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Antimicrobial reverse-thermal gel for surgical applications

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Title:
Antimicrobial reverse-thermal gel for surgical applications
Creator:
Bortot, Maria Belen ( author )
Place of Publication:
Denver, Colo.
Publisher:
University of Colorado Denver
Publication Date:
Language:
English
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1 electronic file (88 pages). : ;

Thesis/Dissertation Information

Degree:
Master's ( Master of Science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Bioengineering, CU Denver
Degree Disciplines:
Bioengineering
Committee Chair:
Park, Daewon
Committee Members:
Patel,Vikas
Vazquez-Torres, Andres

Subjects

Subjects / Keywords:
Surgical wound infections ( lcsh )
Anti-infective agents -- Effectiveness ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
A surgical site infection (SSI) is defined as an infection that is developed during a surgical procedure or up to 30 days after. In the US, 2-5% of patients undergoing surgery suffer from SSIs. These patients have 2-11 times higher risk of death 1 . A main aspect of SSI prevention is the use of an antiseptic impregnated plastic surgical incision drape. However, these drapes present numerous pitfalls. Amongst the most severe, are that the placement process of the drapes is time consuming, they often do not remain well-attached and they only provide temporary protection. Polymers functionalized with quaternary ammonium groups have shown high inherent antimicrobial properties. The antimicrobial activity is due to the disruption of the negatively charged cell wall or membrane. We developed a polymer-based antimicrobial surgical coating that can act as a surgical incision drape. This polymer is specifically designed to possess a reverse thermal gelling property to ease the application process. It can be sprayed onto the skin and turns into a thin adhesive gel when it comes into contact with the body and can then be removed by washing with soap/water at low temperatures (less than 20°C). Methods: Poly(serinol hexamethylene urea) (PSHU) was
Thesis:
Thesis (M.S.)--University of Colorado Denver. Bioengineering
Bibliography:
Includes bibliographic references.
System Details:
System requirements: Adobe Reader.
General Note:
Department of Bioengineering
Statement of Responsibility:
by Maria Belen Bortot.

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University of Colorado Denver
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|Auraria Library
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903536522 ( OCLC )
ocn903536522

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Full Text
ANTIMICROBIAL REVERSE-THERMAL GEL FOR SURGICAL APPLICATIONS
by
MARIA BELEN BORTOT
M.S., Universidad Nacional de Cuyo, Instituto Balseiro, Argentina, 2012
B.E., The University of Sydney, Australia, 2008
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Science
Bioengineering
2014


This thesis for the Master of Science degree by
Maria Belen Bortot
has been approved
by
Daewon Park, Chair
Vikas Patel
Andres Vazquez-Torres
September 2, 2014


Maria Belen Bortot (M.S., Bioengineering)
Antimicrobial reverse-thermal gel for surgical applications
Thesis directed by Assistant Professor Daewon Park.
ABSTRACT
A surgical site infection (SSI) is defined as an infection that is developed during a surgical
procedure or up to 30 days after. In the US, 2-5% of patients undergoing surgery suffer from
SSIs. These patients have 2-11 times higher risk of death. During the surgery, one of the main
sources for pathogens is often thought to be the skin surface. Skin antiseptic preparations
along with antiseptic-impregnated surgical incision drapes (SIDs) are used as a barrier to
prevent re-colonization and immobilize the organisms that might have survived the treatment.
However, these drapes present numerous pitfalls. Amongst the most severe are that the
placement process of the drapes is time consuming, they often do not remain well-attached
and they only provide temporary protection. Similarly, there are risks associated with
epidermal cell layer detachment during removal, exposing bacteria found beneath the skin,
thereby increasing risk of secondary bacterial infection.
Polymers functionalized with quaternary amine groups have shown high inherent
antimicrobial properties. The positive charges on these functional groups cause disruption of
the negatively charged bacterial cell wall.
In order to overcome the limitations associated with SIDs, we developed a polymer-based
antimicrobial surgical coating that can act as a SID. This polymer is specifically designed to
possess a reverse thermal-gelling property to ease the application process. It can be sprayed
in


onto the skin, turns into a thin adhesive gel when it comes into contact with the body, and can
then be removed by washing with soap/water at low temperatures (less than 20C).
The form and content of this abstract are approved. I recommend its publication.
Approved: Daewon Park
IV


ACKNOWLEDGMENTS
I would like to thank my friends, family, professors, teachers, and mentors that contributed to
my academic and personal development throughout this process; without them, this would
not have been possible.
First and foremost, I want to thank my academic and thesis advisor, Dr. Park, for taking me
on as a student and being extremely patient and excellent guide. Thanks, Dr. Park. I learned
lots!
I would also like to thank the other members of my Committee, Dr. Vazquez-Torres and Dr.
Patel, for being so responsive every time I reached out and for encouraging and motivating
me.
At the same time, I want to thank Melissa Laughter and Lindsay Hockensmith, two amazing
friends that shared this whole experience with me. I learned so much from them in and
outside of the lab. My time in Colorado would not have been the same without them, and I
am extremely lucky and thankful for having met them and shared so many great experiences.
Similarly, I want to thank all the Translational Biomaterial Research Laboratory members,
the ones that have left and the ones that are still with us.
Finally, I would like to dedicate this thesis to my family. First and foremost, I want to thank
my sister, as she motivated me to study and believed in me every step of the way, and my
parents, my dad for being an amazing guide and my mom for following me around
everywhere Fve moved to. And I would also like to thank Paschal Duru, my boyfriend, for
sharing this experience with me and encouraging me every day.
v


TABLE OF CONTENTS
Chapter
1. Introduction..........................................................................1
1.1. Background...................................................................1
1.2. Surgical Site Infections.....................................................2
1.3. Preventative Measures for SSIs...............................................3
1.4. Objective of This Study......................................................6
2. Literature Review.....................................................................8
2.1. Antimicrobial Mechanisms of Action...........................................8
2.2. Antimicrobial Polymers......................................................11
2.3. Reversible-gelling Properties...............................................17
3. A reverse-thermal gel with antimicrobial activity....................................22
3.1. New Approach: Polyethyleneimine.............................................23
3.2. New Polymer.................................................................25
4. Materials and Methods................................................................26
4.1. Materials...................................................................26
4.2. Polymer Synthesis...........................................................26
4.3. Polymer Characterization....................................................29
4.4. Antimicrobial Tests.........................................................30
4.5. Functional Tests............................................................32
4.6. Cytotoxicity Test of Q-PEI-PNIPAAm and Q-PSHU-PNIPAAm.......................34
5. Results and Discussion...............................................................36
5.1. Preliminary Studies.........................................................36
vi


5.1.1. Antimicrobial Tests Using Q-PSHU-PNIPAAm...................36
5.1.2. Cytotoxicity Tests of Q-PSHU-PNIPAAm.......................38
5.1.3. Conclusion from Tests Performed with Q-PSHU-PNIPAAm........40
5.2. Development of a New Polymer: Q-PEI-PNIPAAm.....................41
5.2.1. Polymer Characterization...................................41
5.2.2. Antimicrobial Tests using Q-PEI-PNIPAAm....................48
5 .3. Cytotoxicity Tests of Q-PEI-PNIPAAm............................55
5.4. Functional Tests................................................58
6. Conclusions and Future Directions.......................................62
References..................................................................66
Appendix....................................................................73
Vll


Table
LIST OF TABLES
1.LCST values of each polymer.
48
vm


LIST OF FIGURES
Figure
Figure 1.A: Epidermal cell detachment during the removal of a SID, possibly leading to
secondary infection by bacteria found beneath the skin. B: Surgical incision drape on the skin
before surgery. C: Surgical incision drape during surgery..............................6
Figure 2. Mode of action of various antimicrobial compounds............................9
Figure 3. Outer membrane/cell wall differences between Gram-positive and Gram-negative
bacteria [21] [22].....................................................................11
Figure 4. PNIPAAm structure and behavior above and below the LCST......................20
Figure 5. Polymer backbone with quatemized amine groups................................23
Figure 6. Structure of branched Polyethyleneimine......................................24
Figure 7. Results from the first antibacterial test against S. aureus using different polymer and
bacterial concentrations...............................................................37
Figure 8. Results from the first MTT assay conducted using Q-PSHU-PNIPAAm at different
concentrations. The only statistically significant difference was observed between the
experimental samples and positive control (cells cultured with 5% DMSO). indicates p >
0.01,and *** indicates p < 0.01.).................................................39
Figure 9. Results from the second MTT assay conducted using Q-PSHU at different
concentrations....................................................................40
Figure 10. !H NMR (500 MHz, CDC13) analysis of the PEL Peak assignments were
corroborated by NMR modeling (Advanced Chemistry Development).....................42
Figure 11.!H NMR (500 MHz, CDC13) analysis of the PEI-PNIPAAm (30% conjugation).
Peak assignments were corroborated by NMR modeling (Advanced Chemistry
Development)......................................................................43
Figure 12. 4 NMR (500 MHz, CDC13) analysis of the Q-PEI. Peak assignments were
corroborated by NMR modeling (Advanced Chemistry Development).....................44
Figure 13. Zeta potential measurements of quatemized and non-quatemized polymers.
Significant statistical difference was noted between the values indicates p<0.01).45
Figure 14. LCST of PEI-PNIPAAm (20% and 50% conjugation) and Q-PEI-PNIPAAm.......46
Figure 15. LCST of PEI-PNIPAAm and Q-PEI-PNIPAAm (both 30% conjugation)..........47
IX


Figure 16. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% conjugation) were added to
stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120
minutes. Numbers indicate reductions in logi CFU/ml......................................49
Figure 17. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to
stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120
minutes. Numbers indicate reductions in logi CFU/ml......................................50
Figure 18. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to
stationary-phase cultures of Staphylococcus aureus subsp. aureus MuS (MRSA/hetero-VISA).
Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in logi
CFU/ml................................................................................52
Figure 19. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% and 30% conjugation) were added to
stationary-phase cultures of Staphylococcus epidermidis. Samples were taken after 30, 60,
and 120 minutes. Numbers indicate reductions in logi CFU/ml.........................53
Figure 20. Q-PEI-PNIPAAm and PEI-PNIPAAm (30 and 20% conjugation) were added to
stationary-phase cultures of E. coli. Samples were taken after 30, 60, and 120 minutes.
Numbers indicate reductions in logi CFU/ml...........................................54
Figure 21.Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to log-phase
cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes.
Numbers indicate reductions in logi CFU/ml...........................................55
Figure 22. No statistical difference between negative control and cells exposed to PNIPAAm
and PEI-PNIPAAm. Statistically significant difference was observed between the
experimental samples, negative and positive control (cells cultured with 5% DMSO)
indicates p > 0.05, and indicates p < 0.05.). No statistical difference between cells exposed
to Chlorhexidine (FDA approved) and Q-PEI-PNIPAAm....................................57
Figure 23. Bacterial growth after inoculation with used SIDs. For the test, pieces of SIDs
were taken right after a surgery, inoculated on 5 spots on an agar plate. Surprisingly, a
formation of bacterial colony was observed on 2 spots, indicating bacterial recolonization.. 59
Figure 24.1 x 1 cm glass slides coated with polymer and then sprayed with bacteria...60
Figure 25. Glass slide that was half coated with the polymer and half uncoated. The left insert
shows colonies that grew on the non-coated edges of the slide.........................60
Figure 26. Image of the glass slide sprayed with the polymer-ethanol solution before soaking
in water..............................................................................61
Figure 27. A) Image of the glass slide after it was soaked for 24 hours in water. B) A razor
blade was used to remove part of the polymer on the pre-soaked slide................61


A 1.4 NMR (500 MHz, CDC13) analysis of the PEI. Peak assignments were corroborated by
NMR modeling (Advanced Chemistry Development)73
B 1.Agar plates showing bacterial colonies. Dilutions were performed and each samples
triplicates..............................................................................73
B 2. Agar plates showing bacterial colonies. Time points at t= 60 and 120 minutes. Dilutions
were performed and each samples triplicates..............................................74
B 3. Agar plates showing bacterial colonies subjected to PEI-PNIPAAm ...................74
B 4. Agar plates showing no bacterial growth after 30 minute antibacterial test with Q-PEI-
PNIPAAm (30% conjugation)................................................................75
B 5. Agar plates showing no bacterial growth. The bacteria had been subjected to Q-PEI-
PNIPAAm (30% conjugation) for 60 minutes.................................................75
B 6. Agar plates showing no bacterial growth. The bacteria had been subjected to Q-PEI-
PNIPAAm (30% conjugation) for 120 minutes................................................75
xi


LIST OF ABBREVIATIONS
ACA 4,4,-AZOBIS(4-CYANOVALERIC ACID)
AMHP 2,2'-AZOBIS[2-METHYL-N-(2- HYDROXYETHYL)PROPIONAMIDE]
DMEM DULBECCO'S MODIFIED EAGLE'S MEDIUM
DMF DIMETHYLFORMAMIDE
DMSO DIMETHYL SULFOXIDE
EDC TV-P-DIMETHYLAMINOPROPYLVA^-ETHYLCARBODIIMIDE HYDROCHLORIDE
FT-IT FOURIER TRANSFORM INFRARED
GPC GEL PERMEATION CHROMATOGRAPHY
HDI HEXAMETHYLENE DIISOCYANATE
LCST LOWER CRITICAL SOLUTION TEMPERATURE
MTT MW 3-(4,5-DIMETHYLTHIAZOL-2-YL)-2,5- DIPHENYLTETRAZOLIUM BROMIDE MOLECULAR WEIGHT
NHS 7V-HYDROXYSUCCINIMIDE
NMR NUCLEAR MAGNETIC RESONANCE
PBS PHOSPHATE-BUFFERED SALINE
PEI POLYETHYLENIMINE
PNIPAAM POLY(7V-ISOPROPYLACRYLAMIDE)
PSHU POLY (SERINOL HEXAMETHYLENE UREA)
QUATs QUATERNARY AMMONIUM COMPOUNDS
RTG REVERSE THERMAL GEL
SID SURGICAL INCISION DRAPES
XII


TFA
TRIFLUOROACETIC ACID
uv
ULTRAVIOLET
XIII


1.Introduction
1.1. Background
Human microbiota is the term used to refer to the vast number of microbes that reside in and
on the human body. These organisms play both beneficial and harmful roles. Gut
flora/microbiota, for example, aid in the digestion of certain foods and play a role in the
production of some vitamins (e.g., B and K). Similarly, human skin is also host to microbes
that are regarded as pathogens, potential pathogens, or innocuous symbiotic organisms [1].
Four main species of bacteria predominate on the superficial layer of the epidermis:
Diphtheroids (Q.g., corynebacteria), Staphylococci (Q.g., S. aureus), Micrococci, Streptococci
(either alpha (a) or gamma (y) hemolytic), and the Enterococci [2] [3][4]. Depending on
various factors, such as moisture and body area, the species that predominates will vary. The
number of bacteria may range from approximately 1,000 to more than 10 million organisms
per square centimeter of skin surface, with Gram-positive organisms usually predominating
[2][5].
The intact surface of a healthy epidermis prevents bacteria from penetrating. However, this
protection is lost if the integrity of the epidermis is broken, for example, by cuts or abrasions.
During surgeries, the skin barrier is broken and patients are at risk of bacteria invading the
surgical site, leading to infections. Accordingly, the use of antimicrobial agents is required
and protocols to reduce risk of infection are a part of common clinical practice. The risk of
hospital-acquired infections, along with the emergence of multi-drug-resistant bacteria such
as methicillin-resistant strains of Staphylococcus aureus (MRSA), vancomycin-resistant
enterococci^ and Pseudomonas aeruginosa^ has led to a growing interest in the development
of alternative antimicrobial agents.
1


Advances in polymer science have enabled the synthesis of novel material structures with
added functionalities. Advances in the area of microscopy (transmission electron microscopy,
scanning electron microscopy, and fluorescence microscopy) facilitated the study of
interactions between materials, biological molecules, and cells. This allowed the development
of new polymeric materials with specific functionalities, such as antimicrobial properties.
The literature pertaining to this topic is vast, and the new materials developed to either kill or
inhibit bacterial colonization vary depending on the application (food, animal feed, water
purification systems, or medical industry).
1.2. Surgical Site Infections
A surgical site infection (SSI) is defined as an infection that develops in the body as a
consequence of a surgical procedure during and up to 30 days after the procedure. They
manifest as pus or swab with >106 colony-forming units (CFU) per mm3 tissue [6]. As
mentioned previously, they are generally caused by Staphylococci, Streptococci, Diphtheroid
organisms^ Pseudomonas, and Propionibacterium, which are constantly present on patients5
skin |7].
In the United States, SSIs account for 14-16% of all hospital-acquired infections.
Approximately 70% of SSIs are superficial infections; the remaining 30% involve implanted
materials, deeper tissues, or organs. The Institute for Healthcare Improvement stated that
SSIs in the United States lead to an average 7.5-day increase in patient hospitalization periods
[8]. This nas an estimated cost of $130 million to $845 million per year [9]. At the same time,
the rising incidence of hospital-acquired, antibiotic-resistant Staphylococcus aureus
infections is a major concern. Almost 50% of hospital-acquired Staphylococcus aureus
infections are methicillin resistant [10][11].
2


1.3. Preventative Measures for SSIs
There are numerous Protocols and Practice Recommendation documents established to
prevent and reduce the incidence of SSIs [12][7][13]. During surgery, the main source of
pathogens is thought to be the skin surface; hence, the skin preparation step before the
procedure is critical. Standard surgical pre-operative care includes hair removal followed by
' skin prepping^ to achieve asepsis of the surgical area through the use of general topical
antiseptics. The underlying mechanism of antimicrobial activity varies depending on the
agent used; the main agents are [14]:
1.70% isopropyl alcohol, which acts by denaturing proteins. It is a fast-acting fungicidal and
virucidal liquid and is also effective against Gram-positive and Gram-negative organisms.
The only drawback to using it is its short-term action.
2.0.5% chlorhexidine is a quaternary ammonium compound and acts by disrupting the
bacterial cell wall. It is most effective against Gram-positive organisms and, although it is
bactericidal, does not kill spore-forming organisms. However, the antimicrobial activity is
long-lasting (up to 6 hours).
3.70% povidone-iodine is an antibacterial agent that acts via oxidation/substitution of free
iodine. It is effective against spore-forming, Gram-positive, and Gram-negative organisms.
However, it is rapidly inactivated by organic material such as blood. Additionally, patient
skin sensitivity is occasionally a problem, and in some cases chlorhexidine has been shown to
be more effective than iodine.
The use of general antiseptics can reduce bacterial counts by approximately 80% [6];
however, some microorganisms found deep in hair follicles can survive the treatment.
Because of this, and because surgeries last for several hours, once asepsis of the surgical area
3


is achieved an extra protective barrier is put in place. Surgical incision drapes (SIDs) are used
as a barrier to prevent re-colonization and immobilize the organisms that might have survived
the treatment.
Current Practices: The Role of Surgical Incision Drapes in Surgery
The use of SIDs has been a common practice in surgeries for approximately 50 years. There
are several types of commercialized antimicrobial drapes that are impregnated with different
antimicrobial agents. Most common SIDs include:
l.IOBAN, which was commercialized by the company 3M and is an iodophor-impregnated
drape;
2.Tiburon (ISO-BAC(), developed by Cardinal Health, which is a microfiber drape that
consists of three layers: an absorbent fluid-control layer, an impermeable membrane, and a
patient-comfort layer (this material has antimicrobial properties as a result of the presence of
ISO-BAC, a 3-trimethoxysilylpropylocta-decyldimethyl ammonium chloride (silanequat)
compound); and
3.InteguSeal, which is a cyanoacrylate semi-adhesive commercialized by Kimberly Clark;
the specifics of this product will be discussed later in this review.
Although many measures are taken to reduce the risk of SSIs, statistics indicate that current
practices need to be improved. The orthopedic SSI literature pertaining to iodophor-
impregnated drapes suggested that their use achieves a reduction in wound contamination but
without a concurrent decrease in wound infection [15]. Similarly, in seven trials Webster et
al.found no evidence that plastic adhesive drapes reduced SSI rates, and even encountered
some evidence showing that they increased infection rates [16].
4


Current SIDs pose numerous drawbacks. Some patients show an allergic reaction to iodine,
which makes IOBAN drapes an unviable option. Some SIDs contain leachable
antimicrobial agents, and as a result lose their antimicrobial activity after a short period of
time. With regard to clinical applications, medical professionals complain that the placement
process of the drapes is time consuming and that they often do not remain properly attached
to the skin surface, leading to increased infection risk. At the same time, occlusion of the skin
by SIDs generates a moist and warm skin surface that encourages microbial growth as well as
loss of drape adhesion [17]. Similarly, the formation of air pockets and wrinkles creates
locations where microbes can proliferate. Another concern involves drape removal. This step
can cause skin tearing close to the surgical site, increasing the risks of an infection. Even if
there are no apparent skin tears, a layer of epidermal cells can be detached, exposing bacteria
found beneath the skin, thereby increasing risk of secondary bacterial infection (see Figure
1).
5


