance for the dendrimers to exhibit broad-

spectrum antibacterial activity, a structural motif analogous to that of HDPs. To confirm that these

dendrimers exhibit antibacterial activity by acting on bacterial membranes, Florescence

microscopy (FM)36,37,38 and transmission electron microscopy (TEM)6 experiments were

conducted for Gram-positive strain (MRSA) and a Gram-negative strain (E. coli). In FM assay,

4′,6-diamidino-2-phenylindole (DAPI) is used as a membrane permeable dye that stains all live

and dead cells, whereas propidium-iodide (PI) is the non-membrane permeable dye that stains only

the dead cells. The most active dendrimer D-A-2 was chosen for the study, which revealed its

ability to disrupt the membrane of MRSA and E. coli, respectively, as PI stain was observed only

with the bacteria which were treated with D-A-2 but not in the control bacteria (Figure 3.3).

MRSA E. coli

Figure 3.3 Fluorescence micrographs of MRSA and E. coli that were treated or not treated with 25

μg/mL of D-A-2 for 2 h: (C1) control, no treatment, DAPI stained; (C2) control, no treatment, PI

stained; (D1) treatment with D-A-2, DAPI stained; (D2) treatment with D-A-2, PI stained.

30

To further prove our hypothesis that lipidated dendrimers mimic the mechanism of action

of HDPs, TEM microscopy images were used to analyze the morphology of the drug treated and

non-drug treated bacteria. As shown in Figure 3.4 the membranes of both MRSA and E. coli were

compromised when treated with the dendrimer D-A-2.

Figure 3.4 TEM micrographs of MRSA and E. coli treated with 10 µg/mL of D-A-2 for 2 h: (a)

control, no treatment; (b) treatment with D-A-2, initial rupture of the bacterial membrane; (c)

treatment with D-A-2, complete cell rupture leading to bacterial cell death.

3.3.3 Bacterial Kinetic Study

To future investigate the time of bactericidal action of the dendrimers, bacterial kinetics

assay was conducted. The most active dendrimer D-A-2 was investigated and the time required to

show its bactericidal action was analyzed for Gram-positive and Gram-negative bacteria. Colony

forming units of the Gram-positive and Gram-negative bacteria on the agar plate were counted for

three different concentrations (50 µg/mL, 25 µg/mL, 12.5 µg/mL) of the D-A-2 treated bacteria

and the control bacteria at regular intervals of 10 min, 30 min, 1 h, 2 h. As shown in Figure 3.5, it

is evident that at the concentration of 50 µg/mL, 25 µg/mL 12.5 µg/mL, the Gram-positive bacteria

MRSA were eradicated completely after 1 h, whereas the Gram-negative bacteria, E. coli was also

eradicated after 2 h at the concentration of 50 µg/mL, 25 µg/mL, 12.5 µg/ml. This delay in

bactericidal action on the Gram-negative bacteria might be due to the presence of the extra outer

membrane layer, which is not present in the Gram-positive bacterial strains. Overall, the Time kill

31

studies deomonstrated that the dendrimer could rapidly kill bacteria.

MRSA E. coli

Figure 3.5 Time kill study of D-A-2 against MRSA and E. coli.

3.3.4 Bacterial Biofilm Assay

The infections caused by biofilms are a great threat to human life. Recent reports have

shown that 80% of the bacterial infections are caused due to biofilms.39 Bacterial biofilms have

become a severe problem specially in the cases of patients who suffer from infections that occur

from the antibiotic resistant bacteria.40 Biofilms formed by MRSA and E. coli are currently the

major concern in hospitals as they contaminate the surgical tools and organ catheters.41 Therefore,

development of new antibiotic agents that act against biofilm formation has become a major

strategy to treat bacterial infections. We thus analyzed the biofilm inhibiting efficiency of the most

active dendrimer D-A-2 against MRSA and E. coli. As shown in Figure 3.6, even at 0.17 μg/mL,

D-A-2 was able to inhibit 85% of biofilm formation of E. coli and was able to completely eradicate

the biofilm formation of MRSA. At the concentration of 0.8 μg/mL, D-A-2 was able to eradicate

biofilm formation of E. coli completely. The above stated results confirm that the dendrimers can

act as biofilm inhibitors.

