application of nanotechnology for targeted delivery of antibacterial enzymes and for enzyme-based

223 Pages·2017·3.93 MB·English

Clemson University
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5-2011
APPLICATION OF NANOTECHNOLOGY
FOR TARGETED DELIVERY OF
ANTIBACTERIAL ENZYMES AND FOR
ENZYME-BASED COATINGS ON MEDICAL
DEVICES AND IMPLANTS
Rohan Satishkumar
Clemson University, [email protected]
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Recommended Citation
Satishkumar, Rohan, "APPLICATION OF NANOTECHNOLOGY FOR TARGETED DELIVERY OF ANTIBACTERIAL
ENZYMES AND FOR ENZYME-BASED COATINGS ON MEDICAL DEVICES AND IMPLANTS" (2011).All Dissertations. 740.
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APPLICATION OF NANOTECHNOLOGY FOR TARGETED
DELIVERY OF ANTIBACTERIAL ENZYMES AND FOR ENZYME-
BASED COATINGS ON MEDICAL DEVICES AND IMPLANTS

A Dissertation
Presented to
the Graduate School of
Clemson University

In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Bioengineering

by
Rohan Satishkumar
May 2011

to
Dr. Alexey Vertegel, Committee Chair
Dr. Naren Vyavahare
Dr. Ken Webb
Dr. Antony Guiseppi-Elie

i
ABSTRACT

The frequency of S. aureus infection and subsequent biofilm formation associated with
vascular catheterization has been increasing in recent years and often begins as a local
colonization at the site of the catheter insertion. Antimicrobial enzymes and peptides, which are
effective against a broad range of pathogens and low rates of resistance, have attracted attention
as promising alternative candidates in treatment of infections caused by antibiotic resistant
bacteria. The use of nanoparticles as carriers for enzymes, in addition to their size, charge, high
surface area per volume etc. offers targeted delivery of enzymes to pathogenic bacteria. We
proposed to use nanoparticles as surfaces for targeted delivery of antibacterial enzymes and as
„surrogate‟ surface coatings on indwelling central venous catheters (CVCs) to inhibit bacterial
colonization and subsequent biofilm formation.
It was shown that nanoparticle charge can be used to enhance delivery and increase
bactericidal activity of an antimicrobial enzyme, lysozyme. In the case of bacterial lysis assay
with a Gram-positive bacterium Micrococcus lysodeikticus, activity of lysozyme conjugated to
positively charged nanoparticles was approximately twice as high as that of free lysozyme. This
was believed to occur through charge-directed targeting of enzyme-nanoparticle conjugates to
negatively charged bacterial cell walls through enhanced electrostatic interactions. In a clinically
more relevant model, we studied antimicrobial activity of lysostaphin against S. aureus for both
lysostaphin-coated and lysostaphin-antibody coated nanoparticle conjugates at different enzyme:
antibody: nanoparticle ratios. At the highest antibody loading, bacterial lysis rates for antibody-
lysostaphin-coated samples were significantly higher than for plain lysostaphin-coated samples
and free enzyme due to multiple-ligand directed targeting of antibody molecules to bacterial cell
walls (p<0.05).
ii
We also performed in vitro experiments to evaluate the inhibition of bacterial colonies
adhering to a surface. Bacterial infections by S. aureus strains are among the most common
postoperative complications in surgical hernia repair with synthetic polymer meshes. Colony
counting data from the broth count (model for bacteria in wound fluid) and wash count (model
for colonized bacteria) for the enzyme-coated samples showed significantly decreased number of
colony forming units (CFU) when compared to uncoated samples (p< 0.05). A pilot in vivo study
showed a dose-dependent anti-S. aureus efficacy of lysostaphin-coated meshes in a rat model.
Finally, we observed that that coating of nanoparticles overall did not significantly
improve binding yield, leaching, durability and antibiofilm activity of enzymes adsorbed on
catheter segments (p>0.05). Alternatives to coating catheter surfaces using covalent chemistry
through functional groups on nanoparticles either directly or through appropriate crosslinkers
could result in significantly higher enzyme loadings, better stability and long term durability for
future applications. The approach developed here is universal and can potentially be used for
treatment of other medical device-associated infections. Moreover, use of antibacterial enzyme-
NP conjugates can eventually be expanded for intravenous administration, which will further
broaden its range of application.

iii
DEDICATION
I would like to dedicate this work to my parents who have always been there for me with their
unwavering support, guidance and strength. Thank you for all your love and prayers!

iv
ACKNOWLEDGEMENTS
I would like to sincerely thank my advisor, Dr. Alexey Vertegel, for his guidance and
patience through the past 5.5 years of my graduate studies at Clemson University. His high
expectations, continuous support and mentorship are greatly appreciated. I am grateful to the
other committee members, Dr. Naren Vyavahare, Dr. Antony Guiseppi-Elie and Dr. Ken Webb
for their time and active support in the completion of this dissertation. I would like to thank Dr.
Dan Simionescu and Dr. Martine Laberge for their support and guidance from the very
beginning of my graduate study.
A special thanks to the all the members of the Nanobiomaterials laboratory for being
there during my study at Clemson, sharing all those great moments and engaging in all the
discussions with each and every one of you has been memorable. I would like to acknowledge
Gary Thompson, Sriram Sankar and Vladimir Reukov specifically for their help and support. I
would also like to thank Sravya Durugu for her encouragement and constant support. Dr. David
Bruce and Iris Cordova (BET study), Dr. Meredith Morris (Flow cytometry), Dr. Terry Bruce
(Confocal microscope), Dr. Joann Hudson, Donald Musvee and Dayton Cash (SEM imaging),
Dr. Yuliya Yurko and Dr. Todd Heniford ( In vivo studies at Carolinas Medical Center). To all
the staff in the Bioengineering department for their help and cheerful disposition – Thank you so
much! I would like to personally thank all my friends and colleagues who have studied with me
during my years at Clemson for their help and support.
Finally, I would like to acknowledge the financial support and assistance provided by
Carolinas Medical Center, ViMedrx and the Department of Bioengineering.