Figure 1.A: Epidermal cell detachment during the removal of a SID, possibly leading to
secondary infection by bacteria found beneath the skin. B: Surgical incision drape on the skin
before surgery. C: Surgical incision drape during surgery.
Other disadvantages include the fact that SIDs are designed to fit specific sizes and shapes
and sometimes do not adapt well to the patient5s shape, thereby increasing the risk of drape
lift during surgery. Additionally, the current SIDs are designed for single use, increasing
clinical waste. In one study it was reported that 53% of medical waste is comprised of single-
use disposable items, and the biggest source of the waste is the operation room, which
generates up to 30% of the waste [12]. Lastly, the use of SIDs is recommended to prevent
postoperative infections, but they are only in place during the surgery and, hence, do not
protect the patient after removal.
1.4. Objective of This Study
The main objective of this study was to develop an antimicrobial reverse-thermal gel that
could be used to replace current SIDs. To achieve this goal,a polymer consisting of a
6


polyurea-polyurethane backbone functionalized with poly(N-isopropylacrylamide)
(PNIPAAm) and quatemized amine groups was initially developed. However, the biocidal
capacity of the polymer was limited, so an alternative polymer was proposed. The final
copolymer was synthesized by grafting PNIPAAm-COOH to a poly(ethyleneimine)
backbone through carbodiimide-linking chemistry. The antimicrobial properties were
obtained by quatemizing the primary amine groups on the polymer side chains. Quaternary
amine groups possess positive charges and are able to disrupt the bacterial cell
wall/membrane though electrostatic interactions.
Aims of this study
The aims of the study can be summarized as follows:
Specific aim 1:design a reverse-thermal gel the quatemize to obtain antimicrobial properties
Specific aim 2: characterize the antimicrobial activity of the polymer, and
Specific aim 3: test the cytotoxicity of Q-PSHU-PNIPAAm and Q-PEI-PNIPAAm.
Thesis Layout
Chapter 1 contains an introduction to the scope of this study and the main objectives of this
thesis.
Chapter 2 is used to present an overview of antimicrobial polymers used for medical
applications and their mechanisms of action.
Chapter 3 comprises an introduction to the polymer that was developed at the beginning of
this study and the alternatives that were found to overcome the limitations encountered.
Chapter 4 covers materials and experimental methods used in this study
Chapter 5 contains the results and discussion.
Chapter 6 is used to present conclusions and recommendations for future research.
7


2. Literature Review
The field of antimicrobial compounds is vast and has grown during the past decade as a result
of increasing concerns about hospital-acquired infections and the emergence of drug-resistant
bacteria. The need for materials that prevent bacteria colonization exists in many industries,
such as water treatment, food and packaging, and the medical industry, to name a few.
Specific to the healthcare industry, SSIs are a current issue. Several preventative measures
have been proposed and followed to reduce the incidence of these infections. Traditionally,
antiseptic preparations along with surgical drapes have been used to prevent SSIs. However,
there is room for improvement in these products.
This review will first introduce several antimicrobial mechanisms of action as a context to
explain the reasons behind choosing the mechanism targeted in this study. Then, different
approaches that can be followed to develop an antimicrobial polymer will be discussed, along
with the factors affecting the antimicrobial activity of the polymer developed in this work.
Different modifications, such as reversible-thermal gelling properties, will be discussed, and
an overview of how these can improve the polymer application process will be presented.
Finally, the polymer proposed will be introduced and described.
2.1. Antimicrobial Mechanisms of Action
Several antimicrobial mechanisms have been explored in the design of bacteriostats (see
Figure 2). Generally, the underlying antimicrobial mechanism targets biochemical pathways
that cause disruption in protein synthesis, cell membrane synthesis, or nucleic acid synthesis;
or inhibit cell membrane function.
8


Figure 2. Mode of action of various antimicrobial compounds.
Most conventional antibiotics penetrate the cell without damaging the bacteria cell
wall/membrane and target biochemical pathways within the cell. Pathogens sensitive to
antibiotics may become resistant by acquiring genes from resistant microorganisms in the
same niche. Microorganisms develop resistance by: (a) producing enzymes that inactivate the
antimicrobial agent, (b) developing efflux pumps that remove an antimicrobial agent from the
cell, and (c) up regulating/down regulating/altering genes encoding an outer membrane
protein channel[18]. Hence, the use of antimicrobials has led to the emergence of resistant
9


superbugs and mutant antimicrobial-resistant pathogen species. In response, scientists have
focused on developing antibacterial treatments that work through mechanisms to which
bacteria are less likely to develop resistance. Many researchers have used positively charged
moieties to disrupt the cell membrane/wall structure, and because alteration of this
configuration is less likely, this mechanism ultimately reduces the risk associated with
antibacterial resistance.
At the same time, biocidal agents used in biomedical applications need to be selective in
terms of toxicity; they should have no/low mammalian cell biotoxicity. Fortunately, there are
several differences between eukaryotic and prokaryotic cells. These differences can be used
as targets to achieve selective cell death. The cell membrane/wall is the first layer of
protection for these organisms; therefore, cell death can be attained by disruption of this
structure. The outer leaflet of mammalian cell membranes is composed mainly of
phosphatidylcholine (PC), sphingomyelin, and cholesterol, all of which have no net charge.
Conversely, bacteria outer cell structures consist of anionic lipids, such as
phosphatidylglycerol (PG), lipopolysaccharides, and cardiolipin [19]. In short, bacteria cell
walls/membranes are known to be more negatively charged than mammalian cell membranes.
This difference has allowed the use of cationic polymers as antimicrobials. These positively
charged polymers preferentially bind and interact with the outer structure of bacteria as a
result of electrostatic attraction, resulting in the selective targeting of bacterial cells over
human cells [20]. It is also important to mention that bacteria can be broadly classified into
Gram-positive and Gram-negative. Gram-positive bacteria (e.g., S. aureus) are characterized
by a cell wall composed of lipoteichoic acid molecules, whereas Gram-negative bacteria
(e.g., E. coli) have an outer membrane structure in the cell wall that acts as an additional
10


barrier to foreign molecules. Although the structures of Gram-positive and Gram-negative
outer wall/membranes differs significantly, both exhibit the same negative charge on their
outer wall/membranes (see Figure 3).

Copynght t M4CrW-Ni Inc rquir*tf 1r rpf educnon r dply

Oranv-Nmtiv
Figure 3. Outer membrane/cell wall differences between Gram-positive and Gram-negative
bacteria [21][22].
This common outer wall/membrane structural characteristic among bacteria has led
researchers to use electrostatic forces as a means to target bacterial cell membranes, thereby
applying one strategy to induce cell death of both types of bacteria. All cationic antimicrobial
molecules, regardless of their structure (detergents, peptide, natural, or synthetic polymers),
achieve bacteria cell death through the same mechanism.
2.2. Antimicrobial Polymers
In biomedical applications, long-lasting antimicrobial activity, ease of synthesis, and
biocompatibility are generally requirements of antimicrobial materials. Accordingly,
polymers have been used as matrices to hold and control the release of antimicrobial agents.
However, sometimes release is not desired, and the development of polymers that possess
11


antimicrobial activity themselves has become an important area of research. When compared
to small amphiphilic molecules, antimicrobial polymers generally show enhanced and longer-
lasting microbicidal activity as well as lower mammalian cell biotoxicity because of their
non-leaching characteristics [23].
The polymer synthesis approach chosen depends on the application and the mechanism of
action desired. In their review, Kenawy et al.discussed different approaches researchers have
taken to develop cationic polymers with biocidal properties [24]. The main approaches
include: (a) adding an inorganic or organic biocide during the synthesis, (b) modifying the
polymer after it is synthesized, (c) polymerizing a monomer that contains antimicrobial
moieties, and (d) grafting an antimicrobial agent to a synthetic or naturally occurring polymer
[25] . Several non-leaching polymers that possess antimicrobial properties because of the
presence of positive charges on the polymer structure have been reported in the literature
[26] . These polymers cause cell death by disrupting the bacteria cell wall/membrane,
consequently leading to leakage of the intracellular components. The primary polymers in
this group are:
1. Polymers with quaternary nitrogen atoms (quatemized groups can be found on side chain,
polymer backbone, or end groups);
2. Guanidine-containing polymers;
3. Antimicrobial peptides (AMPs), such as defensins, that are positively charged, possess
both hydrophilic and hydrophobic side groups that allow the molecule to be soluble in
aqueous environments, can penetrate through the cell membrane, and have biocidal activity
through several mechanisms (however, AMPs are susceptible to proteolysis, their
pharmacokinetics are not fully understood, and their production involves high manufacturing
12


costs. Synthetic antimicrobial peptides have been developed to overcome the limitations, and
these mainly act by disrupting the outer cell structure);
4. Halogen polymers, including fluorine or chlorine-containing polymers;
5. Polymers containing phospho and sulfo derivatives; and
6. Organometallic polymers containing metals in their pendant groups or backbone [27].
To date, scientists have been focused on researching polycationic systems because these are
synthetically flexible and the development process is more straightforward [27]. Most of the
antimicrobial polymers available are cationic hydrophilic-hydrophobic macromolecular
systems that exhibit surface-activity properties [19]. Two main types of polymers [24], block
polymers that consist of a hydrophilic block that holds a cationic charge and a hydrocarbon
non-polar hydrophobic block, as well as random copolymers that contain a hydrophilic
monomer attached to functional group and a hydrophobic co-monomer, have been reported in
the literature. These polymer structures contain adsorption/absorption abilities along with a
high binding affinity for bacterial cells. The cell membrane disruption mechanism is still not
fully understood; however, studies have shown that the process is first driven by electrostatic
force interactions between the cationic charges of the antimicrobial agent and the negative
sites of the lipid bilayer membrane. This is followed by Van der Waals interactions between
the hydrophobic moieties of the antimicrobial agent and the phospholipids, leading to
disruption of the bacterial cell membrane, thereby causing leakage of cytoplasmic contents
and cell lysis [28].
Cationic polymers: Quaternized amine groups
Polymers with quatemized amine groups are probably the most studied and used type of
cationic polymeric biocides [27]. Quaternary ammonium compounds (QUATs) are organic
13


molecules that have been widely used in healthcare, domestic, personal care products,
agricultural, and industrial applications as surfactants, emulsifiers, disinfectants, and
pesticides.
QUATs can be obtained through a process called quatemization. This chemistry involves the
reaction of an amine group with alkyl halides, such as methyl iodide. This reaction modifies
the molecule, leading to a higher degree of alkylation. Some studies report that as a result of
steric effects, tertiary amines react much more slowly than secondary or primary amines.
Primary and secondary amines usually result in a mixture of amines and ammonium salts,
whereas tertiary amines undergo a one-step reaction [29]. This structure contains a central
nitrogen atom (R4N+) covalently attached to four functional groups (R). The functional
groups generally consist of one (or more) long alkyl chains, and the rest are methyl or benzyl
groups [30].
Figure 4. Quatemization of tertiary amines with alkyl halides and molecular structure of a
QUAT (central positive nitrogen, X represents a counter ion, such as N03-, C1-, or Br-, and R
represents a functional group) [30].
QUATs have several advantages over other antibacterial agents. They exhibit significant cell
membrane penetration while generating low toxicity and lack of skin irritation. At the same
time, they are very good in terms of environmental stability and have proven to have
extended biological activity [28][19] [31].
14


Effect of Structural Parameters on Antibacterial Properties
Different polymer structural parameters affect the overall antimicrobial activity of the
cationic macromolecule. Antimicrobial efficacy is largely dependent upon the specific
polymer system used. However, it has been shown that for cationic polymers structural
parameters, such as alkyl chain length, charge density, molecular weight, hydrophilic-
hydrophobic balance, and counterions play a role in antimicrobial capacity [24].
Effect of molecular weight
The influence of molecular weight on the overall antimicrobial activity of a polymer depends
on the particular polymer system used. Cationic polymers consist of a greater hydrophobic
mass and greater net charge per molecule when compared to their monomer counterparts. The
greater net charge is suspected to increase the interaction with the bacterial cell
membrane/wall, while the higher hydrophobic mass induces better membrane penetration,
thereby improving antimicrobial performance [19].
Cooper et al.conducted structure-activity studies of quaternary ammonium functionalized
poly(propylene imine) and found that a molecular weight within the range of 5xl4 to
1.2xl5 Da is best for antimicrobial activity against Gram-negative (e.g., E. coli) and Gram-
positive (e.g., S. aureus) bacteria [32]. Klibanov et al.studied the mechanism of bactericidal
and fungicidal activities of textiles covalently modified with alkylated polyethylenimine.
Their work focused on S. aureus and the antimicrobial properties of N-alkylated
polyethyleneimine (PEI) polymer systems immobilized on amino-glass slides [33]. They
found that the polymers they tested of molecular weights 25 kDa and 750 kDa had excellent
antibacterial activity, while the two tested with lower molecular weights (0.8k Da and 2kDa)
did not exhibit antibacterial properties.
15


Effect of alkyl chain length
The antimicrobial mechanism of action of QUATs requires the cationic sites of the polymer
to be adsorbed into the anionic sites of the cell wall by electrostatic interactions and then the
diffusion of alkyl chains through the cell wall. Hence, the alkyl chains act as a surfactant by
disrupting the cytoplasmic membrane and causing the release of electrolytes and bacterial cell
materials, leading to cell death [34]. Due to this, the length of the hydrophobic chains
correlates with the antimicrobial activity. Klibanob et al.found that alkyl chain lengths
between C6 and C12 have performed best against both Gram-negative and Gram-positive
bacteria [34]. Xu et al.studied the effects of quaternary ammonium chain length on
antibacterial bonding agents and their results showed increasing antibacterial activity with
chain length increasing from 3C to 16C. However, they observed that when the chain length
was increased to 18C, the antibacterial efficacy decreased. They suggested that if the alkyl
chain is too long it may aggregate, thereby altering the electrostatic interactions with bacteria
cell/membrane and diminishing the antibacterial potency. Hence, very long alkyl chains can
have an unfavorable effect on the overall antimicrobial properties of the polymer [35].
Further studies are required to understand the exact mechanism that leads to the reduction of
antimicrobial activity when longer carbon chains are used. Cooper et al.studied the
antimicrobial activity of quatemized poly (propylene imine) dendrimer systems and reported
that a hydrophobic alkyl chain of CIO exhibited the best performance in terms of
antimicrobial activity. They also mentioned that the antimicrobial properties were highly
influenced by the length of alkyl chains and that the results followed a parabolic trend [32].
The alkyl chain length also has an impact on the overall hydrophilic/hydrophobic balance and
charge density of the polymer, which impacts other properties of the material, such as the
16


solubility. Therefore, the polymer must meet other requirements depending on the
application.
In efforts to design a water-soluble polymer, Nonaka et al.synthesized a thermosensitive
polymer containing phosphonium groups. They developed the polymer by copolymerization
on N-isopropylacrylamide (NIPAAm) with methacryloyloxyethyl trialkyl phosphium
chlorides (METRs), and they studied the properties of the polymer with varying alkyl chain
lengths. They found that the thermosensitivity of the copolymer was highly affected by the
addition of salts and by the hydophobicity of the alkyl groups [36]. They also noted that the
relative viscosity of the copolymers increased with increasing phosphium content; they stated
that this change occurred as a result of the expansion of polymer chains in water because of
the repulsion among cationic charges introduced by the presence of phosphonium groups
[36]. In conclusion, the quatemization process will affect the overall behavior of the polymer,
and this must be considered when designing a material that needs to fulfill several
requirements.
2.3. Reversible-gelling Properties
Placement of surgical incision drapes
Aside from developing a polymer with antimicrobial properties to replace current SIDs, it
was also the aim of this study to develop a system that simplifies the application process. As
mentioned previously, SIDs placements are complicated and issues can be encountered
during the placement. The skin needs to be disinfected and the drape then needs to be placed
very carefully to avoid contamination of the area and the formation of "air bubbles/1 as seen
in Figure 5.
17


Figure 5. Placement of SID [37].
As an alternative to current SIDs scientists at Kimberly Clark developed a cyanoacrylate -
based polymer called Interguseal Microbial Sealant. This product can be applied to the skin
as a liquid during the preoperative skin preparation step, and moisture on the epidermis
triggers polymerization of the n-butyl cyanoacrylate [38]. During this process, the compound
bonds to the skin to form a barrier intended to immobilize the bacteria that still remain on the
skin [39]. Unfortunately, Vierhout Bastiaan et al.compared the use of a cyanoacrylate skin
sealant to conventional procedures and were unable to confirm a reduction in the incidence of
SSIs [40]. Dohmen et al.also tested the incidence of SSIs and found no statistical difference
between the control group and the group treated with cyanoacrylate adhesives [41].Aside
from the contradictory literature regarding clinical tests, one of the advantages cyanoacrylate
adhesives possess is the rapid setting on the skin surface (5-60 seconds) and the capacity for
forming a waterproof barrier. However, among the major disadvantages discussed in the
literature were polymerization shrinkage, brittleness, and erratic polymerization set times.
18


Additionally, patients experienced unpleasant shredding of a thick polymer days after the
surgery.
A reverse-thermal gel: Poly(N-isopropylacrylamide)
A possible alternative to polymerization of cyanoacrylates is the use of smart
polymers. These are materials that exhibit reversible, sharp property changes in response to
environmental cues such as electric field, pH, temperature, or light.
Temperature can be a stimulus that is relatively simple to control; because of this, it has been
central in the development of smart polymers with thermally reversible properties. For a
polymer to possess reversible thermal gelling properties, at least one component of the
system needs to have temperature-dependent solubility in a solvent (generally water), and the
constituents must be insoluble above or below a certain temperature, called the lower critical
solution temperature (LCST) [42]. There are many thermo-responsive polymers used for
biomedical applications. However, because of its acute phase transition near human body
temperature one of the most prominent and well-studied examples is Poly(N-
isopropylacrylamide)(PNIPAAm). The reverse-thermal gelling properties of PNIPAAm are a
direct result of the chemical groups that form this polymer. The structure is shown in Figure
4.
19


Hydrophobic Group
Hydrophobic Group
Hydrophilic Group
Figure 4. PNIPAAm structure and behavior above and below the LCST.
In a solvent such as water at low temperature, the hydration of the polymer is dominant as a
result of hydrogen bonding. However, as the temperature increases, hydrophobicity begins to
dominate because of the presence of the methyl groups and the hydrophobic backbone, which
contributes to gelling of the polymer. Hence, the polymer solution undergoes a phase
transition as the temperature is increased. The changes go from random coil form (soluble
state) to a collapsed or globule form (insoluble state) [43]. Depending on the requirements of
the application, the LCST of thermo-responsive polymers can also be modified by adjusting
the ratio of hydrophilic and hydrophobic segments of the polymer [44]. Modification of
LCST polymers (e.g., PNIPAAm) with more hydrophilic monomers favors hydrogen
bonding over hydrophobic interactions and increases the LCST of the copolymer [44] [45].
Although the biomedical applications of PNIPAAm have been studied extensively, especially
concerning drug delivery, not much work has been done regarding epidermal applications.
Kubota et al.designed a gauze coated with PNIPAAm as an alternative to current gauzes,
with the aim of reducing the risk of skin abrasions encountered when a gauze is peeled from a
20


wound. They showed that the new gauze held and peeling of the polymer could easily be
done after several days without facing the risks of epidermal cell layer detachment [46].
Dincer et al.[47] studied 7V-isopropylacrylamide-ethyleneimine block copolymers as possible
gene delivery vectors. They reported that an increase in molecular weight of PNIPAAm
increased the overall temperature sensitivity of the polymer as a result of the dominating
hydrophobic interactions between the PNIPAAm chains at higher temperatures. They also
showed that copolymerization of PNIPAAm chains with more hydrophilic polyethyleneimine
(PEI) chains caused significant changes in the LCST behavior. However, this change was
smaller when higher molecular weight PNIPAAm was used because the PNIPAAm chains
dominated LCST behavior. They found that PEI-PNIPAAm copolymer solubility was higher
in the aqueous medium when a lower molecular weight PNIPAAm was used. This was
expected because the hydrophilicity of the copolymer chain increases with the addition of
more hydrophilic PEI chains. They also showed that the relative size of the blocks of
PNIPAAm and PEI on the copolymer chains were important factors in the hydrophobocity of
the copolymer. Finally, their results showed that the copolymerization of PNIPAAm with
more hydrophilic PEI chains caused an increase in the LCST of the PNIPAAm chains from
31C to around body temperature (36-39C) [47].
21


3. A reverse-thermal gel with antimicrobial activity
Ideally, an antimicrobial polymer designed to replace current SIDs should fulfill the
following requirements:1)non-toxic or irritating; 2) biocompatible; 3) biocidal to a broad
spectrum of pathogenic microorganisms; 4) antimicrobial activity should be long lasting; 5)
inexpensively synthesized; 6) increased ease of application when compared to current SIDs;
and 7) ease of removal.
Poly(Serinol Hexamethylene Urea) (PSHU)
This study began with the characterization of a polymer consisting of a poly (urethane urea)
backbone functionalized with quatemized amine groups and PNIPAAm.
The initial polymer structure proposed can be observed in Figure 5Figure 1.The polymer
backbone structure resembled a peptide bond due to the presence of urea which is an organic
compound that consists of two amine (NH2) groups joined by a carbonyl (C=0) functional
group. The side chains of Poly (Serinol Hexamethylene Urea) initially contained amine
groups protected by Boc-groups that after chemical modifications were removed to reveal the
primary amines. Then, PSHU-NH2 was functionalized with a) PNIPAAm to achieve reverse-
thermal gelling properties and improve the ease of application and removal b) with alkylation
agents to obtain quatemized amine groups and, hence, antimicrobial properties.
22