32

0.0 0.3 0.6 0.9

0

50

100

MRSA

E. Coli

Rela

tiv

e B

iofi

lm B

iom

ass

(%

)

Compound D-A-2 (g/mL)

Figure 3.6. Inhibition of biofilms of MRSA and E. coli by D-A-2

3.4 Materials and methods

3.4.1 Antimicrobial Assay

Lipidated dendrimers were tested against different strains of bacteria to determine their

Minimum inhibitory concentration (MIC). Three clinically threatening Gram-positive bacterial

strains including methicillin-resistant S. epidermidis (MRSE, RP62A), vancomycin-resistant E.

faecalis (VREF, ATCC 700802), and methicillin-resistant Staphylococcus aureus (MRSA, ATCC

33591) and two Gram-negative bacterial strains, E. coli (ATCC 25922), P. aeruginosa (ATCC

27853) were used for testing. A serial dilution method was performed to determine the

antimicrobial activities. In 4 mL of TSB solution a single colony of bacteria was grown and

incubated at 37 °C overnight after which the cultured bacteria were diluted by 100-fold and were

shaken until their mid logarithmic phase was obtained. 50 μL of medium containing different

concentrations of the lipidated dendrimers was made in each vial of the 96 well plate. 50 μL

aliquots of bacterial suspension was added to the medium with different drug concentrations. The

96 well plate was incubated at 37 °C overnight and the cell growth was monitored using Biotek

synergy HT microtiter plate reader under the 600 nm wavelength absorbance. The assay was

repeated three times.42,42b,43

33

3.4.2 Hemolysis Assay

The selectivity of the lipidated dendrimers was determined through hemolysis assay. To

perform this assay fresh K2EDTA treated human red blood cells (hRBCs) were centrifuged at

1000g for 10 minutes. The step was done three times until the supernatant was clear, then the

desired suspended erythrocytes were collected and washed with PBS buffer a couple of times. The

collected RBCs were then diluted into 5% v/v suspension. From the diluted solution 50 μL was

taken and added to the already prepared serially- diluted lipidated dendrimer solutions and were

left for incubation at 37 °C for 1 hr. The incubated solutions were centrifuged at 3500 rpm for 10

minutes. To measure the absorbance, 30 μL of supernatant was diluted with 100 μL of PBS and

absorbance at a wavelength of 540 nm was recorded. Selectivity of the lipidated dendrimers (%

Hemolysis) was then calculated by applying the formula % hemolysis = (Abs sample − Abs PBS)/

(Abs Triton − Abs PBS) × 100%, where PBS was used as a negative control and 0.1% Triton x-

100 was used as a positive control. Results were repeated two times with duplicates each time.44

3.4.3 Fluorescence Microscopy

To assess whether the lipidated dendrimers act on the bacterial membrane or not, a double

staining Florescence microscopy assay was used. Both 6-diamidino-2-phenylindole

dihydrochloride (DAPI) and propidium iodide (PI) dyes were selected as fluorophores to

differentiate between the dead bacterial cells and viable cells. DAPI is capable of staining both

live and dead cells but PI can only stain dead cells due to its impermeable nature through the

membrane. The bacteria were grown to mid logarithmic phase and were incubated with lipidated

dendrimers at 37 °C for 2 h, the mixture was centrifuged at 1000g for 10 min. Once the bacterial

pellets were collected and washed with PBS three to four times. They were subsequently incubated

with PI (5 μg/mL) in the dark for 15 min at 0 °C, the excessive PI was rinsed using PBS several

34

times. Lastly, the cells were incubated with DAPI (10 μg/mL) for 15 min on ice and the excess

DAPI was removed by washing with PBS buffer. After the samples were ready 10 μL of the

bacteria were used for testing under Zeiss Axio Image Zloptical microscope using 100× oil-

immersion objective.44

3.4.4 Time kill study

The kinetic assay of the lipidated dendrimers was tested against MRSA and E. coli. The

bacteria were grown to midlogarithmic phase in TSB medium and then diluted into 106 CFUmL-1

suspensions. Different concentrations of the lipidated dendrimers were then added to the diluted

suspensions and incubated for 10 min, 30 min, 1 h, and 2 h, respectively. After incubation, the

mixture was diluted into 102 to 104 times, then dispersed on TSB agar plates and incubated

overnight at 37 °C. The colonies on the plates were counted and graphed against colony forming

unit of bacteria and incubation time.45

3.4.5 TEM Study

Similar procedure was used to grow the bacteria to mid logarithmic phase as in the case of