v
TABLE OF CONTENTS

TITLE ......................................................................................................................................... i
ABSTRACT .............................................................................................................................. ii
DEDICATION .......................................................................................................................... iv
ACKNOWLEDGEMENTS.........................................................................................................v
TABLE OF CONTENTS .......................................................................................................... vi
LIST OF FIGURES .................................................................................................................. ix
LIST OF TABLES ...................................................................................................................xiv
CHAPTER ONE .........................................................................................................................1
INTRODUCTION ...................................................................................................................1
CHAPTER TWO ........................................................................................................................4
LITERATURE REVIEW ........................................................................................................4
2.1. Bacteria .........................................................................................................................4
2.2. Antibiotic resistance .................................................................................................... 10
2.3. Antimicrobial proteins and peptides ............................................................................ 16
2.4. Enzyme immobilization ............................................................................................... 24
2.5. Nanotechnology for enzyme immoblization ................................................................ 32
2.6. Nanoparticles .............................................................................................................. 35
2.7. Enzyme-nanoparticle conjugates as antibacterials – Targeted delivery ......................... 41
2.8. Enzymes as antibacterial coatings on medical devices and implants............................. 43
CHAPTER 3 ............................................................................................................................. 45
SPECIFIC AIMS AND SIGNIFICANCE .............................................................................. 45
3.1. Clinical significance .................................................................................................... 45
3.2. Specific aims ............................................................................................................... 48
vi
CHAPTER 4 ............................................................................................................................. 53
CHARGE-DIRECTED TARGETING OF ANTIMICROBIAL ENZYME-NANOPARTICLE
CONJUGATES ..................................................................................................................... 53
4.1. Introduction ................................................................................................................. 53
4.2. Materials and methods ................................................................................................. 54
4.2.2. Methods ................................................................................................................... 56
4.3. Results ........................................................................................................................ 63
4.4. Discussion ................................................................................................................... 73
CHAPTER 5 ............................................................................................................................. 77
ANTIBODY-DIRECTED TARGETING OF LYSOSTAPHIN ADSORBED TO
POLYLACTIDE NANOPARTICLES INCREASES ITS ANTIMICROBIAL ACTIVITY
AGAINST S.AUREUS IN VITRO ........................................................................................ 77
5.1. Introduction ................................................................................................................. 77
5.2. Materials and methods ................................................................................................. 79
5.3. Results and Discussion ................................................................................................ 85
CHAPTER 6 ........................................................................................................................... 100
EVALUATION OF ANTIMICROBIAL ACTIVITY OF LYSOSTAPHIN-COATED
HERNIA REPAIR MESHES ............................................................................................... 100
6.1. Introduction ............................................................................................................... 100
6.2. Materials and methods ............................................................................................... 102
6.3. Results ...................................................................................................................... 110
6.4. Discussion ................................................................................................................. 120
CHAPTER 7 ........................................................................................................................... 125
APPLICATION OF ENZYMES AS ANTIBACTERIAL COATINGS ON PLAIN AND
NANOPARTICLE COATED CATHETER SEGMENTS .................................................... 125
7.1. Introduction ............................................................................................................... 125
vii
7.2. Materials and methods ............................................................................................... 127
7.3. Results and Discussion .............................................................................................. 139
7.4. Conclusion ................................................................................................................ 163
CHAPTER 8 ........................................................................................................................... 165
CONCLUSIONS AND FUTURE RECOMMENDATIONS ................................................ 165
8.1 Conclusions ................................................................................................................ 165
CHAPTER 9 ........................................................................................................................... 169
CHAPTER 10 ......................................................................................................................... 177
References ........................................................................................................................... 177

viii
LIST OF FIGURES
Page
2.1 A) Biofilm growing on a creek and B) S .aureus biofilm growing on a catheter segment.......5
2.2 Bacterial cell walls for Gram positive and Gram negative bacteria…………………………..7
2.3 Alternatives to antibiotics - AQUACEL® Ag..........................................................................14
2.4 Schematic of affinity enzyme immobilization through the Avidin-Biotin system…………..30
2.5 Nanotechnology for enzyme immobilization - Basic structures of nanoscale biocatalysts.....34
2.6 Reaction scheme showing different covalent immobilization techniques of proteins on
polymer nanoparticles through different functional groups ………………..................................38
4.1 Schematic of lysozyme conjugation to nanoparticles..............................................................60
4.2 Efficiency of covalent attachment of lysozyme to polystyrene latex nanoparticles for
samples prepared by (A) direct conjugation and (B) conjugation via PEG spacers......................64
4.3 Results of bacterial lysis assay analyzing effect of enzyme activity for samples directly
conjugated or through PEG spacers purified by centrifugation or dialysis...................................66
4.4 Results analyzing effect of control nanoparticles on bacterial lysis…………………………68
4.5 Tapping mode AFM images comparing the degree of aggregation for samples purified
by dialysis and centrifugation........................................................................................................69
ix