H
1 H
0
0 0
Q-PSHU
PNIPAAm-NH>PSHU
Figure 5. Polymer backbone with quatemized amine groups
As will be shown in the results section, the outcomes of the antimicrobial tests using Q-
P SHU -PNIP A Am were not as expected. After analyzing and modifying the polymer
synthesis and quatemization protocol, the results regarding the antibacterial tests were not
positive or consistent. The negative results were attributed to the small number of primary
amine groups per repeating unit available to quatemize. Due to this, a new polymer that
possessed a higher number of primary amine groups was proposed.
3.1. New Approach: Polyethyleneimine
Polyethyleneimine (PEI) is a nontoxic, aliphatic, weakly basic, synthetic polymer. It owes its
polycationic nature to the presence of primary, secondary, and tertiary amine groups as can
be observed in Figure 6. The protonated amine groups of PEI are cationic, and the ethylene
backbone provides hydrophobic groups. Hence, the polymer consists of a repeating cationic,
amphiphilic structure at a neutral pH without any further chemical modification by
hydrophobic groups [48].
23


Figure 6. Structure of branched Polyethyleneimine
PEI has been used extensively in antimicrobial applications. Specific to antibacterial
coatings, Lewis et al.discuss in their study the possible limitations encountered when
attaching an antimicrobial molecule to a surface. They explain how the loss of mobility can
make the agent unable to penetrate the cell and loose its antibacterial activity. Due to this,
they explain that quaternary amine groups are most interesting for coating applications
because they are drawn to the bacterial cell due to the membrane potential [49] [50]. Lin et a.
conducted systematic chemical modifications of immobilized PEI and studied their impact on
the overall properties of the polymer. They conducted tests on 7V-alkylated PEIs of different
molecular weights that were covalently attached to amino-glass slides [34]. They observed
that quatemized amine groups are can be obtained by alkylation of the PEI coating. They also
showed that alkylation leads to higher bactericidal activity at carbon chain lengths of 6 and
decreases beyond this point.
Park et al.researched a method of physical deposition of hydrophobic polycations to obtain
an antimicrobial coating. They developed a one-step painting-like procedure to obtain an
antimicrobial surface [51].They prepared several 7V-hexyl,7V-methyl-PEI coated slides and
determined their bactericidal efficiency against S. aureus and E. coli. The results showed that
these surface-deposited polymers alike many covalently immobilized polymers achieve
bacteria cell death on contact and not by leaching from the surface. At the same time, they
24


also discuss that the antimicrobial mechanism involves rupturing the bacteria cell membrane
[51].
Regarding antimicrobial resistance, Klibanov et al.conducted studies using N-hexyl,methyl-
polyethylenimine, and their results showed that the bacteria failed to develop noticeable
resistance to this lethal action over the course of many successive generations [26].
Because of the inherent antibacterial properties of this polymer and the inability of bacteria to
develop resistance to PEI, this polymer was used in this study to replace PSHU.
3.2. New Polymer
In this context, this study proposes, Q-PEI-PNIPAAm, an antimicrobial reverse-thermal gel
that is designed to provide a non-leaching antimicrobial polymer with 99.9% killing
efficiency. When in a solution, the polymer can be sprayed on the skin and will form an
antimicrobial gel layer. The polymer can act as a uniform antimicrobial surface during the
entire surgery. In addition, it can be removed using cold water (or ethanol 70%) after surgery,
thereby significantly reducing risk of skin irritation, blistering, and medical waste. The pre-
surgical process is streamlined because the polymer system is easy to apply and offers a
universally relevant solution for all patients, therefore, bypassing the need to order currently
used SIDs based on the patient5s size and shape.
25


4. Materials and Methods
4.1. Materials
Polyethyleneimine (PEI branched 10 000 g/mol and 1800 g/mol),N-(3-
dimethylaminopropy^-N'-ethylcarbodiimide hydrochloride (EDC), and N-
hydroxysuccinimide (NHS) were obtained from Alfa Aesar (Ward Hill, MA). N-
isopropylacrylamide (NIPAAm) was obtained from Acros Organics (Geel, Belgium).
Hexamethylene diisocyante (HDI), 4,4,-azobis(4-cyanovaleric acid) (ACA), methanol,
trifluoroacetic acid (TFA), serinol, and urea were obtained from Sigma Aldrich (St. Louis,
MO). Sodium hydroxide (NaOH), 2-propanol, hexane, and diethyl ether were obtained from
Fisher Scientific (Pittsburgh, PA). Dimethylformamide (DMF) was obtained from BDH
Chemicals (Poole, UK).
4.2. Polymer Synthesis
PSHU Synthesis
This method was used to synthesize a polymer consisting of a poly (urethane urea) backbone
with side chains that contain amine groups that possess protective Boc-groups. Once the
polymer synthesis was complete, a latter 'deprotection5 step was followed to expose the
primary amines.
The first step was to synthesize N-Boc serinol;5.973 ml of N-Tert Butyl was dissolved in a
vial containing 25 ml of ethanol. Then, this solution was placed at 4C and added drop-wise
while mixing to 1.959 g of serinol that had previously been dissolved in 20 ml of dry ethanol.
Then, the solution was stirred vigorously for an hour at 37C, and the solvent was removed
using a rotary evaporator with the water bath temperature set at 50C. The white powder
26


obtained was dissolved in 25 ml of 1:1 ethyl acetate/hexane and mixed until a clear solution
was obtained. Subsequently, hexane was added drop-wise until the solution appeared to
crystallize. Then, the solution was left overnight for the crystals to settle to the bottom, and
then a vacuum filter was used to separate the N-Boc serinol.
After N-BOC serinol was obtained, 6.0 mmol were dissolved along with 6.0 mmol of urea in
6 ml of anhydrous DMF under a nitrogen atmosphere. Once both chemicals were dissolved,
12 mmol of HDI were added, and the reaction was continued for a period of 7 days at a
temperature of 90C. Subsequently, it was removed from the hot plate and left until it reached
room temperature before precipitating it in diethyl ether four times and once in water. After
the required precipitations, the polymer was lyophilized and a yellowish powder was
obtained.
PSHU Deprotection
The protocol followed was designed to achieve a 100% deprotection of amine groups because
15% were meant to be conjugated to PNIPAAm and 85% were available for quatemization.
PSHU (1 g) was dissolved in 15 ml of methylene chloride in a flask. Then,15 ml of TFA
were added drop-wise, and the solution was left to mix on a stir plate for 45 minutes. The
solvents and acid were removed using a rotary evaporator and a water bath at a temperature
of 45C. Once all the acid was removed,1 ml of anhydrous DMF was added to return the
polymer to solution. The solution was then precipitated in diethyl ether and then the solvent
removed using a rotary evaporator. Then, the deprotected PSHU was dissolved in TFE and
again precipitated in diethyl ether. This step was repeated twice, and a rotor evaporator was
used each time to remove the solvent. The final step allowed obtaining a dry white powder.
27


PNIPAAm Synthesis [52]
NIPAAm (5.0 g) and ACA (0.060 g) were dissolved in 25 ml of anhydrous methanol and
exposed to nitrogen bubbling for 30 minutes at room temperature. Then, a reflux system was
set, and the temperature was raised to 68C and left for three hours. Subsequently, the
solution was precipitated in warm (60C) water and then left to dissolve in 5 ml of dH20
water at 4C. The solution obtained was then added to a 3500 kDa MWCO dialysis tube that
was placed in a beaker with dH20 water for 48 hours. Finally, once the polymer was purified,
it was lyophilized for a period of 48 hours.
Molecular weight of PNIPAAm-COOH was confirmed by titrating for the carboxylic acid
end groups as in previous studies [53]. PNIPAAm (0.5% w/v) was dissolved in 10 mL dH20.
Then 10 |iL of phenolphthalein solution (2 wt% in absolute ethanol) was added. The solution
was titrated to the end point by adding 0.01 N NaOH.
Conjugation of PNIPAAm to PEI
PNIPAAm-PEI conjugations were performed at 20, 30, and 50% using Ethyl
(dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide chemistry. Calculations
were performed to determine the number of primary free amines on the PEI structure to be
conjugated. PNIPAAm was first dissolved in 10 ml of DMF, and then, EDC-NHS (1.3 M
excess) was added. At the same time, in a separate flask, PEI was dissolved in 5 ml of DMF,
and both reactions were left overnight. Then, the PEI solution was added drop-wise onto the
PNIPAAm-EDC-NHS and left to conjugate for a period of 24 hours. Subsequently, a rotary
evaporator was used to remove the DMF, and precipitations in ditheyl ether were used to
remove unreacted polymer. Then, the polymer was dissolved in dH20 and added to a 12-14
28


kDa MWCO dialysis tube that was placed in a beaker with dH20 water for 48 hours. Finally,
once the polymer was purified, it was lyophilized for a period of 48 hours.
The same methodology was applied to conjugate PNIPAAm to deprotected PSHU.
Quaternization of Primary Amines
Calculations were performed to determine the number of primary free amines on the PEI-
PNIPAAm structure to be conjugated. PEI-PNIPAAm was first dissolved in 10 ml of DMF,
and sodium bicarbonate (1.3 M excess) was added. The reaction was set to a temperature of
95 C with reflux, and while undergoing vigorous stirring, 1-Bromohexane (20 M excess) was
added and left for 48 hours. Then, the temperature was lowered to 68C, Iodomethane (20 M
excess) was added, and the reaction continued for another 12 hours. Subsequently, the flask
was removed from the hot plate and left to cool before using a rotor evaporator to remove
solvents. Three precipitations in diethyl ether were used to remove unreacted polymer. Then,
the polymer was dissolved in 10 ml of dH20, and 7,ml were added to a 12-14 kDa MWCO
dialysis tube that was placed in a beaker with dH20 water for 48 hours. The remaining 3 ml
of polymer solution were added to a separate 12-14 kDa MWCO dialysis tube that was
placed inside a 50-ml tube with 25 ml of dH20. The tube was then lyophilized and weighed
to calculate the PEI-PNIPAAm conjugation amount.
Finally, once the polymer was purified, it was lyophilized for a period of 48 hours.
4.3. Polymer Characterization
Proton Nuclear Magnetic Resonance NMR)
Polymer samples (3-5 mg) were dissolved in DMSO-d6 (60 |il), and then, CDC13 (540 |il)
was added. The 4 NMR spectra were collected on an INOVA 500 MHz instrument
29


(Varian). Results and spectra were analyzed using ACD ID NMR Processor software
(Advanced Chemistry Development, Inc., Toronto, ON).
Zeta Potential Measurements
Polymer samples (10 mg) were dissolved in 1ml of dH20. The ^-potentials of the polymers
were measured using a Malvern Zeta Sizer 2000 (Malvern Instruments, USA), ^-potentials
were measured at least nine times, and the averages with standard deviation were calculated.
Gelling Properties LCST Determination
The LCST was determined to characterize the gelling properties of the polymer. The polymer
samples were dissolved in dH20 at 1 wt%. Then, transmittance values were measured at 500
nm, starting from temperature of 25C and increasing 0.5C/min up to 50C. The results were
obtained using a Cary 100 UV-visible spectrophotomer (Agilent Technologies, Inc., Santa
Clara, CA).
4.4. Antimicrobial Tests
Antimicrobial Tests Using Q-PSHU-PNIPAAm
The antibacterial property of the Q-PSHU-PNIPAAm was evaluated against Staphylococcus
aureus. All bacteria were cultured in Lysogeny broth (LB). The experiment consisted of
forming gel surfaces with polymer solutions (200 |il) with different concentrations (starting
from 2% to 10% (wt/v) with z% increments). These solutions were added to a 24-well plate
and placed in a 37C incubator to promote the solution-to-physical gel transition.
Subsequently, 500 |il of bacterial suspension at a concentration of lxl8 colony-forming
units (CFU)/ml were added on the gel and cultured at 37C for 24 hours. For better results,
each gel-bacteria sample was triplicated, and standard deviation values were calculated. For
controls, 500 |il of bacterial suspension of equal concentrations were to be cultured on a plain
30


24-well plate. After the culture period,100 |il of the suspension were taken and diluted with
900 |il of DI water. The antibacterial activity was determined by measuring optical density
(OD) of a bacterial suspension at 600 nm using a microplate reader (SYNERGY Mx,
BioTek). The percentage increase in the optical density was calculated by comparing optical
densities of bacterial suspensions before and after the incubation. Then, the numbers were
normalized to the control group.
Antimicrobial Tests Using Q-PEI-PNIPAAm
The main objective of these tests was to quantify the bactericidal activity of the
polymer/antimicrobial using time-kill curves. This was achieved by measuring the decrease
in bacterial population at different time intervals. The general methodology followed
consisted of dissolving the polymer in a suspension of known bacterial concentration. Then,
the bactericidal activity of the sample was tested at different time intervals by extracting
aliquots in 30-minute time intervals. The aliquots were then diluted and placed on agar plates,
where the bacteria were cultured during a period of 24 hours before colonies were counted.
The antibacterial property of Q-PEI-PNIPAAm was evaluated against 4 clinical, relevant
species of bacteria: Staphylococcus aureus (ATCC 6538), Staphylococcus aureus subsp.
aureus MuS (MRSA strain with heterogeneous vancomycin-intermediate resistance) (ATCC
700698), Staphylococcus epidermidis (Ron Gill Collection), w&E. Coli (ATCC 15597).
Bacterial Stock Preparation
Bacterial streaking was used to identify and isolate bacterial colonies. One colony was
swabbed from an agar plate and added to 5 ml of Lysogeny Broth. The suspension was left
for 16 hours in a shaker incubator at 37C and at 250 RPM. Then, 920 microliters of bacterial
31


suspension were removed and mixed with 80 microliters of filtered DMSO and flash frozen
at -80C.
Antibacterial Test
The bacterial suspension was prepared by extracting one crystal from the flash-frozen
bacterial stock and mixing it with 5 ml of LB. The new stock was left for 16 hours at 37C in
a shaker incubator (250 RPM). Subsequently, the bacterial concentration was calculated
using optical density measurements of absorbance at 600 nm using a microplate reader
(SYNERGY Mx, BioTek). This measurement was taken to have an initial estimate of the
bacterial concentration; these values were then confirmed with dilutions that were inoculated
on agar plates.
Dilutions were performed using PBS to obtain a stock of 108 cells/ml. Then, the polymer was
added to obtain a suspension of 10% w/w and was placed at 37C in a shaker incubator (250
RPM). Aliquots (20 |iL) were taken at 0, 30, 60, 90, and 120 minutes, and dilutions were
performed to have samples at different concentrations. Aliquots (40 |iL) were taken from
these samples and plated on agar plates that were left at 37C during 24 hours. Then,
colonies were counted, and the values were used to extrapolate the initial number of CFUs.
All results were compared with the values obtained at time zero to quantify the killing power.
4.5. Functional Tests
Preliminary tests using surgical incision drapes
To asses if bacterial recolonization occurs during a surgical procedure while using SIDs a
used drape was carefully used to inoculate an agar plate and cultured for a period of 24 hours
at37C.
32


Antimicrobial Activity of Polymer-Coated Glass Slips
The polymer was used to coat glass slips to assess the antimicrobial properties of the surface
and the durability of the polymer after being soaked in water. The method involved coating
15 glass slides of 1cm x 1cm, which were cut and cleaned with alcohol. Then, they were
placed on a hot plate at 37C and left until a stable temperature was obtained. Four different
10% w/v polymer solutions were prepared: PEI-PNIPAAm (30% conjugation), PEI-
PNIPAAm (20% conjugation), QPEI-PNIPAAm (30% conjugation), and QPEI-PNIPAAm
(20% conjugation). Glass slips were then painted with 100 |il of each polymer solution.
Triplicates were done for each case. Also, three glass slips were cleaned and left as controls.
The bacterial suspension was prepared as previously described. After the polymers gelled, the
bacterial suspension (108 cells/ml) was sprayed on the glass slides. Subsequently, the glass
slides were placed on agar plates and left at 37C for 24 hours. As in the previous
antibacterial tests, the bacterial stock was diluted and also plated to extrapolate the initial
number of CFUs. All results were compared with the values obtained at time zero to quantify
the killing power.
Finally, the same process was followed with a glass slide that was half coated with the
polymer solution consisting of 10% w/v Q-PEI-PNIPAAm (30%) in 70% ethanol and half
left uncoated.
Since there is water present during a surgery, a preliminary test was designed to evaluate
whether the polymer could remain on the surface of a glass slide after being subjected to
water.
Polymer solutions,10% w/v of QPEI-PNIPAAm (30% conjugation) and QPEI-PNIPAAm
(20% conjugation), were dissolved in 70% ethanol and sprayed on a glass slide. The glass
33


slide was then left until the alcohol evaporated and was placed in a water bath at 37C
overnight. Images were taken to register changes.
4.6. Cytotoxicity Test of Q-PEI-PNIPAAm and Q-PSHU-PNIPAAm
The cytotoxicity of the Q-PEI-PNIPAAm was examined by performing a MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using human smooth muscle
cells. The MTT assay is commonly used as a measure for cell viability and provides a general
indication of cell health. The MTT assay was performed by supplier's instructions, with
absorbance (570 nm) and values collected on a microplate reader.
To assess the cytotoxicity of Q-PSHU-PNIPAAm, polymer solutions (10% (wt/v)) were
obtained by dissolving the reverse-thermal gel in SMC culture medium. Smooth muscle cells
were cultured for a period of a week until they were confluent. Then, the cells were counted,
and 10,000 cells/well were plated in a 96-well plate and left to attach. Subsequently, polymer
solutions (200 |il) were added to each well. As a negative control, cells were also cultured
with plain media, and as positive control, cells were exposed to media with 5% DMSO. All
samples were triplicated for statistical significance. The absorbance (directly related to
cellular metabolic activity) was measured after 1,3, and 5 days. Final results were
normalized to the 1-day control group.
To assess the cytotoxicity of Q-PEI-PNIPAAm, a different approach was taken to ensure that
the polymer was in direct contact with the cells at all times. In this case, fibroblasts
(Cardiovascular Pulmonary Research Cell Repository Nana Bums and Sandy Walchak)
were used since these are more sensitive and relevant for the application. Polymer solutions
(1,3, and 5% (wt/v)) were obtained by dissolving the reverse-thermal gel in ethanol 70%.
Several controls were used, including plain media, PEI-PNIPAAm (20% conjugation),
34


PNIPAAm, Chlorhexidine 2%, and media with 5% DMSO. Fibroblasts cells were cultured
for a period of a week until they were confluent. Then, a 96-well plate was coated with the
different polymer solutions. Cells were counted, and 7,000 cells/well were placed on top of
each polymer coating. Then, they were left to culture at 37C for a period of 2 hours. All
samples were triplicated for statistical significance. Subsequently, the MTT was performed
by supplier's instructions, with absorbance (570 nm) and values collected on a microplate
reader. All results were normalized and expressed as a percentage of the negative control.
35


5. Results and Discussion
The Results and Discussion section consists of three components:
- Antimicrobial and cytotoxicity test results concerning the first polymer developed,
Q-PSHU-PNIPAAm. Limitations of the tests are discussed along with the results.
- Characterization, antimicrobial and cytotoxicity results obtained from tests using the
new polymer, Q-PEI-PNIPAAm are shown.
- Functional tests: Short preliminary test regarding current surgical incision drapes,
water resistance of the polymer coating, antibacterial tests of the polymer coating
5.1. Preliminary Studies
5.1.1. Antimicrobial Tests Using Q-PSHU-PNIPAAm
A UV-visible spectrophotometer was initially used to obtain OD values and evaluate the
antimicrobial activity of Q-PSHU-PNIPAAm.
The OD method can be performed automatically in a high throughput manner using a
microtiter plate reader [55]. Researchers[48][56][57] have relied on UV-visible
spectrophotometers to test the antibacterial properties of different materials. The underlying
principle is that the light scattered by the cells does not reach photoelectric cell and hence the
readings of absorbance change. However, this occurs as a result of light scattering, not due to
molecular absorption because cells of most bacteria are close to colorless and real light
absorption is marginal[58]. Since the reading obtained when using the absorbance setting
on the spectrophotometer is not truly the absorbance, the term used to describe this value is
Optical Density or O.D. The main limitation faced when using this technique to assess the
biocidal activity of a material is that it cannot distinguish between dead or live bacteria [55].
36


Three antimicrobial tests were conducted using this technique. Bacteria cell cultures were
adjusted to 108 -104 colony forming units CFU/ml by PBS solution. Only results from the
first test were included in this section since they were representative of all three cases. The
data can be observed in Figure 7.
0.6 i
104cells/ml _105cells/ml 106cells/ml 107cells/ml^ 108cells/ml
Control NQPSHU 2%QPSHU NQPSHU 4%QPSHU NQPSHU 6%QPSHU
Sample
Figure 7. Results from the first antibacterial test against S. aureus using different polymer and
bacterial concentrations.
The results showed a reduction in the OD values when compared to the control. Due to this,
the initial findings led to think that the polymer had antibacterial properties. However, these
results were not conclusive. As can be seen on the graph, both the quatemized polymer and
the non-quatemized polymer showed similar results and, as mentioned previously, OD values
do not measure cell death. To clarify the results the test was repeated and aliquots from the
samples that had been subjected to OD measurements were used to inoculate agar plates for
colony counting.
37


Unfortunately, the agar tests performed to confirm cell death showed bacterial growth after
24 hours at 37C at all polymer and bacterial concentrations. The changes in OD values were
attributed to presence of the polymer within the samples affecting the absorbance
measurements. This observation led to modify the antibacterial test protocol.
Similarly, the negative results let to conclude that more quatemized amine groups were
required to possess significant antimicrobial activity.
5.1.2. Cytotoxicity Tests of Q-PSHU-PNIPAAm
Although the preliminary results from antibacterial tests with Q-PSHU-PNIPAAm did not
seem promising, there was interest in confirming the biocompatibility of PNIPAAm and Q-
PSHU before designing the next polymer. The aim was to evaluate the biocompatibility of
quatemized amine groups and to confirm that PNIPAAm was non-cytotoxic. An MTT assay
was conducted to assess the cytotoxicity of Q-PSHU-PNIPAAm.
Figure 8 shows data normalized to the day 1 media-only control sample. The results show
that the positive control samples (10% DMSO) were the only samples that were significantly
different (p<0.0001).The samples containing Q-PSHU-PNIPAAm were statistically
insignificant from the negative control sample of pure culture media.
38