MIC study. Two samples were made, one batch has only bacteria and the other batch was the

bacteria treated with D-A-2. Samples were incubated at 37 °C for 2 h and the solution was

centrifuged at 1000g for 10 min. Bacterial pellets were collected at the bottom of the tube. Pellets

were washed with PBS three to four times before dissolving in water to a 10-6 M.32 A drop of the

solution was added on the carbon-coated Cu grid and the excess sample was wiped off with the

filter paper to avoid any aggregation. The grid was then left for 30 min on the bench top so that

the sample can be absorbed onto the grid. The samples on the grids were analyzed and images

were taken with FEI Morgagni 268D TEM instrument.46,47

35

3.4.6 Biofilm assay

The bacteria were grown and the treated with the dendrimers in 96 well plate in a similar

fashion as MIC study. The 96 well plates were left for incubation at 37 °C for 48 hours. The plate

was shaken over an empty tray to remove all the planktonic bacteria. The 96-well plate was then

rinsed in a large tray of water and shaken. Rinsing of the plate in water was done a couple of times.

The plate was placed on the paper towel to get rid of the water from the wells and was laid on

another paper towel and left to dry overnight. The wells were stained with 125 μL 0.1% crystal

violet solution for 10 minutes. The plate was shaken over the empty tray to remove the solution

and the plate was rinsed with water a couple of times until the wells are free of any liquid crystal

violet and left to dry overnight. 200 μL of 30% acetic acid were added to the wells to solubilize

the stained crystal violet and left for 10 min. 125 μL of the solution was transferred from each well

into a flat-bottom 96-well plate. The plate was read at OD 595 nm with a plate reader.43 The

average of the blank wells was subtracted from the OD of each sample that contained sample and

the average of the wells with samples were calculated. The averages of the sample wells were

normalized to the average of the control wells.4

3.5 Conclusion

We have developed a new class of antimicrobial lipidated dendrimers that can mimic the

HDPs. The design was straightforward which allows further development and optimization at ease.

Our findings suggest that amphiphilicity is required for the dendrimer to display potent and broad-

spectrum activity against a range of multidrug-resistant Gram-negative and Gram-positive

bacteria. These lipidated dendrimers were also proven to act as good biofilm inhibitors, further

augmenting their potential of practical applications. Furthermore, these dendrimers could be

36

developed for other biological applications to treat various fungal, viral infections and their activity

is yet to be explored.

3.6 References

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Mimics: Beyond Hydrophobic and Cationic Subunits. Journal of the American Chemical Society

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S.; Kuroda, K., Block versus Random Amphiphilic Copolymers as Antibacterial Agents.

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Hydrophobicity Using a Same-Centered Polymer Approach. Biomacromolecules 2013, 14 (12),

4331-4339; (b) Nederberg, F.; Zhang, Y.; Tan, J. P. K.; Xu, K.; Wang, H.; Yang, C.; Gao, S.; Guo,

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44

CHAPTER 4: UNIMOLECULAR NANOPARTICLES AS POTENT ANTIMICROBIAL

AGENTS

4.1 Introduction

Antibiotic resistance is emerging to be one of the three greatest threats to global public

health in the 21st century.1,2 As a result, significant interest has been devoted to the development

of the new strategies combating drug resistance. Among them, natural cationic host-defense

peptides (HDPs) are believed to be most promising due to their minimum risk of resistance

development.3,4 It is known that conventional antibiotics generally target proteins involved in

bacterial cell wall biosynthesis, or intracellular targets such as nucleic acids.3,5 Such mechanisms

involving defined targets can frequently lead to mutations in bacteria, thereby inducing significant

antibiotic resistance.6 However, HDPs have existed universally in vertebrates for thousands of

years, and they still possess broad-spectrum antibacterial activity. It is intriguing that HDPs have

diverse three dimensional structures, such as α-helices and β-sheets,3 however they are all rich in

cationic and hydrophobic residues. The cyclic and linear forms of the HDP are due to the presence

of disulfide bonds and presence of multiple arginine and lysine amino acids in the sequences. This

also gives them a basic property and amphiphilic nature.7

Bacterial cell membranes are composed mainly of proteins and phospholipids. The

phospholipid layer has a polar hydrophobic glycerol and hydrophobic fatty acid tails which gives

the amphiphilic nature of the membrane. HDPs directly acts on the bacterial membrane. The

positively charged nature of HDPs allows its electrostatic interaction with the negatively charged

45

membrane as the glycoprotein present in the membrane is the binding sites for the HDPs.8, 9 HDPs

are believed to use their hydrophobic domain to penetrate and disrupt membrane lipid layers,

which subsequently leads to leakage of cellular contents and cell death.7,10 This explains why

HDPs have diverse three dimensional structures, because this mechanism of membrane disruption

is based on non-specific biophysical force of hydrophobic and hydrophilic interactions, and

therefore no defined secondary or tertiary structures are necessary.11

Although any therapeutic agents would inevitably develop resistance in their targets,

including cells and organs of the organisms, HDPs may be less prone to the trend because the

membrane disruption by HDPs is non-specific and HDPs lack defined membrane targets.9 It should

also be noted that the action of HDPs may also involve other mechanisms, such as targeting

intracellular and cell wall targets, much as traditional antibiotics do, however, the electrostatic

interaction and/or disruption of bacterial membranes still generally exist in these HDPs, which are

critical for their permeation into bacterial cells.8

One of the successful examples of HDPs is magainin-2,12 which shows moderate

antimicrobial activity against bacteria. Synthetic peptide analogs such as Pexiganan (also known

as MSI-78) have shown much improved activity and entered Phase III clinical trials for the

treatment of diabetic foot ulcers. However, it eventually failed in the clinical trial, largely due to

its moderate antimicrobial activity and high production cost.13 Furthermore, although the

selectivity of HDPs is good, it is still not ideal yet, as they display toxicity such as hemolytic

activity.

The scientific and clinical expansions in the antibiotic development is to advance agents

which can mimic the mechanism of HDP and with relatively low bacterial resistance and

maximum efficacy as well as minimum toxicity. Recently many research studies are launched to

46

develop synthetic peptidomimetics including β-peptides,14-16 peptoids,17 oligoureas,18

AApeptides.19-23 Though the ongoing development in the area of peptidomimetics is promising,

the selectivity problem, manufacturing cost and the efficiency of the synthesis method are

challenging.24-26

Another innovative approach to developing antimicrobial agents is mimicking the HDPs

through biodegradable polymers. Like HDPs, they contain both hydrophobic and hydrophilic

cationic groups in their sequences, which are synthesized by amphiphilic building blocks through

homo-polymerization, or by both hydrophobic monomers and cationic monomers through block-

by-block or random copolymerization. The antibacterial mechanism of action of polymers is

similar to that of HDPs. Some polymers function through interaction with bacterial membranes by

single random sequences,10,27-31 while others are designed to self-assemble into nanoparticles, then

act on the surface of bacterial membranes.27,32,33 It is suggested that through the formation of

nanoparticles, there is an increase of local mass and cationic charges on the bacterial surface which

facilitates disintegration of bacterial membranes.33 However, this limits the mechanism of action

and activity of self-assembled nanoparticles to be higher than their critical micelle concentration

(CMC).33

4.2 Overview of Unimolecular Micelle Hyperbranched Polymers

Unimolecular micelles are a class of small entities of micelles with a discrete core

surrounded by shells which are formed by a covalent bond chain. With the current innovations in

polymer chemistry, unimolecular micelles are functionalized in various architectural designs,

including amphiphilic dendrimers, amphiphilic star polymers and hyperbranched polymers called

polymeric micelles.34 Unimolecular micelle hyperbranched polymers are built from a dendrimer

core where the arms of the core are composed of both hydrophobic and cationic polymers. Bearing

47

special structural and functional properties, biodegradable-unimolecular polymeric micelles are

becoming imperative candidates for drug delivery applications, encapsulating and solubilizing a

hydrophobic guest molecule and antimicrobial applications in the biomedical era.35