Figure 8. Results from the first MTT assay conducted using Q-PSHU-PNIPAAm at different
concentrations. The only statistically significant difference was observed between the
experimental samples and positive control (cells cultured with 5% DMSO). indicates p >
0.01and *** indicates p < 0.01.)
However, as mentioned previously, when PNIPAAm is exposed to a temperature above the
LCST, it exhibits shrinking; hence, the polymer didn5t seem to be in contact with the cells
during the entire period. PNIPAAm and PSHU are biocompatible; as has been shown in
previous studies, the only moieties that could cause cytotoxicity are the quatemized amine
groups. Due to this, the MTT assay was repeated with Q-PSHU.
Positive Control Negative Control Polymer 1%
Polymer 3% Polymer 5%
300 -i------------------------------------------------------

39


Day 3
Time (days)
Figure 9. Results from the second MTT assay conducted using Q-PSHU at different
concentrations.
Figure 9 shows data normalized to the day 1 media-only control sample. The results show
that the positive control samples (5% DMSO) were the only samples that were significantly
different (p<0.01).The samples containing Q-PSHU-PNIPAAm were statistically
insignificant from the negative control sample of pure culture media.
5.1.3. Conclusion from Tests Performed with Q-PSHU-PNIPAAm
The antimicrobial and cytotoxicity tests conducted using Q-PSHU-PNIPAAm allowed to
reach to several conclusions. First, PSHU contained one primary amine and this was assumed
to be the limiting factor to the antibacterial activity. Moving forward, a new polymer with
numerous primary amine groups available for quatemization was thought to be required to
obtain significant antimicrobial activity. Although the literature states that quatemized amine
groups disrupt the bacterial cell wall causing cell death [59] [60][51][34], there is no specific
knowledge regarding the minimum number of primary amine groups per repeating unit
400
350
Positive Control Negative Control
10.12 mg/ml
10.012 mg/ml

PJSOOJO c/0:3UBqJ0sqv
40


required to have significant bacterial cell death. Similarly, for future tests a new protocol was
required to test the antibacterial properties of the polymer because the method using OD
values presented several limitations.
However, some positive conclusions were reached. The cytotoxicity tests involving Q-
PNIPAAm, PNIPAAm and Q-PSHU showed statistically insignificant from the negative
control sample of pure culture media. Hence, although it was concluded that more
quatemized groups were required to obtain antibacterial activity, the polymer was
biocompatible. Similarly, the reverse-thermal gelling properties were satisfactory.
Due to the conclusions from the initial results and new polymer system was developed. As
mentioned in the literature review, Polyethyleneimine (PEI) is a nontoxic, aliphatic, weakly
basic, synthetic polymer. It owes its polycationic nature to the presence of primary,
secondary, and tertiary amine groups and has been used extensively in antimicrobial
applications. Branched as well as linear PEI structures can be obtained. However, the
branched configuration contains a combination of primary, secondary and tertiary amines
while the linear structure only contains secondary amines. Due to these structural differences
branched PEI was selected to design a new polymer.
5.2. Development of a New Polymer: Q-PEI-PNIPAAm
The new polymer PEI-PNIPAAm and Q-PEI-PNIPAAm was synthesized as described in the
method section. After obtaining the new polymer different approaches were taken to
characterize its structure.
5.2.1. Polymer Characterization
Proton Nuclear Magnetic Resonance NMR)
41


The structure of PEI was confirmed by !H NMR (500 MHz) in CDC13 (8, ppm):D 1.5-2
[CH2-CH2-NH2], 2.5-2.7 [N-CH2-CH2-N]. The results agree with the literature[48] and peak
assignments were corroborated by NMR modeling (Advanced Chemistry Development). The
spectra showed the presence of primary amine groups and confirmed the polymer backbone
structure.
Figure 10. !HNMR (500 MHz, CDC13) analysis of the PEL Peak assignments were
corroborated by NMR modeling (Advanced Chemistry Development).
The spectra of PEI-PNIPAAm (30% conjugation) was also obtained with !H NMR
spectroscopy (500 MHz, CDC13) and is shown on Error! Reference source not found.jError!
No se encuentra el origen de la referenda..
42


Figure 11.!H NMR (500 MHz, CDC13) analysis of the PEI-PNIPAAm (30% conjugation).
Peak assignments were corroborated by NMR modeling (Advanced Chemistry
Development).
^ NMR (500 MHz) in CDC13(5, ppm):D 0.85-1.4 [N-CH2-(CH3)2], 3.6-4.2 [N-CH2-(CH3)2
and -NH- of PEI]. Peak assignments were corroborated by NMR modeling (Advanced
Chemistry Development).
Unfortunately, as expected, PNIPAAm dominated the spectra due to its molecular weight and
high conjugation ratio. However, some changes were observed and the spectra can be used as
supporting information proving the conjugation of PEI-PNIPAAm. As mentioned
previously, PNIPAAm-COOH was conjugated to PEI through carbodiimide linking
43


chemistry leading to the formation of an amide bond. The spectra for PNIPAAm (found in
Appendix A) shows that the area 5 5.85-6.8 ppm changed from one peak corresponding to an
amide bond to two peaks in the spectra corresponding to PEI-PNIPAAm. This change,
although small, shows the presence of a new amide bond, possibly due to the conjugation of
PEI-PNIPAAm. However, since the %mol of the amide bond of interest is small in the
overall polymer, it is hard to detect.
Due to the limitations encountered with PNIPAAm, to ensure that the quatemization
chemistry was appropriate PEI was quatemized and the structure was the analyzed using !H
NMR.
^ NMR (500 MHz) in CDC13 (5, ppm):D 0.85-1.0 [N-CH2-(CH2)4-CH3], 1.6-1.9 [N-CH2-
(CH2)4-CH3], 3.25-3.4 [N-CH2-(CH2)4-CH3], 2.7-2.98 [N-CH3, alkylated PEI]. The spectra
was as expected and the peaks agree with the literature [51].Peak assignments were
corroborated by NMR modeling (Advanced Chemistry Development)
44


Zeta Potential Measurements
Charges surrounding the surface of a particle in suspension can be quantified using zeta
potential. The zeta potential measures the electrophoretic mobility of the particles in an
electrical field [61].Soluble polymers do not have a two-dimensional interface as solid
nanoparticles do; therefore, there is no zeta potential in a conventional sense. However,
cationic polymers do exhibit electrokinetic mobility due to the presence of charges. In the
case of soluble polymers, there is no strict relation between charge density and electric
potential. When zeta potential is used to measure the charges of a particle, no fluid flows
through the solid particle; hence, it is a two-dimensional surface charge density. In the case of
polyelectrolytes, the fluid can flow through the coils-branched structure of the polymer. The
results of zeta potential in this case are not conclusive but indicative of the presence of
positive charges.
8
7
6
5

3
2
1
0
PEI-PNIPAAM (20%) BQ-PEI-PNIPAAM (20%)
Figure 13. Zeta potential measurements of quatemized and non-quatemized polymers,
significant statistical difference was noted between the values indicates p<0.01).
45


The results for PEI-PNIPAAm 30% and 50% conjugation were not included because they
displayed substantial variability. Measurements were taken with lower molecular weight
PNIPAAm (2000 g/mol), and these exhibited positive results. However, when measuring
samples with PNIPAAm molecular weight greater than 9000 g/mol and conjugations above
20%, the error associated with the measurements made the results unreliable. The
hydrophobic ends of PNIPAAm were probably aggregating with the long alkyl chains of Q-
PEI-PNIPAAm; therefore, the zeta potential results exhibited substantial variations after
mixing and every run.
Lower Critical Solution Temperature (LCST)
An aqueous solution of a thermo-responsive polymer becomes turbid at temperatures above
the LCST, leading to changes in light transmittance and scattering. UV-visible spectroscopy
is a common tool used to study LCSTs because it allows users to measure the changes in
amount of light that is transmitted through the sample.
Solutions of PEI-PNIPAAm (20, 30, and 50% conjugation) as well as the quatemized
versions were subjected to LCST measurements. The results below show that the
transmittance decreases with increasing temperature.
l
| 80
60
I 40
^ 20
0

-Q-PEI-PNIPAAm (20%)
-PEI-PNIPAAm (20%)
20 30 40
Temperature (C)
50
120

80
1 60
1 40
20
0
-Q-PEI-PNIPAAm (50%)
-PEI-PNIPAAm (50%)
20
30 40 50
Temperature (C)
Figure 14. LCST of PEI-PNIPAAm (20% and 50% conjugation) and Q-PEI-PNIPAAm.
46


100
Q-PEI-PNIPAAm (30%)
PEI-PNIPAAm(30%)
Temperature (C)
Figure 15. LCST of PEI-PNIPAAm and Q-PEI-PNIPAAm (both 30% conjugation).
All transitions were thermally reversible, and the turbid copolymer solutions became
transparent when they returned to temperatures below the LCST. As mentioned in the
literature review, PNIPAAm itself exhibits an LCST at 32C[62][63]. The thermo-
responsive behavior depends on the solvent interaction with the polymer and the
hydrophilic/hydrophobic balance within the polymer molecules. The introduction of
hydrophilic and hydrophobic co-monomers can cause and increase or decrease in the
LCST[64] +
In this study the LCST of the polymers was defined as the temperature reducing the change in
light transmittance by a half. Table 1 shows the increasing LCST value with increasing
hydrophilic PEI content for the different copolymers. In all three cases, the polymers
exhibited higher transmittance after quatemization.
In conclusion, the addition of PEI increased the LCST the alkylation-quatemization process
decreased the LCST. Studies [63] mention that incorporation of more hydrophobic groups
will lead to a lower LCST. So, we can attribute the decrease of LCST of the quatemized
polymers to the presence of the long hydrophobic alkyl chains that contribute to the overall
hydrophobicity of the polymer.
47


Polymer LCST(C) Polymer LCST(C)
PEI-PNIPAAm (Conj 20%) 35 Q-PEI-PNIPAAm (Conj 20%) 32.5
PEI-PNIPAAm (Conj 30%) 34.5 Q-PEI-PNIPAAm (Conj 30%) 32.6
PEI-PNIPAAm (Coni 50%) 34.1 Q-PEI-PNIPAAm (Conj 50%) 33.7
Table l.LCSTval ues of each polymer.
5.2.2. Antimicrobial Tests using Q-PEI-PNIPAAm
In a clinical setting, non-multiplying (stationary phase) and multiplying (log phase) bacteria
coexist. The mechanisms of action of several antibiotics require ongoing cell division and are
less efficient at killing non-multiplying bacteria [65]. For example, beta lactams need
ongoing bacterial cell wall synthesis for bactericidal activity. Many studies have been
performed on log-phase bacteria (rapidly dividing bacteria); however, bacteria seldom
encounter optimal conditions that enable logarithmic growth [65]. S. aureus has been shown
to modulate gene expression, thereby survive long periods in the stationary phase under
unfavorable growth conditions [66] [67].
In vitro antibacterial studies were conducted to obtain time-kill curves to quantify the killing
capacity of the polymers developed. Due to reasons previously mentioned, most tests
involved bacteria in the stationary phase. However, the killing of S. aureus during the log
phase was also tested to ensure the antimicrobial activity of this material under different
conditions.
Initially, the antimicrobial activity of PEI-PNIPAAm (20%) was tested against S. aureus.
The polymer (concentration 10% w/v in PBS) exhibited more than a 8-logi CFU/ml bacteria
reduction in 30 minutes, as can be observed in Figure 16.
For sample images showing agar plates and colony counting, please refer to the Appendix B.
48


9
Bacteria Sample-PEI-PNIPAAm (20%) Q-PEI-PNIPAAm (20%)
0 1------------------^---------------------------------------------------------
0 30 60 90 120
Time (minutes)
Figure 16. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% conjugation) were added to
stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120
minutes. Numbers indicate reductions in log10 CFU/ml.
Unfortunately, although the antimicrobial activity of the polymer was outstanding and the
LCST results showed a transition, the gel obtained at 37C was unstable. Since increasing the
polymer concentration in water resulted in very slow gelling and de-gelling, the alternative
chosen was to repeat the tests using a higher PNIPAAm conjugation ratio. As explained in
the method, PEI-PNIPAAm (50% conjugation) and Q-PEI-PNIPAAm (50% conjugation)
were synthesized. The antimicrobial tests did not exhibit significant bacterial reductions even
after 2 hours. Several factors were attributed to this, first the large content of PNIPAAm
affecting the quatemization process. Also, as mentioned in the literature review, increasing
the molecular weight will substantially affect the antibacterial properties; by conjugating 30%
more primary amines to PNIPAAm (total of 50% conjugation), the molecular weight would
increase substantially since the PNIPAAm chains have a higher molecular weight than the
7 6 5 4 3 2 1
(US) JunoulBI sohj
49


0 30 60 90 120
Time (minutes)
Figure 17. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to
stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120
minutes. Numbers indicate reductions in log10 CFU/ml.
alkyl chains. At the same time, longer chains can also play a role m inhibiting the positive
charges of the polymer to destabilize the negative charges associated with the bacterial celFs
outer structure.
Due to this, the polymer was modified once again, and PEI-PNIPAAm (30% conjugation)
and Q-PEI-PNIPAAm (30% conjugation) were synthesized.
As shown in Figure 17, the first antimicrobial test against S. aureus showed promising
results, over an 8-logi CFU/ml bacterial reduction. This test was conducted two more times
to ensure the killing capacity of the polymer. The tests were carried out using different
polymer samples and different bacteria cultures. Standard deviations were calculated,
accounting for every sample.
----Bacteria Sample ---PEI-PNIPAAm (30%) Q-PEI-PNIPAAm (30%)
(US unoulapBa sohj
50


The results shown in Figure 17 also show that PEI-PNIPAAm (30% conjugation) itself
possesses antibacterial activity. This is consistent with the literature; PEI itself has been
shown to kill S. aureus [48] [27]. This is due to the presence of protonated ammonium groups
and non-protonated amine groups. The ethylene backbone serves as hydrophobic groups,
which create repeating cationic, amphiphilic structures along the polymer backbone at a
neutral pH without any further chemical modification. At the same time, it can be observed
that this polymer exhibited a 2-, 3-, and 4-log reduction after 30, 60, and 120 minutes, which
is slightly lower than what the PEI-PNIPAAm (20% conjugation and 3-, 4-, and 5-log
reduction after 30, 60, and 120 minutes), likely due to having more PNIPAAm conjugated
and less primary free amines.
Tests with Staphylococcus aureus subsp. aureus Mu3 (MRSA/hetero-VISA)
Once the antibacterial properties against S. aureus were confirmed, a new strain was tested.
Tests were conducted to measure the killing capacity of the polymer against S. aureus subsp.
aureus MuS (MRSA/hetero-VISA). Once again, the test was conducted three times using
different polymer samples and different bacteria cultures. Standard deviations were
calculated, accounting for every sample.
In this case, PEI-PNIPAAm (30% conjugation) also exhibited an 8-logi bacterial reduction.
Two tests showed a 6-log10 reduction in 30 minutes, while the third test showed an 8-log
reduction was achieved in the same period of time. This variability is normal, and it was
concluded that in 1 hour, the polymer was able to achieve an 8-logi bacteria reduction in all
cases. One of the controls previously used, PEI-PNIPAAm (30% conjugation) that had
previously shown to possess antibacterial activity, did not show the same behavior in this
case. The reason for this is because it appears that vancomycin resistance inS. aureus is
51


0 30 60 90 120
Time (minutes)
Figure 18. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to
stationary-phase cultures of Staphylococcus aureus subsp. aureus MuS (MRSA/hetero-VISA).
Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in logi
CFU/ml.
Tests with S. epidermidis
S. epidermidis is a gram-positive organism and a major inhabitant of the skin, and in some
areas, it makes up more than 90% of the resident aerobic flora [71].Due to this, antibacterial
tests were conducted to ensure that the polymer would be able to kill this strain. The results,
as shown in
conferred by alterations m the bacterial cell wall [68][69]. Specific to Mu3, Hanaki et Al.
studied the activated cell-wall synthesis associated with vancomycin resistance in methicillin-
resistant S. aureus clinical strains Mu3 and Mu50. They demonstrated that cell-wall synthesis
and turnover are upregulated in VRSA isolates, leading to thicker and more disorganized cell
walls [70]. Hence, since the cell wall is thicker, the long alkyl chains penetrate the cell wall to
achieve cell death. The quatemization of PEI-PNIPAAm is strictly required to achieve cell
death.
----Bacteria Sample ----PEI-PNIPAAm (30%)
Q-PEI-PNIPAAm (30%) ----PNIPAAm
(US unoulapBa sohj
52


Figure 19, confirm that Q-PEI-PNIPAAm (30% conjugation) also exhibited an 8-log10
0 30 60 90 120
Time (minutes)
Figure 19. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% and 30% conjugation) were added to
stationary-phase cultures of Staphylococcus epidermidis. Samples were taken after 30, 60,
and 120 minutes. Numbers indicate reductions in logi CFU/ml.
Tests with E. coli
Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-
positive and gram-negative bacteria. Since the antibacterial properties of Q-PEI-PNIPAAm
(30% conjugation) were confirmed against gram-positive bacteria, the next step was to test it
against gram-negative bacteria. As mentioned in the literature review, gram-positive bacteria
have a thick cell wall, containing several layers of peptidoglycan, while gram-negative
bacteria contains a similar a cell wall, but is also surrounded by an outer membrane. This
outer membrane can be harder to destabilize at times. Although gram-positive bacteria are
bacterial reduction.
---Bacteria Sample ----Q-PEI-PNIPAAm (Conj 30%)
Q-PEI-PNIPAAm (Conj 20%) ----PEI-PNIPAAm
7 6 2 1
UOUKUUaouo3--uuapugsoq
53


prevalent on the skin, gram-negative bacteria can also be present in a clinical setting, and due
to this, the polymer antibacterial activity against E. coli was assessed.
Q-PEI-PNIPAAm (30% conjugation) exhibited a 2-, 3-, and 8-log10 bacteria reduction after
30, 60, and 120 minutes, respectively. The lower bactericidal action, when compared to the
previous tests, can be explained from the cell wall structural differences already mentioned
between gram-positive and gram-negative bacteria. Q-PEI-PNIPAAm (20% conjugation)
was also tested in this case but only exhibited slightly higher bactericidal action. On the other
hand, one of the controls, PEI-PNIPAAm (30% conjugation), had very little antibacterial
activity, similar to the effect it had over the Mu3 strain of S. aureus.
Bacteria Sample
Q-PEI-PNIPAAm (Conj 30%)
I
P
U

I
s
09
x
o
0 30 60 90 120
Time (minutes)
Figure 20. Q-PEI-PNIPAAm and PEI-PNIPAAm (30 and 20% conjugation) were added to
stationary-phase cultures of E. coli. Samples were taken after 30, 60, and 120 minutes.
Numbers indicate reductions in log 10 CFU/ml.
Test with S. aureus during Log Phase
Finally, the last agar test conducted was to ensure the antibacterial properties of the polymer
against bacteria in the log phase. As mentioned previously, some antibiotics require the cells
54


0 30 60 90 120
Time (minutes)
Figure 21.Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) were added to log-phase
cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes.
Numbers indicate reductions in log 10 CFU/ml.
5.3. Cytotoxicity Tests of Q-PEI-PNIPAAm
An MTT assay was conducted to assess the biocompatibility of Q-PEI-PNIPAAm. Several
methods were used to conduct this test. Previously, the polymer had been placed on top of the
cells mixed with the media, however, due to the gelling nature of the sample the polymer was
not in contact with the cells for long period of time. Due to this, the methodology was
modified and the cells were plated on top of the polymer and then tripsinized and re-plated
before conducting the test. This method also was proven faulty because tripsinizing.
to be multiplying m order to kill the bacteria. The mechanism of action of this polymer does
not require this. But it is important to ensure that the polymer can still be effective when
subjected to continuously dividing cells. As can be observed in Figure 21,Q-PEI-PNIPAAm
(30% conjugation) achieved a 6-log bacteria reduction in 30 minutes.
---Bacteria Sample ---PEI-PNIPAAm Q-PEI-PNIPAAm
(hJUI/SIUu) JunoulB-capBa sohj
55


centrifuging and re-plating cells lead to higher variability in number of cells per well and
since the cells were not left to attached on the plates for long enough the results were
inaccurate. Finally the polymer was dissolved in alcohol, left to evaporate and then the cells
were plated on top. Since the MTT measures absorbance, blanks were required for each
sample. However, the layer of polymer on the plate needed to be very thin so the polymer
concentration tested wasl,3 and 5%. Q-PEI-PNIPAAm (20% Conjugation) was chosen
since it had the highest number of quatemized amine groups and was assumed to be most
toxic. At the same time, chlorhexidine 2%, a common skin preparation solution was tested as
well.
56


u

0
1
oT
fl

Xi
o
jx
<
Positive control Negative Control 1% 3% 5%
120-----------------------------------------------------------------------------


QPP (20% PEI-PNIPAAm (20% PNIPAAm Chlorhexadine
Conjugation; Conjugation;
Samples
figure z2. No statistical difference between negative control and cells exposed to PNIPAAm
and PEI-PNIPAAm. Statistically signiricant difference was observed between the
experimental samples, negative and positive control (cells cultured with 5% DMSO)
indicates p > 0.05, and indicates p < 0.05.). No statistical difference between cells exposed
to Chlorhexidine (FDA approved) and Q-PEI-PNIPAAm
57


The cells exposed to Q-PEI-PNIPAAm showed statistically significant difference in
metabolic activity when compared to cells cultured with complete growth medium alone and
to the ones exposed to 5%DMSO. Hence, a reduction in metabolic activity was observed but
the same difference was obtained in the samples with cells subjected to an FDA approved
skin preparation, chlorhexidine 2%. At the same time, it must be mentioned that the cells
showed higher metabolic activity than the positive control as well. MTT-assays asses
metabolic activity and live-dead staining would need to have more conclusive information
regarding toxicity.
Similarly, the toxicity of the cells was assessed when skin sensitization and irritation tests
would probably be more appropriate when considering the polymer application.
5.4. Functional Tests
Antimicrobial Activity of Commercially Available Surgical Incision Drapes
The preoperative skin preparations that are currently used before surgeries disinfect the
superficial layer of the skin. However, some remaining bacteria may cause recolonization
during surgery [11].3M, one of the major companies producing SIDs, states that surgeons
use antimicrobial SIDs as an added protection to lower the risk of potential recolonization.
However, the literature concerning risk of recolonization whilst using a drape is controversial.
Many studies publish faster recolonization of skin with plastic drape than without plastic
drape[54].
To evaluate whether bacteria does recolonize the skin surface, agar plates were inoculated
with a used surgical drape. Colonies were observed after a 24 hour period as can be observed
in Figure 23.
58


Figure 23. Bacterial growth after inoculation with used SIDs. For the test, pieces of SIDs
were taken right after a surgery, inoculated on 5 spots on an agar plate. Surprisingly, a
formation of bacterial colony was observed on 2 spots, indicating bacterial recolonization
Antimicrobial Activity of Polymer-Coated Glass Slides
Since the polymer will be used in a similar manner to a coating, it was tested after coating
glass slides. All the different polymers were dissolved in water and placed in a spray bottle to
assess the application method. Unfortunately, the viscosity of the samples was too high, and
the coating seemed uneven. Due to this, a new technique was developed. Instead of
dissolving the polymer in dH20, the new approach involved dissolving the polymer samples
(1% w/v) in 70% ethanol and then spraying it and allowing the ethanol to evaporate. This
new approach also could potentially mean that the 'skin-prepping5 step using ethanol could
be skipped and, hence, the steps before the surgery reduced.
The glass slides coated with Q-PEI-PNIPAAm (20% and 30%) showed no bacterial growth,
while the controls, did as can be seen in Figure 24. The coating with Q-PEI-PNIPAAm has an
orange/yellowish color, but no colonies were seen, while PEI-PNIPAAm (20 and 30%)
showed colonies. The bacterial dilutions performed showed that the initial bacterial
concentration was 7.96 x 107CFU/ml.
59


-
Figure 24.1 x 1 cm glass slides coated with polymer and then sprayed with bacteria.
The half-coated/half-non-coated slide showed no bacterial growth on the coated region, as
can be observed in Figure 25.

Figure 25. Glass slide that was half coated with the polymer and half uncoated. The left insert
shows colonies that grew on the non-coated edges of the slide.
Images were taken of the glass slide coated with the polymer-ethanol solution before and
after soaking in dH20 for 24 hours.
60


Figure 26. Image of the glass slide sprayed with the polymer-ethanol solution before soaking
in water.
Figure 27. A) Image of the glass slide after it was soaked for 24 hours in water. B) A razor
blade was used to remove part of the polymer on the pre-soaked slide.
Figure 26 and Figure 27 do not show any significant changes, and the coating is still present
on the slide.
61


6. Conclusions and Future Directions
Conclusion of specific aim 1:Design and synthesize a reverse-thermal gel the
quaternized to obtain antimicrobial properties
An antimicrobial reverse-thermal gel was designed and characterized. Initially, PSHU-
PNIPAAm and Q-PSHU-PNIPAAm were developed and tested. Although both polymers
exhibited reverse-thermal gelling properties, they did not display good antimicrobial
properties. Based on the knowledge gathered from the initial development, a new polymer,
PEI-PNIPAAm and its quaternized version were designed and synthesized. First, PEI-
PNIPAAm (20% conjugation) and Q-PEI-PNIPAAm (20% conjugation) were obtained, and
the zeta potential was measured. The results indicated the presence of positive charges on the
macromolecule. Further characterization was conducted through NMR that confirmed the
conjugation of PEI to PNIPAAm and the quatemization step.
At the same time, UV-visible spectroscopy was used to verify the LCST. However, although
this polymer exhibited a transition temperature, the gel obtained was unstable. Finally, two
variations of this polymer were obtained by varying the conjugation ratio to 30% and 50%.
The detection of positive charges using zeta potential was impeded by the presence of
PNIPAAm that also dominated the NMR spectra at 50% conjugation.
In conclusion, three polymers were designed and characterized with one exhibiting properties
suitable for antimicrobial applications. However, in terms of future work, further analysis
should be conducted to quantify the number of quaternized amine groups and analyze the
effect of these on the overall properties of the polymer. A possible option would be to use
confocal Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), or a solution of
fluorescein (sodium salt) that can be used in a similar manner to a dye. Then, the conversion
62


of the dye concentration to surface charge density can be determined, assuming that one
surface quaternary ammonium group complexes with one dye molecule. However, functional
antibacterial tests were prioritized in this study, and a more thorough analysis is suggested as
part of future work.
Conclusion of specific aim 2: Characterize the antimicrobial activity of the polymer
The antimicrobial properties of the polymers synthesized were assessed. As mentioned
previously, initially, PSHU-PNIPAAm exhibited antimicrobial properties when evaluated
using optical density tests. However, agar tests confirmed the opposite. The antibacterial test
protocols and approach were modified, and a new set of polymer-quatemized PEI-PNIPAAm
polymers of different conjugation ratios were tested. All samples were exposed to bacterial
suspensions of 108 CFU/ml. The results showed that although a conjugation ratio of 20%
exhibits an 8-logl0 bacteria reduction, the polymer did not satisfy other requirements. Later,
the 50% conjugation ratio proved to have insufficient antibacterial activity, while the 30%
conjugation ratio showed an 8-logi bacterial reduction.
At the same time, Q-PEI-PNIPAAm (30% conjugation) was tested against different bacterial
species. The polymer exhibited 8-log10 bacterial reductions when testing gram-positive and
gram-negative bacteria. The time-kill graphs showed differences in each case, and these were
related to the outer cell wall structural differences of the strains tested.
In conclusion, a polymer exhibiting excellent antibacterial properties and good gelling
behavior was obtained and tested. The next step is to conduct in vivo test using Q-PEI-
PNIPAAm (30% conjugation) and compare it to SIDs. An animal protocol has been
submitted and accepted for this next test.
63


Conclusion of specific aim 3: Cytotoxicity test of Q-PSHU-PNIPAAm and Q-PEI-
PNIPAAm
The cytotoxicity of Q-PSHU-PNIPAAm was assessed by means of an MTT assay using
smooth muscle cells. The results showed no significant statistical difference with the negative
control.
The tests were then repeated using Q-PEI-PNIPAAm (20% conjugation) because this sample
exhibited the highest antibacterial properties. Fibroblasts that are more sensitive were used in
this case. At the same time, chlorhexidine was used because it is the golden standard. The
results showed a decrease in metabolic activity when compared to the negative control.
However, the metabolic activity of the fibroblasts subjected to Q-PEI-PNIPAAm was
comparable to the cells that were exposed to chlorhexidine. Since MTT assays are indicative
of the celFs metabolic activity, it can be concluded that the polymer does affect the celFs
metabolic activity; however, the results observed show no statistical difference to those
obtained with Chlorhexidine, an FDA-approved product of clinical use. However, as part of
future work, skin sensitivity/irritation and sensitization tests, such as the Bowman Berger
tests recommended by the FDA, should be conducted in vivo.
Future work
Finally, although further characterization would be interesting in order to correlate the
number of quatemized amine groups to antibacterial properties, it can be concluded that a
reverse-thermal gel with excellent antimicrobial properties was developed in this study.
However, a major point that was not targeted in this study involves the analysis of adhesive
and mechanical properties of the polymer. During surgery, this material has to be able to
withstand water and tough pulling of the skin. Although preliminary tests showed that the
64


polymer can remain attached to a surface during a period of 24 hours in water, further tests
need to be conducted.
Research was conducted on possible methods to assess the adhesive properties of the polymer
and an interesting option was explored by Vemengo et al.[72]. They tested the adhesive
properties of a PNIPAAm-PEG/PEI polymer blend using an Instron mechanical testing
system. They placed the polymer on fresh porcine skin and then removed it at a rate of 2 mm
min"1 whilst obtaining load-displacement data that they then used to obtain stress-strain
values. Using this method they then calculated the maximum force required to detach the gel
form a skin surface. An assay similar to the one previously described would be of interest to
gain insight regarding the adhesive properties of the polymer.
Finally, future work concerns in vivo studies initially on mice (protocol and method
approved) and comparing the polymer to current SIDs.
65


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1.^NMRofPNIPAAm
APPENDIX
Figure A 1. NMR (500 MHz, CDCI3) analysis of the PEI. Peak assignments were
corroborated by NMR modeling (Advanced Chemistry Development)
2. Agar test example images
Images
Figure B 1.Agar plates showing bacterial colonies. Dilutions were performed and each
samples triplicated. Data was collected from counting colonies in E and extrapolating to
obtain the initial bacterial concentration
73


Figure B 2. Agar plates showing bacterial colonies. Time points at t= 60 and 120 minutes.
Dilutions were performed and each samples triplicated. Data was collected from counting
colonies in E and extrapolating to obtain the initial bacterial concentration
Figure B 3. Agar plates showing bacterial colonies subjected to PEI-PNIPAAm (30%
conjugation). Dilutions were performed and each samples triplicated. Data was collected
from counting colonies in E and extrapolating to obtain the initial bacterial concentration
74


Figure B 4. Agar plates showing no bacterial growth after 30 minute antibacterial test with Q-
PEI-PNIPAAm (30% conjugation)
Figure B 5. Agar plates showing no bacterial growth. The bacteria had been subjected to Q-
PEI-PNIPAAm (30% conjugation) for 60 minutes
Figure B 6. Agar plates showing no bacterial growth. The bacteria had been subjected to Q-
PEI-PNIPAAm (30% conjugation) for 120 minutes
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Full Text

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ANTIMICROBIAL REVERSE-THERMAL GEL FOR SURGICAL APPL ICATIONS by MARIA BELEN BORTOT M.S., Universidad Nacional de Cuyo, Instituto Balse iro, Argentina, 2012 B.E., The University of Sydney, Australia, 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Bioengineering 2014

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ii This thesis for the Master of Science degree by Maria Belen Bortot has been approved by Daewon Park, Chair Vikas Patel Andres Vazquez-Torres September 2, 2014

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iii Maria Belen Bortot (M.S., Bioengineering) Antimicrobial reverse-thermal gel for surgical appl ications Thesis directed by Assistant Professor Daewon Park. ABSTRACT A surgical site infection (SSI) is defined as an in fection that is developed during a surgical procedure or up to 30 days after. In the US, 2-5% o f patients undergoing surgery suffer from SSIs. These patients have 2-11 times higher risk of death. During the surgery, one of the main sources for pathogens is often thought to be the sk in surface. Skin antiseptic preparations along with antiseptic-impregnated surgical incision drapes (SIDs) are used as a barrier to prevent re-colonization and immobilize the organism s that might have survived the treatment. However, these drapes present numerous pitfalls. Am ongst the most severe are that the placement process of the drapes is time consuming, they often do not remain well-attached and they only provide temporary protection. Similar ly, there are risks associated with epidermal cell layer detachment during removal, exp osing bacteria found beneath the skin, thereby increasing risk of secondary bacterial infe ction. Polymers functionalized with quaternary amine group s have shown high inherent antimicrobial properties. The positive charges on t hese functional groups cause disruption of the negatively charged bacterial cell wall. In order to overcome the limitations associated wit h SIDs, we developed a polymer-based antimicrobial surgical coating that can act as a SI D. This polymer is specifically designed to possess a reverse thermal-gelling property to ease the application process. It can be sprayed

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iv onto the skin, turns into a thin adhesive gel when it comes into contact with the body, and can then be removed by washing with soap/water at low t emperatures (less than 20C). The form and content of this abstract are approved. I recommend its publication. Approved: Daewon Park

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v ACKNOWLEDGMENTS I would like to thank my friends, family, professor s, teachers, and mentors that contributed to my academic and personal development throughout thi s process; without them, this would not have been possible. First and foremost, I want to thank my academic and thesis advisor, Dr. Park, for taking me on as a student and being extremely patient and exc ellent guide. Thanks, Dr. Park. I learned lots! I would also like to thank the other members of my Committee, Dr. Vazquez-Torres and Dr. Patel, for being so responsive every time I reached out and for encouraging and motivating me. At the same time, I want to thank Melissa Laughter and Lindsay Hockensmith, two amazing friends that shared this whole experience with me. I learned so much from them in and outside of the lab. My time in Colorado would not h ave been the same without them, and I am extremely lucky and thankful for having met them and shared so many great experiences. Similarly, I want to thank all the Translational Bi omaterial Research Laboratory members, the ones that have left and the ones that are still with us. Finally, I would like to dedicate this thesis to my family. First and foremost, I want to thank my sister, as she motivated me to study and believe d in me every step of the way, and my parents, my dad for being an amazing guide and my m om for following me around everywhere IÂ’ve moved to. And I would also like to thank Paschal Duru, my boyfriend, for sharing this experience with me and encouraging me every day.

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vi TABLE OF CONTENTS Chapter 1. Introduction ................................... ................................................... ........................................ 1 1.1. Background .................................. ................................................... ............................ 1 1.2. Surgical Site Infections .................... ................................................... ........................ 2 1.3. Preventative Measures for SSIs .............. ................................................... .................. 3 1.4. Objective of This Study ..................... ................................................... ....................... 6 2. Literature Review .............................. ................................................... .................................... 8 2.1. Antimicrobial Mechanisms of Action .......... ................................................... ............ 8 2.2. Antimicrobial Polymers ...................... ................................................... ................... 11 2.3. Reversible-gelling Properties ............... ................................................... .................. 17 3. A reverse-thermal gel with antimicrobial activit y ................................................. ................. 22 3.1. New Approach: Polyethyleneimine ............. ................................................... ........... 23 3.2. New Polymer ................................. ................................................... ......................... 25 4. Materials and Methods .......................... ................................................... .............................. 26 4.1. Materials ................................... ................................................... .............................. 26 4.2. Polymer Synthesis ........................... ................................................... ....................... 26 4.3. Polymer Characterization .................... ................................................... ................... 29 4.4. Antimicrobial Tests ......................... ................................................... ....................... 30 4.5. Functional Tests ............................ ................................................... ......................... 32 4.6. Cytotoxicity Test of Q-PEI-PNIPAAm and Q-PSHU -PNIPAAm ............................ 34 5. Results and Discussion ......................... ................................................... ............................... 36 5.1. Preliminary Studies ......................... ................................................... ....................... 36

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vii 5.1.1. Antimicrobial Tests Using Q-PSHU-PNIPAAm .. ........................................... 36 5.1.2. Cytotoxicity Tests of Q-PSHU-PNIPAAm ...... ............................................... 38 5.1.3. Conclusion from Tests Performed with Q-PSHU -PNIPAAm ......................... 40 5.2. Development of a New Polymer: Q-PEI-PNIPAAm ............................................... 41 5.2.1. Polymer Characterization .................. ................................................... ........... 41 5.2.2. Antimicrobial Tests using Q-PEI-PNIPAAm ... ............................................... 48 5.3. Cytotoxicity Tests of Q-PEI-PNIPAAm .......... ................................................... ....... 55 5.4. Functional Tests ............................. ................................................... ......................... 58 6. Conclusions and Future Directions............... ................................................... ....................... 62 References .................................................. ................................................... ................................. 66 Appendix .................................................. ................................................... ................................... 73

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viii LIST OF TABLES Table 1. LCST values of each polymer. ................... ................................................... ...................... 48

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ix LIST OF FIGURES Figure Figure 1. A: Epidermal cell detachment during the r emoval of a SID, possibly leading to secondary infection by bacteria found beneath the s kin. B: Surgical incision drape on the skin before surgery. C: Surgical incision drape during s urgery. ........................................... ............ 6 Figure 2. Mode of action of various antimicrobial c ompounds. ......................................... ...... 9 Figure 3. Outer membrane/cell wall differences betw een Gram-positive and Gram-negative bacteria [21] [22]. ............................... ................................................... .................................. 11 Figure 4. PNIPAAm structure and behavior above and below the LCST. .............................. 20 Figure 5. Polymer backbone with quaternized amine g roups ............................................. .... 23 Figure 6. Structure of branched Polyethyleneimine ................................................... ............ 24 Figure 7. Results from the first antibacterial test against S. aureus using different polymer and bacterial concentrations. ......................... ................................................... .............................. 37 Figure 8. Results from the first MTT assay conducte d using Q-PSHU-PNIPAAm at different concentrations. The only statistically significant difference was observed between the experimental samples and positive control (cells cu ltured with 5% DMSO). (** indicates p > 0.01, and *** indicates p < 0.01.) ................ ................................................... ........................ 39 Figure 9. Results from the second MTT assay conduct ed using Q-PSHU at different concentrations. ................................... ................................................... ................................... 40 Figure 10. 1H NMR (500 MHz, CDCl3) analysis of the PEI. Peak assignments were corroborated by NMR modeling (Advanced Chemistry De velopment). ................................ 42 Figure 11. 1H NMR (500 MHz, CDCl3) analysis of the PEI-PNIPAAm (30% conjugation). Peak assignments were corroborated by NMR modeling (Advanced Chemistry Development). ..................................... ................................................... ................................. 43 Figure 12. 1H NMR (500 MHz, CDCl3) analysis of the Q-PEI. Peak assignments were corroborated by NMR modeling (Advanced Chemistry De velopment). ................................ 44 Figure 13. Zeta potential measurements of quaterniz ed and non-quaternized polymers. Significant statistical difference was noted betwee n the values (** indicates p<0.01). .......... 45 Figure 14. LCST of PEI-PNIPAAm (20% and 50% conjuga tion) and Q-PEI-PNIPAAm. .... 46 Figure 15. LCST of PEI-PNIPAAm and Q-PEI-PNIPAAm (b oth 30% conjugation). ........... 47

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x Figure 16. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% conju gation) were added to stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. .......................................... ............... 49 Figure 17. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conju gation) were added to stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. .......................................... ............... 50 Figure 18. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conju gation) were added to stationary-phase cultures of Staphylococcus aureus subsp. aureus Mu3 (MRSA/heter o-VISA). Samples were taken after 30, 60, and 120 minutes. N umbers indicate reductions in log10 CFU/ml. ........................................... ................................................... ..................................... 52 Figure 19. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% and 3 0% conjugation) were added to stationary-phase cultures of Staphylococcus epidermidis. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. .......................................... 53 Figure 20. Q-PEI-PNIPAAm and PEI-PNIPAAm (30 and 20 % conjugation) were added to stationary-phase cultures of E. coli. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. .......................................... ............................. 54 Figure 21. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conju gation) were added to log-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. .......................................... ............................. 55 Figure 22. No statistical difference between negati ve control and cells exposed to PNIPAAm and PEI-PNIPAAm. Statistically significant differen ce was observed between the experimental samples, negative and positive control (cells cultured with 5% DMSO) (*** indicates p > 0.05, and ** indicates p < 0.05.). No statistical difference between cells exposed to Chlorhexidine (FDA approved) and Q-PEI-PNIPAAm ................................................... .. 57 Figure 23. Bacterial growth after inoculation with used SIDs. For the test, pieces of SIDs were taken right after a surgery, inoculated on 5 s pots on an agar plate. Surprisingly, a formation of bacterial colony was observed on 2 spo ts, indicating bacterial recolonization .. 59 Figure 24. 1 x 1 cm glass slides coated with polyme r and then sprayed with bacteria. .......... 60 Figure 25. Glass slide that was half coated with th e polymer and half uncoated. The left insert shows colonies that grew on the non-coated edges of the slide. ....................................... ...... 60 Figure 26. Image of the glass slide sprayed with th e polymer-ethanol solution before soaking in water. ......................................... ................................................... ....................................... 61 Figure 27. A) Image of the glass slide after it was soaked for 24 hours in water. B) A razor blade was used to remove part of the polymer on the pre-soaked slide. ................................ 61

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xi A 1. 1H NMR (500 MHz, CDCl3) analysis of the PEI. Peak assignments were corrob orated by NMR modeling (Advanced Chemistry Development)73 B 1. Agar plates showing bacterial colonies. Diluti ons were performed and each samples triplicates ....................................... ................................................... ....................................... 73 B 2. Agar plates showing bacterial colonies. Time p oints at t= 60 and 120 minutes. Dilutions were performed and each samples triplicates ....... ................................................... ................ 74 B 3. Agar plates showing bacterial colonies subject ed to PEI-PNIPAAm ............................ 74 B 4. Agar plates showing no bacterial growth after 30 minute antibacterial test with Q-PEIPNIPAAm (30% conjugation) ......................... ................................................... ..................... 75 B 5. Agar plates showing no bacterial growth. The b acteria had been subjected to Q-PEIPNIPAAm (30% conjugation) for 60 minutes .......... ................................................... ........... 75 B 6. Agar plates showing no bacterial growth. The b acteria had been subjected to Q-PEIPNIPAAm (30% conjugation) for 120 minutes ......... ................................................... .......... 75