The antimicrobial application of the unimolecular micelle is hypothesized to be more

potent and selective compared with the regular antimicrobial polymers, because of the spatial

architectural design, which improves their interaction with the bacterial membrane. Each

unimicelle is acting as a nanoparticle. Such a design is devoid of potential de-assembly of

assembled nanoparticles under low concentrations, ensuring constant high local mass and cationic

charges which is crucial for antimicrobial application mechanism. In addition, the hydrophobic

groups present in the structure may be sequestered in the interior of the micelles, which decreases

the probability of non-specific interaction of nanoparticles with mammalian cells, thereby

enhancing the selectivity towards bacteria. Indeed, these unimicelle nanoparticles can also be

scaled-up to make large quantities, and they are biodegradable, augmenting their potential in vitro

and in vivo applications.33,35 Herein, we present the design and application of unimolecular micelle

hyperbranched polymers as antimicrobial agents.

4.3 Experimental Section

4.3.1 Synthesis of Hyperbranched Polyester Polyacid (HBPP) (Figure4.1)

A weighed portion of 1 equiv of Boltorn H311 was heated to 140 OC for 2h under nitrogen

blowing to break the hydrogen bonds and make it anhydrous. Then it was cooled to 50 OC and

dissolved in 1,4-dioxane. To the heated solution of the Boltorn H311, a stoichiometric amount 5

equiv of maleic anhydride to each dendrimer arm (5 equiv*23) and catalytic amount of stannous

chloride (SnCl2) (1% with respect to the Boltorn weight) were added simultaneously. The mixture

was stirred at a temperature of 100 OC for 36 h. After cooling, the solvent was removed in vacuo

48

and the product was purified by dialysis against methanol for 2 days and distilled water for 2 days

using dialysis tubing MWCO 3500 where methanol and water being replaced twice a day.36 The

product was then freeze dried and characterized by 1HNMR (Figure 4.2).

Figure 4.1 Synthesis of HBPP

Figure 4.2 1H NMR of HBPP

4.3.2 Synthesis of hydroxyl terminated hyperbranched polyester (Figure 4.3)

To synthesize hydroxyl terminated hyperbranched polyester, 1 equiv of hyperbranched

polyester polyacid (HBPP, Figure 4.1) was dissolved in 20 ml DMF and then (5 equiv*23) of

ethanolamine (2-aminoethan-1-ol), (5 equiv*23) EDC, (2 equiv*23) HOBT and (2 equiv*23) DIPEA

base were added. The solution mixture was stirred at room temperature overnight. After the reaction

was completed, work up was done using 100 ml of ethyl acetate and the product was washed with 1N

49

HCl (100 mL×3) and Brine (50 mL×1). The product was then dried over anhydrous sodium sulfate

and the solvent was removed in vacuo to give yellowish oily product, it was then purified in a dialysis

tubing for 4 days using methanol and water.36 The product was then freeze dried and characterized by

1HNMR (Figure 4.4).

Figure 4.3 Synthesis of hydroxyl terminated hyperbranched polyester

Figure 4.4 1H NMR of hydroxyl terminated hyperbranched polyester

4.3.3 Synthesis of Hydrophilic monomer (M1) (Figure 4.5)

Monomer 1 (2-((tert-butoxycarbonyl) amino) ethyl 5-methyl-2-oxo-1,3-dioxane-5-

carboxylate) was synthesized following the published paper in our lab by Jianfeng Cai and Alekhya

Nimmagadda.37

50

Figure 4.5 Monomer 1 (M1) synthesis.

4.3.4 Synthesis of Hydrophobic monomer (M2) (Figure 4.6)

Monomer 2 was prepared according to the previously reported protocol by Yang and

Hedrick.38

Figure 4.6 Monomer 2 (M2) synthesis

4.3.5 Synthesis of Unimolecular micelle hyperbranched polymers (Figure 4.7 & Figure 4.8)

We propose to synthesize both random and diblock unimolecular micelles hyperbranched

polymers (Figure 4.7 & Figure 4.8). As shown in Figure 4.5 & Figure 4.6 first both hydrophilic and

hydrophobic monomers were synthesized following the protocols reported in the published paper of

our lab and a paper from Yang and Hedrick et al.37,38 Hyperbranched polymers were then synthesized

through ring-opening polymerization in the presence of the two complementary monomers and the

unimolecular micelle core.39 To synthesize random unimicelle polycarbonate P6, 1 equiv of hydroxyl

terminated hyperbranched polyesters as a macro initiator and 20 equiv of hydrophilic monomer (M1)

and 20 equiv of hydrophobic monomer (M2) were mixed together and dissolved in 10ml of anhydrous

51

DCM in a N2 purged round bottom flask. To this mixture 1 equiv of 1,8-diazabicyclo undec-7-ene

(DBU) and 1 equiv of 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2- thiourea (TU) catalyst

were added and the reaction was left overnight and the next day the reaction was quenched by 1.1

equiv of benzoic acid and purification of the polymer was done by dialyzing against methanol in a

MWCO 3500 dialysis tubing for 3 days, after that the polymers were then treated with 15 ml of a 1:1

TFA:DCM mixture for 3 h to deprotect the Boc protecting group in order to obtain the free amine

functional groups. And then the polymer was purified again in a dialysis tubing (MWCO 3500) for 2

days in methanol and 2 days in water and it was then freeze dried and a brownish semisolid product

was attained. As such, a series of hyperbranched polymers containing varying numbers of hydrophobic

and hydrophilic groups were prepared in the same manner (Table 4.1).

Figure 4.7 Synthesis of unimolecular nanoparticles with random chains of polycarbonates.

Similarly, Di-block hyperbranched polycarbonate armed unimicelle polymers (Table 4.1)

were synthesized in the same way as the random block polymers. However, the two monomers were

added in two batches, first monomer 1 was added along with the macro initiator, TU catalyst and DBU

base and the reaction runs for 6 h and then monomer 2 was added and the reaction was left for

polymerization for 6 h (Figure 4.8).37

52

Figure 4.8 Synthesis of unimolecular nanoparticles with diblock chains of polycarbonates.

Table 4.1 Unimolecular micelle hyperbranched polymers.

Compound

Type of

Unimicelle polymer

Hydrophobic arms

Hydrophilic arms

P1 Di-block 10 10

P2 Random 10 10

P3 Di-block

15 15

P4 Random

15 15

P5 Di-block

20 20

P6 Random

20 20

P7 Di-block

25 25

P8 Random

25 25

P9 Di-block

30 30

P10 Random

30 30

P11 Di-block

40 40

P12 Random

40 40

4.3.6 Antimicrobial Assay

Antimicrobial assay of the synthesized unimicelle polymers was carried out following similar

protocols as reported in the published papers of our lab.40, 41Two clinically relevant bacterial strains,

Gram-positive Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 33591) and Gram-

negative E. coli (ATCC 25922) bacteria were used to perform the MIC assay. The minimum inhibitory

53

concentration (MIC) is the lowest concentration that completely inhibits the growth of bacteria in 20

h. The MIC was made in sterile 96 well plate, first a single colony of bacteria was isolated and allowed

to grow in 4 ml TSb solution for 12 h at 37 °C in a shaking incubator. Next day the grown culture was

diluted by 100-fold and was shaken for 6 h to attain mild-logarithm phase. To the 96-wellplate 50 µL

of the unimicelle polymer solution in 2-fold serial dilution of TSB were added, then 50 µL diluted

bacterial in TSB medium (1 × 106 CFU/mL) was added to each well. The well-plate was incubated for

20 h at 37 °C and MIC was determined from the Biotek Synergy HT microtiter plate reader absorption

at 600 nm wavelength.

4.4 Results and Discussion

Dendritic polymers, including HBPs in which polar amines, hydroxyl or amide groups

presented in their terminal ends were found to be a highly selective and potent antimicrobial

compounds in addition to the straight forward synthesis method and high yield production.42 We are

here presenting a contemporary design of unimolecular micelle hyperbranched polymers by

incorporating polycarbonate monomers in the arms of a dendrimer core (Boltorn™ H311) to function

as antimicrobial agents in biomedical era. We chose polycarbonates to be incorporated in the arms of

unimolecular micelle as polycarbonates are biocompatible, biodegradable, and can be polymerized

easily through ring-opening polymerization (ROP). Moreover, Yang and Hedrick et al have already

demonstrated the polycarbonates potential for antimicrobial applications.38

Polyol Boltorn™ (aliphatic hyperbranched polyester) are dendritic polymers which have a

highly branched flexible backbone that can embrace more than 20 terminal hydroxyl groups. In our

study, Boltorn™ H311 (MW 5300 g/mole) which has 23 terminal hydroxyl branches of functional

groups was used for the synthesis of the unimolecular nanoparticle as it can be adapted easily to

dendrimers as well as unimicelles.43,44 As per our design the polycarbonate monomers have to be