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XII LIST OF ABBREVIATIONS ACA 4,4 -AZOBIS(4-CYANOVALERIC ACID) AMHP 2,2'-AZOBIS[2-METHYL-N-(2HYDROXYETHYL)PROPIONAMIDE] DMEM DULBECCO'S MODIFIED EAGLE'S MEDIUM DMF DIMETHYLFORMAMIDE DMSO DIMETHYL SULFOXIDE EDC N -(3-DIMETHYLAMINOPROPYL)N -ETHYLCARBODIIMIDE HYDROCHLORIDE FT-IT FOURIER TRANSFORM INFRARED GPC GEL PERMEATION CHROMATOGRAPHY HDI HEXAMETHYLENE DIISOCYANATE LCST LOWER CRITICAL SOLUTION TEMPERATURE MTT 3-(4,5-DIMETHYLTHIAZOL-2-YL)-2,5DIPHENYLTETRAZOLIUM BROMIDE MW MOLECULAR WEIGHT NHS N -HYDROXYSUCCINIMIDE NMR NUCLEAR MAGNETIC RESONANCE PBS PHOSPHATE-BUFFERED SALINE PEI POLYETHYLENIMINE PNIPAAM POLY( N -ISOPROPYLACRYLAMIDE) PSHU POLY (SERINOL HEXAMETHYLENE UREA) QUATs QUATERNARY AMMONIUM COMPOUNDS RTG REVERSE THERMAL GEL SID SURGICAL INCISION DRAPES

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XIII TFA TRIFLUOROACETIC ACID UV ULTRAVIOLET

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1 1. Introduction 1.1. Background Human microbiota is the term used to refer to the vast number of mi crobes that reside in and on the human body. These organisms play both benefi cial and harmful roles. Gut flora/microbiota, for example, aid in the digestion of certain foods and play a role in the production of some vitamins (e.g., B and K). Simila rly, human skin is also host to microbes that are regarded as pathogens, potential pathogens or innocuous symbiotic organisms [1]. Four main species of bacteria predominate on the su perficial layer of the epidermis: Diphtheroids (e.g., corynebacteria ), Staphylococci (e.g., S. aureus ), Micrococci Streptococci (either alpha ( ) or gamma ( ) hemolytic), and the Enterococci [2][3][4] Depending on various factors, such as moisture and body area, th e species that predominates will vary. The number of bacteria may range from approximately 1,0 00 to more than 10 million organisms per square centimeter of skin surface, with Gram-po sitive organisms usually predominating [2][5]. The intact surface of a healthy epidermis prevents bacteria from penetrating. However, this protection is lost if the integrity of the epidermi s is broken, for example, by cuts or abrasions. During surgeries, the skin barrier is broken and pa tients are at risk of bacteria invading the surgical site, leading to infections. Accordingly, the use of antimicrobial agents is required and protocols to reduce risk of infection are a par t of common clinical practice. The risk of hospital-acquired infections, along with the emerge nce of multi-drug-resistant bacteria such as methicillin-resistant strains of Staphylococcus aureus (MRSA), vancomycin-resistant enterococci and Pseudomonas aeruginosa has led to a growing interest in the development of alternative antimicrobial agents.

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2 Advances in polymer science have enabled the synthe sis of novel material structures with added functionalities. Advances in the area of micr oscopy (transmission electron microscopy, scanning electron microscopy, and fluorescence micr oscopy) facilitated the study of interactions between materials, biological molecule s, and cells. This allowed the development of new polymeric materials with specific functional ities, such as antimicrobial properties. The literature pertaining to this topic is vast, an d the new materials developed to either kill or inhibit bacterial colonization vary depending on th e application (food, animal feed, water purification systems, or medical industry). 1.2. Surgical Site Infections A surgical site infection (SSI) is defined as an infection that develops in the body as a consequence of a surgical procedure during and up t o 30 days after the procedure. They manifest as pus or swab with >106 colony-forming units (CFU) per mm3 tissue [6]. As mentioned previously, they are generally caused by Staphylococci, Streptococci, Diphtheroid organisms Pseudomonas, and Propionibacterium, which are constantly present on patientsÂ’ skin [7]. In the United States, SSIs account for 14-16% of al l hospital-acquired infections. Approximately 70% of SSIs are superficial infection s; the remaining 30% involve implanted materials, deeper tissues, or organs. The Institute for Healthcare Improvement stated that SSIs in the United States lead to an average 7.5-da y increase in patient hospitalization periods [8]. This has an estimated cost of $130 million to $845 million per year [9]. At the same time, the rising incidence of hospital-acquired, antibiot ic-resistant Staphylococcus aureus infections is a major concern. Almost 50% of hospit al-acquired Staphylococcus aureus infections are methicillin resistant [10][11].

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3 1.3. Preventative Measures for SSIs There are numerous Protocols and Practice Recommend ation documents established to prevent and reduce the incidence of SSIs [12][7][13 ]. During surgery, the main source of pathogens is thought to be the skin surface; hence, the skin preparation step before the procedure is critical. Standard surgical pre-operat ive care includes hair removal followed by “skin prepping” to achieve asepsis of the surgical area through the use of general topical antiseptics. The underlying mechanism of antimicrob ial activity varies depending on the agent used; the main agents are [14]: 1.70% isopropyl alcohol, which acts by denaturing p roteins. It is a fast-acting fungicidal and virucidal liquid and is also effective against Gram -positive and Gram-negative organisms. The only drawback to using it is its short-term act ion. 2.0.5% chlorhexidine is a quaternary ammonium compo und and acts by disrupting the bacterial cell wall. It is most effective against G ram-positive organisms and, although it is bactericidal, does not kill spore-forming organisms However, the antimicrobial activity is long-lasting (up to 6 hours). 3.70% povidone-iodine is an antibacterial agent tha t acts via oxidation/substitution of free iodine. It is effective against spore-forming, Gram -positive, and Gram-negative organisms. However, it is rapidly inactivated by organic mater ial such as blood. Additionally, patient skin sensitivity is occasionally a problem, and in some cases chlorhexidine has been shown to be more effective than iodine. The use of general antiseptics can reduce bacterial counts by approximately 80% [6]; however, some microorganisms found deep in hair fol licles can survive the treatment. Because of this, and because surgeries last for sev eral hours, once asepsis of the surgical area

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4 is achieved an extra protective barrier is put in p lace. Surgical incision drapes (SIDs) are used as a barrier to prevent re-colonization and immobil ize the organisms that might have survived the treatment. Current Practices: The Role of Surgical Incision Dr apes in Surgery The use of SIDs has been a common practice in surge ries for approximately 50 years. There are several types of commercialized antimicrobial d rapes that are impregnated with different antimicrobial agents. Most common SIDs include: 1.IOBAN™, which was commercialized by the company 3 M and is an iodophor-impregnated drape; 2.Tiburon (ISO-BAC), developed by Cardinal Health, which is a microfiber drape that consists of three layers: an absorbent fluid-contro l layer, an impermeable membrane, and a patient-comfort layer (this material has antimicrob ial properties as a result of the presence of ISO-BAC, a 3-trimethoxysilylpropylocta-decyldimeth yl ammonium chloride (silanequat) compound); and 3.InteguSeal, which is a cyanoacrylate semi-adhesi ve commercialized by Kimberly Clark; the specifics of this product will be discussed lat er in this review. Although many measures are taken to reduce the risk of SSIs, statistics indicate that current practices need to be improved. The orthopedic SSI l iterature pertaining to iodophorimpregnated drapes suggested that their use achieve s a reduction in wound contamination but without a concurrent decrease in wound infection [1 5]. Similarly, in seven trials Webster et al. found no evidence that plastic adhesive drapes reduced SSI rates, and even encountered some evidence showing that they increased infection rates [16].

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5 Current SIDs pose numerous drawbacks. Some patients show an allergic reaction to iodine, which makes IOBAN™ drapes an unviable option. Some SIDs contain leachable antimicrobial agents, and as a result lose their an timicrobial activity after a short period of time. With regard to clinical applications, medical professionals complain that the placement process of the drapes is time consuming and that th ey often do not remain properly attached to the skin surface, leading to increased infection risk. At the same time, occlusion of the skin by SIDs generates a moist and warm skin surface tha t encourages microbial growth as well as loss of drape adhesion [17]. Similarly, the formati on of air pockets and wrinkles creates locations where microbes can proliferate. Another c oncern involves drape removal. This step can cause skin tearing close to the surgical site, increasing the risks of an infection. Even if there are no apparent skin tears, a layer of epider mal cells can be detached, exposing bacteria found beneath the skin, thereby increasing risk of secondary bacterial infection (see Figure 1).

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6 Figure 1. A: Epidermal cell detachment during the r emoval of a SID, possibly leading to secondary infection by bacteria found beneath the s kin. B: Surgical incision drape on the skin before surgery. C: Surgical incision drape during s urgery. Other disadvantages include the fact that SIDs are designed to fit specific sizes and shapes and sometimes do not adapt well to the patientÂ’s shape, thereby i ncreasing the risk of drape lift during surgery. Additionally, the current SIDs are designed for sin gle use, increasing clinical waste. In one study it was reported that 5 3% of medical waste is comprised of singleuse disposable items, and the biggest source of the waste is the operation room, which generates up to 30% of the waste [12]. Lastly, the use of SIDs is recommended to prevent postoperative infections, but they are only in plac e during the surgery and, hence, do not protect the patient after removal. 1.4. Objective of This Study The main objective of this study was to develop an antimicrobial reverse-thermal gel that could be used to replace current SIDs. To achieve t his goal, a polymer consisting of a

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7 polyurea-polyurethane backbone functionalized with poly(N-isopropylacrylamide) (PNIPAAm) and quaternized amine groups was initiall y developed. However, the biocidal capacity of the polymer was limited, so an alternat ive polymer was proposed. The final copolymer was synthesized by grafting PNIPAAm-COOH to a poly(ethyleneimine) backbone through carbodiimide-linking chemistry. Th e antimicrobial properties were obtained by quaternizing the primary amine groups o n the polymer side chains. Quaternary amine groups possess positive charges and are able to disrupt the bacterial cell wall/membrane though electrostatic interactions. Aims of this study The aims of the study can be summarized as follows: Specific aim 1: design a reverse-thermal gel the quaternize to obt ain antimicrobial properties Specific aim 2: characterize the antimicrobial activity of the pol ymer, and Specific aim 3: test the cytotoxicity of Q-PSHU-PNIPAAm and Q-PEIPNIPAAm. Thesis Layout Chapter 1 contains an introduction to the scope of this stud y and the main objectives of this thesis. Chapter 2 is used to present an overview of antimicrobial po lymers used for medical applications and their mechanisms of action. Chapter 3 comprises an introduction to the polymer that was d eveloped at the beginning of this study and the alternatives that were found to overcome the limitations encountered. Chapter 4 covers materials and experimental methods used in this study Chapter 5 contains the results and discussion. Chapter 6 is used to present conclusions and recommendations for future research.

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8 2. Literature Review The field of antimicrobial compounds is vast and ha s grown during the past decade as a result of increasing concerns about hospital-acquired infe ctions and the emergence of drug-resistant bacteria. The need for materials that prevent bacte ria colonization exists in many industries, such as water treatment, food and packaging, and th e medical industry, to name a few. Specific to the healthcare industry, SSIs are a cur rent issue. Several preventative measures have been proposed and followed to reduce the incid ence of these infections. Traditionally, antiseptic preparations along with surgical drapes have been used to prevent SSIs. However, there is room for improvement in these products. This review will first introduce several antimicrob ial mechanisms of action as a context to explain the reasons behind choosing the mechanism t argeted in this study. Then, different approaches that can be followed to develop an antim icrobial polymer will be discussed, along with the factors affecting the antimicrobial activi ty of the polymer developed in this work. Different modifications, such as reversible-thermal gelling properties, will be discussed, and an overview of how these can improve the polymer ap plication process will be presented. Finally, the polymer proposed will be introduced an d described. 2.1. Antimicrobial Mechanisms of Action Several antimicrobial mechanisms have been explored in the design of bacteriostats (see Figure 2). Generally, the underlying antimicrobial mechanism targets biochemical pathways that cause disruption in protein synthesis, cell me mbrane synthesis, or nucleic acid synthesis; or inhibit cell membrane function.

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9 Figure 2. Mode of action of various antimicrobial compounds. Most conventional antibiotics penetrate the cell wi thout damaging the bacteria cell wall/membrane and target biochemical pathways withi n the cell. Pathogens sensitive to antibiotics may become resistant by acquiring genes from resistant microorganisms in the same niche. Microorganisms develop resistance by: ( a) producing enzymes that inactivate the antimicrobial agent, (b) developing efflux pumps th at remove an antimicrobial agent from the cell, and (c) up regulating/down regulating/alterin g genes encoding an outer membrane protein channel [18]. Hence, the use of antimicrobi als has led to the emergence of resistant

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10 superbugs and mutant antimicrobial-resistant pathog en species. In response, scientists have focused on developing antibacterial treatments that work through mechanisms to which bacteria are less likely to develop resistance. Man y researchers have used positively charged moieties to disrupt the cell membrane/wall structur e, and because alteration of this configuration is less likely, this mechanism ultima tely reduces the risk associated with antibacterial resistance. At the same time, biocidal agents used in biomedica l applications need to be selective in terms of toxicity; they should have no/low mammalia n cell biotoxicity. Fortunately, there are several differences between eukaryotic and prokaryo tic cells. These differences can be used as targets to achieve selective cell death. The cel l membrane/wall is the first layer of protection for these organisms; therefore, cell dea th can be attained by disruption of this structure. The outer leaflet of mammalian cell memb ranes is composed mainly of phosphatidylcholine (PC), sphingomyelin, and choles terol, all of which have no net charge. Conversely, bacteria outer cell structures consist of anionic lipids, such as phosphatidylglycerol (PG), lipopolysaccharides, and cardiolipin [19]. In short, bacteria cell walls/membranes are known to be more negatively cha rged than mammalian cell membranes. This difference has allowed the use of cationic pol ymers as antimicrobials. These positively charged polymers preferentially bind and interact w ith the outer structure of bacteria as a result of electrostatic attraction, resulting in th e selective targeting of bacterial cells over human cells [20]. It is also important to mention t hat bacteria can be broadly classified into Gram-positive and Gram-negative. Gram-positive bact eria (e.g., S. aureus ) are characterized by a cell wall composed of lipoteichoic acid molecu les, whereas Gram-negative bacteria (e.g., E. coli ) have an outer membrane structure in the cell wall that acts as an additional

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11 barrier to foreign molecules. Although the structur es of Gram-positive and Gram-negative outer wall/membranes differs significantly, both ex hibit the same negative charge on their outer wall/membranes (see Figure 3). Figure 3. Outer membrane/cell wall differences betw een Gram-positive and Gram-negative bacteria [21] [22]. This common outer wall/membrane structural characte ristic among bacteria has led researchers to use electrostatic forces as a means to target bacterial cell membranes, thereby applying one strategy to induce cell death of both types of bacteria. All cationic antimicrobial molecules, regardless of their structure (detergent s, peptide, natural, or synthetic polymers), achieve bacteria cell death through the same mechan ism. 2.2. Antimicrobial Polymers In biomedical applications, long-lasting antimicrob ial activity, ease of synthesis, and biocompatibility are generally requirements of anti microbial materials. Accordingly, polymers have been used as matrices to hold and con trol the release of antimicrobial agents. However, sometimes release is not desired, and the development of polymers that possess

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12 antimicrobial activity themselves has become an imp ortant area of research. When compared to small amphiphilic molecules, antimicrobial polym ers generally show enhanced and longerlasting microbicidal activity as well as lower mamm alian cell biotoxicity because of their non-leaching characteristics [23]. The polymer synthesis approach chosen depends on th e application and the mechanism of action desired. In their review, Kenawy et al. disc ussed different approaches researchers have taken to develop cationic polymers with biocidal pr operties [24]. The main approaches include: (a) adding an inorganic or organic biocide during the synthesis, (b) modifying the polymer after it is synthesized, (c) polymerizing a monomer that contains antimicrobial moieties, and (d) grafting an antimicrobial agent t o a synthetic or naturally occurring polymer [25]. Several non-leaching polymers that possess an timicrobial properties because of the presence of positive charges on the polymer structu re have been reported in the literature [26]. These polymers cause cell death by disrupting the bacteria cell wall/membrane, consequently leading to leakage of the intracellula r components. The primary polymers in this group are: 1. Polymers with quaternary nitrogen atoms (quatern ized groups can be found on side chain, polymer backbone, or end groups); 2. Guanidine-containing polymers; 3. Antimicrobial peptides (AMPs), such as defensins that are positively charged, possess both hydrophilic and hydrophobic side groups that a llow the molecule to be soluble in aqueous environments, can penetrate through the cel l membrane, and have biocidal activity through several mechanisms (however, AMPs are susce ptible to proteolysis, their pharmacokinetics are not fully understood, and thei r production involves high manufacturing

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13 costs. Synthetic antimicrobial peptides have been d eveloped to overcome the limitations, and these mainly act by disrupting the outer cell struc ture); 4. Halogen polymers, including fluorine or chlorine -containing polymers; 5. Polymers containing phospho and sulfo derivative s; and 6. Organometallic polymers containing metals in the ir pendant groups or backbone [27]. To date, scientists have been focused on researchin g polycationic systems because these are synthetically exible and the development process i s more straightforward [27]. Most of the antimicrobial polymers available are cationic hydro philic-hydrophobic macromolecular systems that exhibit surface-activity properties [1 9]. Two main types of polymers [24], block polymers that consist of a hydrophilic block that h olds a cationic charge and a hydrocarbon non-polar hydrophobic block, as well as random copo lymers that contain a hydrophilic monomer attached to functional group and a hydropho bic co-monomer, have been reported in the literature. These polymer structures contain ad sorption/absorption abilities along with a high binding affinity for bacterial cells. The cell membrane disruption mechanism is still not fully understood; however, studies have shown that the process is first driven by electrostatic force interactions between the cationic charges of the antimicrobial agent and the negative sites of the lipid bilayer membrane. This is follow ed by Van der Waals interactions between the hydrophobic moieties of the antimicrobial agent and the phospholipids, leading to disruption of the bacterial cell membrane, thereby causing leakage of cytoplasmic contents and cell lysis [28]. Cationic polymers: Quaternized amine groups Polymers with quaternized amine groups are probably the most studied and used type of cationic polymeric biocides [27]. Quaternary ammoni um compounds (QUATs) are organic

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14 molecules that have been widely used in healthcare, domestic, personal care products, agricultural, and industrial applications as surfac tants, emulsifiers, disinfectants, and pesticides. QUATs can be obtained through a process called quat ernization. This chemistry involves the reaction of an amine group with alkyl halides, such as methyl iodide. This reaction modifies the molecule, leading to a higher degree of alkylat ion. Some studies report that as a result of steric effects, tertiary amines react much more slo wly than secondary or primary amines. Primary and secondary amines usually result in a mi xture of amines and ammonium salts, whereas tertiary amines undergo a one-step reaction [29]. This structure contains a central nitrogen atom (R4N+) covalently attached to four functional groups (R) The functional groups generally consist of one (or more) long alky l chains, and the rest are methyl or benzyl groups [30]. Figure 4. Quaternization of tertiary amines with al kyl halides and molecular structure of a QUAT (central positive nitrogen, X represents a cou nter ion, such as NO3-, Cl-, or Br-, and R represents a functional group) [30]. QUATs have several advantages over other antibacter ial agents. They exhibit significant cell membrane penetration while generating low toxicity and lack of skin irritation. At the same time, they are very good in terms of environmental stability and have proven to have extended biological activity [28][19] [31].

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15 Effect of Structural Parameters on Antibacterial Pr operties Different polymer structural parameters affect the overall antimicrobial activity of the cationic macromolecule. Antimicrobial efficacy is l argely dependent upon the specific polymer system used. However, it has been shown tha t for cationic polymers structural parameters, such as alkyl chain length, charge dens ity, molecular weight, hydrophilichydrophobic balance, and counterions play a role in antimicrobial capacity [24]. Effect of molecular weight The influence of molecular weight on the overall an timicrobial activity of a polymer depends on the particular polymer system used. Cationic pol ymers consist of a greater hydrophobic mass and greater net charge per molecule when compa red to their monomer counterparts. The greater net charge is suspected to increase the int eraction with the bacterial cell membrane/wall, while the higher hydrophobic mass in duces better membrane penetration, thereby improving antimicrobial performance [19]. Cooper et al. conducted structure-activity studies of quaternary ammonium functionalized poly(propylene imine) and found that a molecular we ight within the range of 5104 to 1.2105 Da is best for antimicrobial activity against Gram -negative (e.g., E. coli ) and Grampositive (e.g., S. aureus ) bacteria [32]. Klibanov et al. studied the mechan ism of bactericidal and fungicidal activities of textiles covalently mo dified with alkylated polyethylenimine. Their work focused on S. aureus and the antimicrobial properties of N-alkylated polyethyleneimine (PEI) polymer systems immobilized on amino-glass slides [33]. They found that the polymers they tested of molecular we ights 25 kDa and 750 kDa had excellent antibacterial activity, while the two tested with l ower molecular weights (0.8k Da and 2kDa) did not exhibit antibacterial properties.