54

complexed to the arms of the Boltorn, so first the hydroxyl terminal of the Boltran has to be lengthened.

To achieve this approach then, first the hydroxyl terminal of Boltran was transformed into carboxyl

terminal to attain a high reactivity for the next reactions by reacting the Boltran H311-OH with maleic

anhydride in the presence of tin(II) chloride in dioxane (Figure 4.1). Once the reactive carboxyl group

was obtained ethanolamine was coupled to it to produce an elongated hydroxyl terminal arm of Boltorn

(Figure 4.3).36

Polymerization was done through ROP method, and 12 unimolecular micelle hyperbranched

polymers were synthesized by incorporating the two monomers M1 and M2 in to the arms of the

hydroxyl terminated hyperbranched polyester H311 macro initiator (Figure 4.7 and Figure 4.8). To

see the disparity in the antimicrobial activity among the 12 polymers, 6 were diblock polymers and

the rest were random block polymers (Table 4.1).

Earlier study in our lab discloses, polycarbonate polymer which has 20 equiv hydrophilic and

20 equiv hydrophobic monomers with benzyl alcohol initiator was the most potent towards gram

positive bacteria but were not active against gram negative bacteria.37 Bearing that in our mind we

hypothesized by manipulating the same polycarbonate polymers in unimolecular micelle core could

enhance the antibacterial activity of the polymers towards broad spectrum.

The antibacterial activity of the synthesized polymers was studied against Gram-positive

Methicillin-resistant Staphylococcus epidermidis (MRSE, RP62A) and Gram-negative E. coli (ATCC

25922) bacteria. As the antimicrobial activity is shown in Table 4.2 both diblock and random polymers

displayed significant antibacterial activity against Gram positive bacteria and to our delight diblock

polymer P5 and random block polymers P6 and P12 presented broad spectrum activity against both

gram positive and gram-negative bacteria.

55

Polymers P1and P2 which have the same number of hydrophilic and hydrophobic units (10

equiv:10 equiv) displayed potent antimicrobial activity (0.75µg /ml) against MRSA but they were not

active to gram negative bacteria, this could be due to the desired amphiphilicity was not obtained to

break the extra outer membrane of the gram-negative bacteria.

Table 4.2 The Antibacterial Activity of Unimolecular micelle hyperbranched polymers.

Polymer

Type of

Copolymer

Hydrophilic

units

Hydrophobic

units

MIC- MRSA

(µg/mL)

MIC- E. coli

(µg/mL)

P1 Di-block 10 10 0.75 NA

P2 Random 10 10 0.75 NA

P3 Di-block 15 15 6~12.5 NA

P4 Random 15 15 3~6 NA

P5 Di-block

20 20 6~12.5 12.5~25

P6 Random

20 20 1.5~3 12.5~25

P7 Di-block

25 25 6~12.5 NA

P8 Random

25 25 6~12.5 NA

P9 Di-block

30 30 6~12.5 NA

P10 Random

30 30 6~12.5 NA

P11 Di-block

40 40 12.5~25 NA

P12 Random

40 40 6~12.5 12.5~25

Next, we synthesized a series of polymers (P3, P4, P5 and P6) by increasing the number of

equivalents of hydrophilic and hydrophobic monomers and they were tested against both strains of

bacteria, among them polymers P6 (random block polymer) displayed potent activity against MRSA

(1.5 µg/mL) and moderate activity (12.5µg/mL) against gram negative E. coli, similarly the diblock

56

polymers P5 displayed broad spectrum activity against MRSA (6 µg/mL) and E. coli (12.5 µg/mL).