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16 Effect of alkyl chain length The antimicrobial mechanism of action of QUATs requ ires the cationic sites of the polymer to be adsorbed into the anionic sites of the cell w all by electrostatic interactions and then the diffusion of alkyl chains through the cell wall. He nce, the alkyl chains act as a surfactant by disrupting the cytoplasmic membrane and causing the release of electrolytes and bacterial cell materials, leading to cell death [34]. Due to this, the length of the hydrophobic chains correlates with the antimicrobial activity. Klibano b et al. found that alkyl chain lengths between C6 and C12 have performed best against both Gram-negative and Gram-positive bacteria [34]. Xu et al. studied the effects of quaternary ammoniu m chain length on antibacterial bonding agents and their results show ed increasing antibacterial activity with chain length increasing from 3C to 16C. However, th ey observed that when the chain length was increased to 18C, the antibacterial efficacy de creased. They suggested that if the alkyl chain is too long it may aggregate, thereby alterin g the electrostatic interactions with bacteria cell/membrane and diminishing the antibacterial pot ency. Hence, very long alkyl chains can have an unfavorable effect on the overall antimicro bial properties of the polymer [35]. Further studies are required to understand the exac t mechanism that leads to the reduction of antimicrobial activity when longer carbon chains ar e used. Cooper et al. studied the antimicrobial activity of quaternized poly (propyle ne imine) dendrimer systems and reported that a hydrophobic alkyl chain of C10 exhibited the best performance in terms of antimicrobial activity. They also mentioned that th e antimicrobial properties were highly influenced by the length of alkyl chains and that t he results followed a parabolic trend [32]. The alkyl chain length also has an impact on the ov erall hydrophilic/hydrophobic balance and charge density of the polymer, which impacts other properties of the material, such as the

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17 solubility. Therefore, the polymer must meet other requirements depending on the application. In efforts to design a water-soluble polymer, Nonak a et al. synthesized a thermosensitive polymer containing phosphonium groups. They develop ed the polymer by copolymerization on N-isopropylacrylamide (NIPAAm) with methacryloyl oxyethyl trialkyl phosphium chlorides (METRs), and they studied the properties of the polymer with varying alkyl chain lengths. They found that the thermosensitivity of t he copolymer was highly affected by the addition of salts and by the hydophobicity of the a lkyl groups [36]. They also noted that the relative viscosity of the copolymers increased with increasing phosphium content; they stated that this change occurred as a result of the expans ion of polymer chains in water because of the repulsion among cationic charges introduced by the presence of phosphonium groups [36]. In conclusion, the quaternization process wil l affect the overall behavior of the polymer, and this must be considered when designing a materi al that needs to fulfill several requirements. 2.3. Reversible-gelling Properties Placement of surgical incision drapes Aside from developing a polymer with antimicrobial properties to replace current SIDs, it was also the aim of this study to develop a system that simplifies the application process. As mentioned previously, SIDs placements are complicat ed and issues can be encountered during the placement. The skin needs to be disinfec ted and the drape then needs to be placed very carefully to avoid contamination of the area a nd the formation of “air bubbles,” as seen in Figure 5.

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18 Figure 5. Placement of SID [37]. As an alternative to current SIDs scientists at Kim berly Clark developed a cyanoacrylatebased polymer called Interguseal Microbial Sealant This product can be applied to the skin as a liquid during the preoperative skin preparatio n step, and moisture on the epidermis triggers polymerization of the n-butyl cyanoacrylat e [38]. During this process, the compound bonds to the skin to form a barrier intended to imm obilize the bacteria that still remain on the skin [39]. Unfortunately, Vierhout Bastiaan et al. compared the use of a cyanoacrylate skin sealant to conventional procedures and were unable to confirm a reduction in the incidence of SSIs [40]. Dohmen et al. also tested the incidence of SSIs and found no statistical difference between the control group and the group treated wit h cyanoacrylate adhesives [41]. Aside from the contradictory literature regarding clinica l tests, one of the advantages cyanoacrylate adhesives possess is the rapid setting on the skin surface (5-60 seconds) and the capacity for forming a waterproof barrier. However, among the ma jor disadvantages discussed in the literature were polymerization shrinkage, brittlene ss, and erratic polymerization set times.

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19 Additionally, patients experienced unpleasant shred ding of a thick polymer days after the surgery. A reverse-thermal gel: Poly(N-isopropylacrylamide) A possible alternative to in situ polymerization of cyanoacrylates is the use of “sm art” polymers. These are materials that exhibit reversib le, sharp property changes in response to environmental cues such as electric field, pH, temp erature, or light. Temperature can be a stimulus that is relatively si mple to control; because of this, it has been central in the development of smart polymers with t hermally reversible properties. For a polymer to possess reversible thermal gelling prope rties, at least one component of the system needs to have temperature-dependent solubili ty in a solvent (generally water), and the constituents must be insoluble above or below a cer tain temperature, called the lower critical solution temperature (LCST) [42]. There are many th ermo-responsive polymers used for biomedical applications. However, because of its ac ute phase transition near human body temperature one of the most prominent and well-stud ied examples is Poly(Nisopropylacrylamide)(PNIPAAm). The reverse-thermal gelling properties of PNIPAAm are a direct result of the chemical groups that form this polymer. The structure is shown in Figure 4.

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20 Figure 4. PNIPAAm structure and behavior above and below the LCST. In a solvent such as water at low temperature, the hydration of the polymer is dominant as a result of hydrogen bonding. However, as the tempera ture increases, hydrophobicity begins to dominate because of the presence of the methyl grou ps and the hydrophobic backbone, which contributes to gelling of the polymer. Hence, the p olymer solution undergoes a phase transition as the temperature is increased. The cha nges go from random coil form (soluble state) to a collapsed or globule form (insoluble st ate) [43]. Depending on the requirements of the application, the LCST of thermo-responsive poly mers can also be modified by adjusting the ratio of hydrophilic and hydrophobic segments o f the polymer [44]. Modification of LCST polymers (e.g., PNIPAAm) with more hydrophilic monomers favors hydrogen bonding over hydrophobic interactions and increases the LCST of the copolymer [44][45]. Although the biomedical applications of PNIPAAm hav e been studied extensively, especially concerning drug delivery, not much work has been do ne regarding epidermal applications. Kubota et al. designed a gauze coated with PNIPAAm as an alternative to current gauzes, with the aim of reducing the risk of skin abrasions encountered when a gauze is peeled from a

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21 wound. They showed that the new gauze held and peel ing of the polymer could easily be done after several days without facing the risks of epidermal cell layer detachment [46]. Dincer et al. [47] studied N -isopropylacrylamide-ethyleneimine block copolymers as possible gene delivery vectors. They reported that an increa se in molecular weight of PNIPAAm increased the overall temperature sensitivity of th e polymer as a result of the dominating hydrophobic interactions between the PNIPAAm chains at higher temperatures. They also showed that copolymerization of PNIPAAm chains with more hydrophilic polyethyleneimine (PEI) chains caused significant changes in the LCST behavior. However, this change was smaller when higher molecular weight PNIPAAm was us ed because the PNIPAAm chains dominated LCST behavior. They found that PEI-PNIPAA m copolymer solubility was higher in the aqueous medium when a lower molecular weight PNIPAAm was used. This was expected because the hydrophilicity of the copolyme r chain increases with the addition of more hydrophilic PEI chains. They also showed that the relative size of the blocks of PNIPAAm and PEI on the copolymer chains were import ant factors in the hydrophobocity of the copolymer. Finally, their results showed that t he copolymerization of PNIPAAm with more hydrophilic PEI chains caused an increase in t he LCST of the PNIPAAm chains from 31C to around body temperature (36-39C) [47].

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22 3. A reverse-thermal gel with antimicrobial activit y Ideally, an antimicrobial polymer designed to repla ce current SIDs should fulfill the following requirements: 1) non-toxic or irritating; 2) biocompatible; 3) biocidal to a broad spectrum of pathogenic microorganisms; 4) antimicro bial activity should be long lasting; 5) inexpensively synthesized; 6) increased ease of app lication when compared to current SIDs; and 7) ease of removal. Poly(Serinol Hexamethylene Urea) (PSHU) This study began with the characterization of a pol ymer consisting of a poly (urethane urea) backbone functionalized with quaternized amine grou ps and PNIPAAm. The initial polymer structure proposed can be obser ved in Figure 5Figure 1. The polymer backbone structure resembled a peptide bond due to the presence of urea which is an organic compound that consists of two amine (NH2) groups joined by a carbonyl (C=O) functional group. The side chains of Poly (Serinol Hexamethyle ne Urea) initially contained amine groups protected by Boc-groups that after chemical modifications were removed to reveal the primary amines. Then, PSHU-NH2 was functionalized with a) PNIPAAm to achieve reve rsethermal gelling properties and improve the ease of application and removal b) with alkylation agents to obtain quaternized amine groups and, henc e, antimicrobial properties.

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23 Figure 5. Polymer backbone with quaternized amine g roups As will be shown in the results section, the outcom es of the antimicrobial tests using QPSHU-PNIPAAm were not as expected. After analyzing and modifying the polymer synthesis and quaternization protocol, the results regarding the antibacterial tests were not positive or consistent. The negative results were a ttributed to the small number of primary amine groups per repeating unit available to quater nize. Due to this, a new polymer that possessed a higher number of primary amine groups w as proposed. 3.1. New Approach: Polyethyleneimine Polyethyleneimine (PEI) is a nontoxic, aliphatic, w eakly basic, synthetic polymer. It owes its polycationic nature to the presence of primary, sec ondary, and tertiary amine groups as can be observed in Figure 6. The protonated amine group s of PEI are cationic, and the ethylene backbone provides hydrophobic groups. Hence, the p olymer consists of a repeating cationic, amphiphilic structure at a neutral pH without any f urther chemical modification by hydrophobic groups [48].

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24 Figure 6. Structure of branched Polyethyleneimine PEI has been used extensively in antimicrobial appl ications. Specific to antibacterial coatings, Lewis et al. discuss in their study the p ossible limitations encountered when attaching an antimicrobial molecule to a surface. T hey explain how the loss of mobility can make the agent unable to penetrate the cell and loo se its antibacterial activity. Due to this, they explain that quaternary amine groups are most interesting for coating applications because they are drawn to the bacterial cell due to the membrane potential [49][50]. Lin et a. conducted systematic chemical modifications of immo bilized PEI and studied their impact on the overall properties of the polymer. They conduct ed tests on N -alkylated PEIs of different molecular weights that were covalently attached to amino-glass slides [34]. They observed that quaternized amine groups are can be obtained b y alkylation of the PEI coating. They also showed that alkylation leads to higher bactericidal activity at carbon chain lengths of 6 and decreases beyond this point. Park et al. researched a method of physical deposi tion of hydrophobic polycations to obtain an antimicrobial coating. They developed a one-ste p painting-like procedure to obtain an antimicrobial surface [51]. They prepared several N -hexyl, N -methyl-PEI coated slides and determined their bactericidal efficiency against S. aureus and E. coli The results showed that these surface-deposited polymers alike many covalen tly immobilized polymers achieve bacteria cell death on contact and not by leaching from the surface. At the same time, they

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25 also discuss that the antimicrobial mechanism invo lves rupturing the bacteria cell membrane [51]. Regarding antimicrobial resistance, Klibanov et al. conducted studies using N-hexyl,methylpolyethylenimine, and their results showed that th e bacteria failed to develop noticeable resistance to this lethal action over the course of many successive generations [26]. Because of the inherent antibacterial properties of this polymer and the inability of bacteria to develop resistance to PEI, this polymer was used in this study to replace PSHU. 3.2. New Polymer In this context, this study proposes, Q-PEI-PNIPAAm an antimicrobial reverse-thermal gel that is designed to provide a non-leaching antimicr obial polymer with 99.9% killing efficiency. When in a solution, the polymer can be sprayed on the skin and will form an antimicrobial gel layer. The polymer can act as a u niform antimicrobial surface during the entire surgery. In addition, it can be removed usin g cold water (or ethanol 70%) after surgery, thereby significantly reducing risk of skin irritat ion, blistering, and medical waste. The presurgical process is streamlined because the polymer system is easy to apply and offers a universally relevant solution for all patients, the refore, bypassing the need to order currently used SIDs based on the patientÂ’s size and shape.

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26 4. Materials and Methods 4.1. Materials Polyethyleneimine (PEI branched 10 000 g/mol and 18 00 g/mol), N-(3dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC), and N hydroxysuccinimide (NHS) were obtained from Alfa Ae sar (Ward Hill, MA). N isopropylacrylamide (NIPAAm) was obtained from Acro s Organics (Geel, Belgium). Hexamethylene diisocyante (HDI), 4,4 -azobis(4-cyanovaleric acid) (ACA), methanol, trifluoroacetic acid (TFA), serinol, and urea were obtained from Sigma Aldrich (St. Louis, MO). Sodium hydroxide (NaOH), 2-propanol, hexane, a nd diethyl ether were obtained from Fisher Scientific (Pittsburgh, PA). Dimethylformami de (DMF) was obtained from BDH Chemicals (Poole, UK). 4.2. Polymer Synthesis PSHU Synthesis This method was used to synthesize a polymer consis ting of a poly (urethane urea) backbone with side chains that contain amine groups that pos sess protective Boc-groups. Once the polymer synthesis was complete, a latter ‘deprotect ion’ step was followed to expose the primary amines. The first step was to synthesize N-Boc serinol; 5.9 73 ml of N-Tert Butyl was dissolved in a vial containing 25 ml of ethanol. Then, this soluti on was placed at 4C and added drop-wise while mixing to 1.959 g of serinol that had previou sly been dissolved in 20 ml of dry ethanol. Then, the solution was stirred vigorously for an ho ur at 37C, and the solvent was removed using a rotary evaporator with the water bath tempe rature set at 50C. The white powder

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27 obtained was dissolved in 25 ml of 1:1 ethyl acetat e/hexane and mixed until a clear solution was obtained. Subsequently, hexane was added drop-w ise until the solution appeared to crystallize. Then, the solution was left overnight for the crystals to settle to the bottom, and then a vacuum filter was used to separate the N-Boc serinol. After N-BOC serinol was obtained, 6.0 mmol were dis solved along with 6.0 mmol of urea in 6 ml of anhydrous DMF under a nitrogen atmosphere. Once both chemicals were dissolved, 12 mmol of HDI were added, and the reaction was con tinued for a period of 7 days at a temperature of 90C. Subsequently, it was removed f rom the hot plate and left until it reached room temperature before precipitating it in diethyl ether four times and once in water. After the required precipitations, the polymer was lyophi lized and a yellowish powder was obtained. PSHU Deprotection The protocol followed was designed to achieve a 100 % deprotection of amine groups because 15% were meant to be conjugated to PNIPAAm and 85% were available for quaternization. PSHU (1 g) was dissolved in 15 ml of methylene chlo ride in a flask. Then, 15 ml of TFA were added drop-wise, and the solution was left to mix on a stir plate for 45 minutes. The solvents and acid were removed using a rotary evapo rator and a water bath at a temperature of 45C. Once all the acid was removed, 1 ml of anh ydrous DMF was added to return the polymer to solution. The solution was then precipit ated in diethyl ether and then the solvent removed using a rotary evaporator. Then, the deprot ected PSHU was dissolved in TFE and again precipitated in diethyl ether. This step was repeated twice, and a rotor evaporator was used each time to remove the solvent. The final ste p allowed obtaining a dry white powder.

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28 PNIPAAm Synthesis [52] NIPAAm (5.0 g) and ACA (0.060 g) were dissolved in 25 ml of anhydrous methanol and exposed to nitrogen bubbling for 30 minutes at room temperature. Then, a reflux system was set, and the temperature was raised to 68C and lef t for three hours. Subsequently, the solution was precipitated in warm (60C) water and then left to dissolve in 5 ml of dH20 water at 4C. The solution obtained was then added to a 3500 kDa MWCO dialysis tube that was placed in a beaker with dH2O water for 48 hours. Finally, once the polymer was purified, it was lyophilized for a period of 48 hours. Molecular weight of PNIPAAm-COOH was confirmed by t itrating for the carboxylic acid end groups as in previous studies [53]. PNIPAAm (0. 5% w/v) was dissolved in 10 mL dH2O. Then 10 L of phenolphthalein solution (2 wt% in ab solute ethanol) was added. The solution was titrated to the end point by adding 0.01 N NaOH Conjugation of PNIPAAm to PEI PNIPAAm-PEI conjugations were performed at 20, 30, and 50% using Ethyl (dimethylaminopropyl) carbodiimide and N-Hydroxysuc cinimide chemistry. Calculations were performed to determine the number of primary f ree amines on the PEI structure to be conjugated. PNIPAAm was first dissolved in 10 ml of DMF, and then, EDC-NHS (1.3 M excess) was added. At the same time, in a separate flask, PEI was dissolved in 5 ml of DMF, and both reactions were left overnight. Then, the P EI solution was added drop-wise onto the PNIPAAm-EDC-NHS and left to conjugate for a period of 24 hours. Subsequently, a rotary evaporator was used to remove the DMF, and precipit ations in ditheyl ether were used to remove unreacted polymer. Then, the polymer was dis solved in dH2O and added to a 12-14

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29 kDa MWCO dialysis tube that was placed in a beaker with dH2O water for 48 hours. Finally, once the polymer was purified, it was lyophilized f or a period of 48 hours. The same methodology was applied to conjugate PNIPA Am to deprotected PSHU. Quaternization of Primary Amines Calculations were performed to determine the number of primary free amines on the PEIPNIPAAm structure to be conjugated. PEI-PNIPAAm was first dissolved in 10 ml of DMF, and sodium bicarbonate (1.3 M excess) was added. Th e reaction was set to a temperature of 95C with reflux, and while undergoing vigorous sti rring, 1-Bromohexane (20 M excess) was added and left for 48 hours. Then, the temperature was lowered to 68C, Iodomethane (20 M excess) was added, and the reaction continued for a nother 12 hours. Subsequently, the flask was removed from the hot plate and left to cool bef ore using a rotor evaporator to remove solvents. Three precipitations in diethyl ether wer e used to remove unreacted polymer. Then, the polymer was dissolved in 10 ml of dH2O, and 7,ml were added to a 12-14 kDa MWCO dialysis tube that was placed in a beaker with dH2O water for 48 hours. The remaining 3 ml of polymer solution were added to a separate 12-14 kDa MWCO dialysis tube that was placed inside a 50-ml tube with 25 ml of dH2O. The tube was then lyophilized and weighed to calculate the PEI-PNIPAAm conjugation amount. Finally, once the polymer was purified, it was lyo philized for a period of 48 hours. 4.3. Polymer Characterization Proton Nuclear Magnetic Resonance (1H NMR) Polymer samples (3-5 mg) were dissolved in DMSO-d6 (60 l), and then, CDCl3 (540 l) was added. The 1H NMR spectra were collected on an INOVA 500 MHz in strument

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30 (Varian). Results and spectra were analyzed using A CD 1D NMR Processor software (Advanced Chemistry Development, Inc., Toronto, ON) Zeta Potential Measurements Polymer samples (10 mg) were dissolved in 1ml of dH2O. The -potentials of the polymers were measured using a Malvern Zeta Sizer 2000 (Malv ern Instruments, USA). -potentials were measured at least nine times, and the averages with standard deviation were calculated. Gelling Properties – LCST Determination The LCST was determined to characterize the gelling properties of the polymer. The polymer samples were dissolved in dH2O at 1 wt%. Then, transmittance values were measure d at 500 nm, starting from temperature of 25C and increasin g 0.5C/min up to 50C. The results were obtained using a Cary 100 UV-visible spectrophotome r (Agilent Technologies, Inc., Santa Clara, CA). 4.4. Antimicrobial Tests Antimicrobial Tests Using Q-PSHU-PNIPAAm The antibacterial property of the Q-PSHU-PNIPAAm wa s evaluated against Staphylococcus aureus. All bacteria were cultured in Lysogeny broth (LB). The experiment consisted of forming gel surfaces with polymer solutions (200 l) with different concentrations (starting from 2% to 10% (wt/v) with 2% increments). These so lutions were added to a 24-well plate and placed in a 37C incubator to promote the solut ion-to-physical gel transition. Subsequently, 500 l of bacterial suspension at a concentration of 11 08 colony-forming units (CFU)/ml were added on the gel and cultured a t 37C for 24 hours. For better results, each gel-bacteria sample was triplicated, and stand ard deviation values were calculated. For controls, 500 l of bacterial suspension of equal concentrations w ere to be cultured on a plain

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31 24-well plate. After the culture period, 100 l of the suspension were taken and diluted with 900 l of DI water. The antibacterial activity was deter mined by measuring optical density (OD) of a bacterial suspension at 600 nm using a mi croplate reader (SYNERGY Mx, BioTek). The percentage increase in the optical den sity was calculated by comparing optical densities of bacterial suspensions before and after the incubation. Then, the numbers were normalized to the control group. Antimicrobial Tests Using Q-PEI-PNIPAAm The main objective of these tests was to quantify t he bactericidal activity of the polymer/antimicrobial using time-kill curves. This was achieved by measuring the decrease in bacterial population at different time intervals The general methodology followed consisted of dissolving the polymer in a suspension of known bacterial concentration. Then, the bactericidal activity of the sample was tested at different time intervals by extracting aliquots in 30-minute time intervals. The aliquots were then diluted and placed on agar plates, where the bacteria were cultured during a period of 24 hours before colonies were counted. The antibacterial property of Q-PEI-PNIPAAm was eva luated against 4 clinical, relevant species of bacteria: Staphylococcus aureus (ATCC 6538), Staphylococcus a ureus subsp. aureus Mu3 (MRSA strain with heterogeneous vancomyc in-intermediate resistance) (ATCC 700698), Staphylococcus epidermidis (Ron Gill Colle ction), and E. Coli (ATCC 15597). Bacterial Stock Preparation Bacterial streaking was used to identify and isolat e bacterial colonies. One colony was swabbed from an agar plate and added to 5 ml of Lys ogeny Broth. The suspension was left for 16 hours in a shaker incubator at 37C and at 2 50 RPM. Then, 920 microliters of bacterial

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32 suspension were removed and mixed with 80 microlite rs of filtered DMSO and flash frozen at -80C. Antibacterial Test The bacterial suspension was prepared by extracting one crystal from the flash-frozen bacterial stock and mixing it with 5 ml of LB. The new stock was left for 16 hours at 37C in a shaker incubator (250 RPM). Subsequently, the bac terial concentration was calculated using optical density measurements of absorbance at 600 nm using a microplate reader (SYNERGY Mx, BioTek). This measurement was taken to have an initial estimate of the bacterial concentration; these values were then con firmed with dilutions that were inoculated on agar plates. Dilutions were performed using PBS to obtain a stoc k of 108 cells/ml. Then, the polymer was added to obtain a suspension of 10% w/w and was pla ced at 37C in a shaker incubator (250 RPM). Aliquots (20 L) were taken at 0, 30, 60, 90, and 120 minutes, and dilutions were performed to have samples at different concentratio ns. Aliquots (40 L) were taken from these samples and plated on agar plates that were l eft at 37C during 24 hours. Then, colonies were counted, and the values were used to extrapolate the initial number of CFUs. All results were compared with the values obtained at time zero to quantify the killing power. 4.5. Functional Tests Preliminary tests using surgical incision drapes To asses if bacterial recolonization occurs during a surgical procedure while using SIDs a used drape was carefully used to inoculate an agar plate and cultured for a period of 24 hours at 37oC.