This result matched with our hypothesis that we propose the unimolecular micelle hyperbranched

polymer with 20 equiv of hydrophilic and hydrophobic monomers might show broad spectrum

activity. The broad-spectrum activity is probably obtained due to more interaction that is created

between the hyperbranched polymers and the outer membrane of the Gram-negative bacteria with the

increase of both hydrophilic and hydrophobic monomers in the arms of the Boltorn.

Encouraged by the MIC result of P5 and P6, we synthesized 6 more polymers by increasing

the hydrophilic and hydrophobic residues. Diblock polymer P11 and random polymer P12 which had

two times more hydrophilic and hydrophobic groups than the potent polymer P6 displayed lower

antimicrobial activity. Diblock polymer P11 displayed 12 µg/mL MIC towards Gram positive bacteria

and its activity totally lost against gram negative E. coli, similarly the random block polymer P12

displayed lower activity towards Gram positive bacteria 6 µg/mL but the activity against Gram

negative bacteria remains the same as polymer P6. The decreased activity of P11 and P12 might be

caused by the reduced interaction of the cationic region of the polymer with the negatively charged

bacterial membrane. From our findings we can propose that obtaining the desired amphiphilicity of

the polymer is crucial for its antimicrobial potency and from the obtained data we were able to identify

the number of equivalence of the hydrophobic and hydrophilic monomers required for potent and

broad-spectrum activity. Consistent with the previous study in our lab,37 polymers developed from 20

equiv of each hydrophilic and hydrophobic monomer showed potent and broad-spectrum activity.

4.5 Conclusion

In conclusion, we reported the design and development of potent antimicrobial unimolecular

micelle hyperbranched polymers. A series of polymers were synthesized and tested for their

antibacterial activity, and the MIC results disclose that the unimolecular unimicelle hyperbranched

57

polymers have broad-spectrum activity against both Gram-positive and Gram-negative bacteria

especially the random block polymers. Furthermore, from our finding we can conclude the potency

of the polymers is determined by its amphiphilicity. This is because the interaction of the bacteria with

a polymer depends on the nature of the amphiphilicity. Supplementary studies including hemolytic

assay, biofilm assay and TEM will be done for the potent polymers to determine the mechanism of

action of the polymers. Moreover, the design of unimolecular micelle is significant for synthesizing

advanced polymers by incorporating distinct kinds of monomers and it may lead to the advance of a

new class of antimicrobial polymers.

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CHAPTER 5: CONCLUSION AND FUTURE DIRECTIONS

Here, in Chapter 5 we sum up the research findings of the three projects presented in the

previous chapters. We also propose some important ideas which could be helpful to further update

those projects or related project. The three projects compiled in this thesis are all aimed to study and

develop broad spectrum antibacterial biodegradable polymers including polycarbonate polymers,

dendrimers and unimolecular nano particle micelles.

We have developed biodegradable polycarbonates from both peptide and non-peptide

analog monomers through a ring opening polymerization technique. Though the activity was not

broad spectrum, the antibacterial activity and selectivity against clinically relevant and drug

resistant Gram-positive bacteria was promising. As our finding reveals, the incorporation of

halogens in the polymer enhances the antibacterial activity, the present polymers could be further

modified and designed so that broad spectrum could be achieved. The contemporary design can

be obtained through incorporating a halogen group into similar bioactive peptides that encompass

cationic and hydrophobic functional groups to make the monomer by itself amphiphilic. This

design could potentially enhance the amphiphilicity of the whole polymer so that broad spectrum

activity can be achieved.

We have also designed and synthesized both lipidated dendrimers and hyperbranched

unimolecular nanoparticle polymers as part of developing broad spectrum acting antimicrobial

polymers. The dendrimers showed potent antibacterial activity towards Gram negative and Gram-

positive bacteria. Furthermore, these dendrimers could be developed for other biological

62

applications to treat various fungal and viral infections. It can also be manipulated as a drug

delivery agent due to the presence of the internal voids in its structure.

Lastly, our research study was focused in developing hyperbranched unimolecular

nanoparticle polymers for antibacterial applications. The spatial architectural design of those micellar

structures allows us to build more branches in the arms of the core and this allowed more interaction

of the polymer and the bacterial membrane. A preliminary study has been done and the obtained result

is promising; the polymers displayed broad spectrum activity.