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33 Antimicrobial Activity of Polymer-Coated Glass Slip s The polymer was used to coat glass slips to assess the antimicrobial properties of the surface and the durability of the polymer after being soake d in water. The method involved coating 15 glass slides of 1cm x 1cm, which were cut and cl eaned with alcohol. Then, they were placed on a hot plate at 37C and left until a stab le temperature was obtained. Four different 10% w/v polymer solutions were prepared: PEI-PNIPAA m (30% conjugation), PEIPNIPAAm (20% conjugation), QPEI-PNIPAAm (30% conjug ation), and QPEI-PNIPAAm (20% conjugation). Glass slips were then painted wi th 100 l of each polymer solution. Triplicates were done for each case. Also, three gl ass slips were cleaned and left as controls. The bacterial suspension was prepared as previously described. After the polymers gelled, the bacterial suspension (108 cells/ml) was sprayed on the glass slides. Subsequ ently, the glass slides were placed on agar plates and left at 37C for 24 hours. As in the previous antibacterial tests, the bacterial stock was dilute d and also plated to extrapolate the initial number of CFUs. All results were compared with the values obtained at time zero to quantify the killing power. Finally, the same process was followed with a glass slide that was half coated with the polymer solution consisting of 10% w/v Q-PEI-PNIPAA m (30%) in 70% ethanol and half left uncoated. Since there is water present during a surgery, a pr eliminary test was designed to evaluate whether the polymer could remain on the surface of a glass slide after being subjected to water. Polymer solutions, 10% w/v of QPEI-PNIPAAm (30% con jugation) and QPEI-PNIPAAm (20% conjugation), were dissolved in 70% ethanol an d sprayed on a glass slide. The glass

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34 slide was then left until the alcohol evaporated an d was placed in a water bath at 37C overnight. Images were taken to register changes. 4.6. Cytotoxicity Test of Q-PEI-PNIPAAm and Q-PSHUPNIPAAm The cytotoxicity of the Q-PEI-PNIPAAm was examined by performing a MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromi de) assay using human smooth muscle cells. The MTT assay is commonly used as a measure for cell viability and provides a general indication of cell health. The MTT assay was perfor med by supplier's instructions, with absorbance (570 nm) and values collected on a micro plate reader. To assess the cytotoxicity of Q-PSHU-PNIPAAm, polym er solutions (10% (wt/v)) were obtained by dissolving the reverse-thermal gel in S MC culture medium. Smooth muscle cells were cultured for a period of a week until they wer e confluent. Then, the cells were counted, and 10,000 cells/well were plated in a 96-well plat e and left to attach. Subsequently, polymer solutions (200 l) were added to each well. As a ne gative control, cells were also cultured with plain media, and as positive control, cells we re exposed to media with 5% DMSO. All samples were triplicated for statistical significan ce. The absorbance (directly related to cellular metabolic activity) was measured after 1, 3, and 5 days. Final results were normalized to the 1-day control group. To assess the cytotoxicity of Q-PEI-PNIPAAm, a diff erent approach was taken to ensure that the polymer was in direct contact with the cells at all times. In this case, fibroblasts (Cardiovascular Pulmonary Research Cell Repository – Nana Burns and Sandy Walchak) were used since these are more sensitive and releva nt for the application. Polymer solutions (1, 3, and 5% (wt/v)) were obtained by dissolving t he reverse-thermal gel in ethanol 70%. Several controls were used, including plain media, PEI-PNIPAAm (20% conjugation),

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35 PNIPAAm, Chlorhexidine 2%, and media with 5% DMSO. Fibroblasts cells were cultured for a period of a week until they were confluent. T hen, a 96-well plate was coated with the different polymer solutions. Cells were counted, an d 7,000 cells/well were placed on top of each polymer coating. Then, they were left to cultu re at 37C for a period of 2 hours. All samples were triplicated for statistical significan ce. Subsequently, the MTT was performed by supplier's instructions, with absorbance (570 nm ) and values collected on a microplate reader. All results were normalized and expressed a s a percentage of the negative control.

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36 5. Results and Discussion The Results and Discussion section consists of thre e components: Antimicrobial and cytotoxicity test results concern ing the first polymer developed, Q-PSHU-PNIPAAm. Limitations of the tests are discus sed along with the results. Characterization, antimicrobial and cytotoxicity re sults obtained from tests using the new polymer, Q-PEI-PNIPAAm are shown. Functional tests: Short preliminary test regarding current surgical incision drapes, water resistance of the polymer coating, antibacter ial tests of the polymer coating 5.1. Preliminary Studies 5.1.1. Antimicrobial Tests Using Q-PSHU-PNIPAAm A UV-visible spectrophotometer was initially used t o obtain OD values and evaluate the antimicrobial activity of Q-PSHU-PNIPAAm. The OD method can be performed automatically in a h igh throughput manner using a microtiter plate reader [55]. Researchers[48][56][5 7] have relied on UV-visible spectrophotometers to test the antibacterial proper ties of different materials. The underlying principle is that the light scattered by the cells does not reach photoelectric cell and hence the readings of absorbance change. However, this occurs as a result of light scattering, not due to molecular absorption because cells of most bacteria are close to colorless and real light absorption is marginal[58]. Since the reading obta ined when using the “absorbance” setting on the spectrophotometer is not truly the absorbanc e, the term used to describe this value is Optical Density or O.D. The main limitation faced w hen using this technique to assess the biocidal activity of a material is that it cannot d istinguish between dead or live bacteria [55].

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37 Three antimicrobial tests were conducted using this technique. Bacteria cell cultures were adjusted to 108 –104 colony forming units CFU/ml by PBS solution. Only results from the first test were included in this section since they were representative of all three cases. The data can be observed in Figure 7. Figure 7. Results from the first antibacterial test against S. aureus using different polymer and bacterial concentrations. The results showed a reduction in the OD values whe n compared to the control. Due to this, the initial findings led to think that the polymer had antibacterial properties. However, these results were not conclusive. As can be seen on the graph, both the quaternized polymer and the non-quaternized polymer showed similar results and, as mentioned previously, OD values do not measure cell death. To clarify the results t he test was repeated and aliquots from the samples that had been subjected to OD measurements were used to inoculate agar plates for colony counting. 0 0.1 0.2 0.3 0.4 0.5 0.6 ControlNQPSHU2% QPSHUNQPSHU4% QPSHUNQPSHU6% QPSHUOD 600 10 4 cells/ml 10 5 cells/ml10 6 cells/ml10 7 cells/ml10 8 cells/ml Sample

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38 Unfortunately, the agar tests performed to confirm cell death showed bacterial growth after 24 hours at 37C at all polymer and bacterial conce ntrations. The changes in OD values were attributed to presence of the polymer within the sa mples affecting the absorbance measurements. This observation led to modify the an tibacterial test protocol. Similarly, the negative results let to conclude tha t more quaternized amine groups were required to possess significant antimicrobial activ ity. 5.1.2. Cytotoxicity Tests of Q-PSHU-PNIPAAm Although the preliminary results from antibacterial tests with Q-PSHU-PNIPAAm did not seem promising, there was interest in confirming th e biocompatibility of PNIPAAm and QPSHU before designing the next polymer. The aim was to evaluate the biocompatibility of quaternized amine groups and to confirm that PNIPAA m was non-cytotoxic. An MTT assay was conducted to assess the cytotoxicity of Q-PSHUPNIPAAm. Figure 8 shows data normalized to the day 1 media-o nly control sample. The results show that the positive control samples (10% DMSO) were t he only samples that were significantly different (p<0.0001). The samples containing Q-PSHU -PNIPAAm were statistically insignificant from the negative control sample of p ure culture media.

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39 Figure 8. Results from the first MTT assay conducte d using Q-PSHU-PNIPAAm at different concentrations. The only statistically significant difference was observed between the experimental samples and positive control (cells cu ltured with 5% DMSO). (** indicates p > 0.01, and *** indicates p < 0.01.) However, as mentioned previously, when PNIPAAm is e xposed to a temperature above the LCST, it exhibits shrinking; hence, the polymer did nÂ’t seem to be in contact with the cells during the entire period. PNIPAAm and PSHU are bioc ompatible; as has been shown in previous studies, the only moieties that could caus e cytotoxicity are the quaternized amine groups. Due to this, the MTT assay was repeated wit h Q-PSHU. 0 50 100 150 200 250 300 Day 1Day 3Day 5Absorbance, % of ControlTime (days) Positive Control Negative Control Polymer 1% Polymer 3% Polymer 5% *** ** ** *** ** ***

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40 Figure 9. Results from the second MTT assay conduct ed using Q-PSHU at different concentrations. Figure 9 shows data normalized to the day 1 media-o nly control sample. The results show that the positive control samples (5% DMSO) were th e only samples that were significantly different (p<0.01). The samples containing Q-PSHU-P NIPAAm were statistically insignificant from the negative control sample of p ure culture media. 5.1.3. Conclusion from Tests Performed with Q-PSHUPNIPAAm The antimicrobial and cytotoxicity tests conducted using Q-PSHU-PNIPAAm allowed to reach to several conclusions. First, PSHU contained one primary amine and this was assumed to be the limiting factor to the antibacterial acti vity. Moving forward, a new polymer with numerous primary amine groups available for quatern ization was thought to be required to obtain significant antimicrobial activity. Although the literature states that quaternized amine groups disrupt the bacterial cell wall causing cell death [59][60][51][34], there is no specific knowledge regarding the minimum number of primary a mine groups per repeating unit 0 50 100 150 200 250 300 350 400 Day 1Day 3Day 5Absorbance % of ControlTime (days) Positive Control Negative Control 0.12 mg/ml 0.012 mg/ml **

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41 required to have significant bacterial cell death. Similarly, for future tests a new protocol was required to test the antibacterial properties of th e polymer because the method using OD values presented several limitations. However, some positive conclusions were reached. Th e cytotoxicity tests involving QPNIPAAm, PNIPAAm and Q-PSHU showed statistically in significant from the negative control sample of pure culture media. Hence, althou gh it was concluded that more quaternized groups were required to obtain antibact erial activity, the polymer was biocompatible. Similarly, the reverse-thermal gelli ng properties were satisfactory. Due to the conclusions from the initial results and new polymer system was developed. As mentioned in the literature review, Polyethyleneimi ne (PEI) is a nontoxic, aliphatic, weakly basic, synthetic polymer. It owes its polycationic nature to the presence of primary, secondary, and tertiary amine groups and has been u sed extensively in antimicrobial applications. Branched as well as linear PEI struct ures can be obtained. However, the branched configuration contains a combination of pr imary, secondary and tertiary amines while the linear structure only contains secondary amines. Due to these structural differences branched PEI was selected to design a new polymer. 5.2. Development of a New Polymer: Q-PEI-PNIPAAm The new polymer PEI-PNIPAAm and Q-PEI-PNIPAAm was s ynthesized as described in the method section. After obtaining the new polymer dif ferent approaches were taken to characterize its structure. 5.2.1. Polymer Characterization Proton Nuclear Magnetic Resonance (1H NMR)

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42 The structure of PEI was confirmed by 1H NMR (500 MHz) in CDCl3 ( ppm): 1.52 [CH2-CH2-N H2], 2.5-2.7 [N-C H2-CH2-N]. The results agree with the literature[48] and peak assignments were corroborated by NMR modeling (Adva nced Chemistry Development). The spectra showed the presence of primary amine groups and confirmed the polymer backbone structure. Figure 10. 1H NMR (500 MHz, CDCl3) analysis of the PEI. Peak assignments were corroborated by NMR modeling (Advanced Chemistry De velopment). The spectra of PEI-PNIPAAm (30% conjugation) was al so obtained with 1H NMR spectroscopy (500 MHz, CDCl3) and is shown on Error! Reference source not found .¡Error! No se encuentra el origen de la referencia.

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43 Figure 11. 1H NMR (500 MHz, CDCl3) analysis of the PEI-PNIPAAm (30% conjugation). Peak assignments were corroborated by NMR modeling (Advanced Chemistry Development). 1H NMR (500 MHz) in CDCl3 ( ppm): 0.851.4 [N-CH2-(CH3)2], 3.6-4.2 [N-CH2-(CH3)2 and –NHof PEI]. Peak assignments were corroborate d by NMR modeling (Advanced Chemistry Development). Unfortunately, as expected, PNIPAAm dominated the s pectra due to its molecular weight and high conjugation ratio. However, some changes were observed and the spectra can be used as supporting information proving the conjugation of P EI-PNIPAAm. As mentioned previously, PNIPAAm-COOH was conjugated to PEI thro ugh carbodiimide linking

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44 chemistry leading to the formation of an amide bond The spectra for PNIPAAm (found in Appendix A) shows that the area 5.85-6.8 ppm changed from one peak corresponding t o an amide bond to two peaks in the spectra correspondin g to PEI-PNIPAAm. This change, although small, shows the presence of a new amide b ond, possibly due to the conjugation of PEI-PNIPAAm. However, since the %mol of the amide b ond of interest is small in the overall polymer, it is hard to detect. Due to the limitations encountered with PNIPAAm, to ensure that the quaternization chemistry was appropriate PEI was quaternized and t he structure was the analyzed using 1H NMR. Figure 12. 1H NMR (500 MHz, CDCl3) analysis of the Q-PEI. Peak assignments were corroborated by NMR modeling (Advanced Chemistry De velopment). 1H NMR (500 MHz) in CDCl3 ( ppm): 0.851.0 [N-CH2-(CH2)4-C H3], 1.61.9 [N-CH2(C H2)4-CH3], 3.253.4 [N-C H2-(CH2)4-CH3], 2.7-2.98 [N-C H3, alkylated PEI]. The spectra was as expected and the peaks agree with the litera ture [51]. Peak assignments were corroborated by NMR modeling (Advanced Chemistry De velopment)

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45 Zeta Potential Measurements Charges surrounding the surface of a particle in su spension can be quantified using zeta potential. The zeta potential measures the electrop horetic mobility of the particles in an electrical field [61]. Soluble polymers do not have a two-dimensional interface as solid nanoparticles do; therefore, there is no zeta poten tial in a conventional sense. However, cationic polymers do exhibit electrokinetic mobilit y due to the presence of charges. In the case of soluble polymers, there is no strict relati on between charge density and electric potential. When zeta potential is used to measure t he charges of a particle, no fluid flows through the solid particle; hence, it is a two-dime nsional surface charge density. In the case of polyelectrolytes, the fluid can flow through the co ils-branched structure of the polymer. The results of zeta potential in this case are not conc lusive but indicative of the presence of positive charges. Figure 13. Zeta potential measurements of quaterniz ed and non-quaternized polymers. Significant statistical difference was noted betwee n the values (** indicates p<0.01). 0 1 2 3 4 5 6 7 8mV PEI-PNIPAAM (20%) Q-PEI-PNIPAAM (20%)

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46 The results for PEI-PNIPAAm 30% and 50% conjugation were not included because they displayed substantial variability. Measurements wer e taken with lower molecular weight PNIPAAm (2000 g/mol), and these exhibited positive results. However, when measuring samples with PNIPAAm molecular weight greater than 9000 g/mol and conjugations above 20%, the error associated with the measurements mad e the results unreliable. The hydrophobic ends of PNIPAAm were probably aggregati ng with the long alkyl chains of QPEI-PNIPAAm; therefore, the zeta potential results exhibited substantial variations after mixing and every run. Lower Critical Solution Temperature (LCST) An aqueous solution of a thermo-responsive polymer becomes turbid at temperatures above the LCST, leading to changes in light transmittance and scattering. UV-visible spectroscopy is a common tool used to study LCSTs because it all ows users to measure the changes in amount of light that is transmitted through the sam ple. Solutions of PEI-PNIPAAm (20, 30, and 50% conjugati on) as well as the quaternized versions were subjected to LCST measurements. The r esults below show that the transmittance decreases with increasing temperature Figure 14. LCST of PEI-PNIPAAm (20% and 50% conjugation) and Q -PEI-PNIPAAm. 0 20 40 60 80 100 120 20304050% TransmittanceTemperature (C) Q-PEI-PNIPAAm (20%) PEI-PNIPAAm (20%) 0 20 40 60 80 100 120 20304050% Transmittance Temperature (C) Q-PEI-PNIPAAm (50%) PEI-PNIPAAm (50%)

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47 Figure 15. LCST of PEI-PNIPAAm and Q-PEI-PNIPAAm (b oth 30% conjugation). All transitions were thermally reversible, and the turbid copolymer solutions became transparent when they returned to temperatures belo w the LCST. As mentioned in the literature review, PNIPAAm itself exhibits an LCST at 32C[62][63]. The thermoresponsive behavior depends on the solvent interact ion with the polymer and the hydrophilic/hydrophobic balance within the polymer molecules. The introduction of hydrophilic and hydrophobic co-monomers can cause a nd increase or decrease in the LCST[64]. In this study the LCST of the polymers was defined as the temperature reducing the change in light transmittance by a half. Table 1 shows the increasing LCST value with increa sing hydrophilic PEI content for the different copolymer s. In all three cases, the polymers exhibited higher transmittance after quaternization In conclusion, the addition of PEI increased the LC ST the alkylation-quaternization process decreased the LCST. Studies [63] mention that incor poration of more hydrophobic groups will lead to a lower LCST. So, we can attribute the decrease of LCST of the quaternized polymers to the presence of the long hydrophobic al kyl chains that contribute to the overall hydrophobicity of the polymer. 0 20 40 60 80 100 20304050% TransmittanceTemperature (C) Q-PEI-PNIPAAm (30%) PEI-PNIPAAm (30%)

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48 Polymer LCST(C) Polymer LCST(C) PEI-PNIPAAm (Conj 20%) 35 Q-PEI-PNIPAAm (Conj 20%) 32.5 PEI-PNIPAAm (Conj 30%) 34.5 Q-PEI-PNIPAAm (Conj 30%) 32.6 PEI-PNIPAAm (Conj 50%) 34.1 Q-PEI-PNIPAAm (Conj 50%) 33.7 Table 1. LCST values of each polymer. 5.2.2. Antimicrobial Tests using Q-PEI-PNIPAAm In a clinical setting, non-multiplying (stationary phase) and multiplying (log phase) bacteria coexist. The mechanisms of action of several antibi otics require ongoing cell division and are less efficient at killing non-multiplying bacteria [65]. For example, beta lactams need ongoing bacterial cell wall synthesis for bacterici dal activity. Many studies have been performed on log-phase bacteria (rapidly dividing b acteria); however, bacteria seldom encounter optimal conditions that enable logarithmi c growth [65]. S. aureus has been shown to modulate gene expression, thereby survive long p eriods in the stationary phase under unfavorable growth conditions [66][67]. In vitro antibacterial studies were conducted to ob tain time-kill curves to quantify the killing capacity of the polymers developed. Due to reasons previously mentioned, most tests involved bacteria in the stationary phase. However, the killing of S. aureus during the log phase was also tested to ensure the antimicrobial a ctivity of this material under different conditions. Initially, the antimicrobial activity of PEI-PNIPAA m (20%) was tested against S. aureus The polymer (concentration 10% w/v in PBS) exhibite d more than a 8-log10 CFU/ml bacteria reduction in 30 minutes, as can be observed in Figu re 16. For sample images showing agar plates and colony co unting, please refer to the Appendix B.

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49 Figure 16. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% conju gation) were added to stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. Unfortunately, although the antimicrobial activity of the polymer was outstanding and the LCST results showed a transition, the gel obtained at 37C was unstable. Since increasing the polymer concentration in water resulted in very slo w gelling and de-gelling, the alternative chosen was to repeat the tests using a higher PNIPA Am conjugation ratio. As explained in the method, PEI-PNIPAAm (50% conjugation) and Q-PEI -PNIPAAm (50% conjugation) were synthesized. The antimicrobial tests did not e xhibit significant bacterial reductions even after 2 hours. Several factors were attributed to this, first the large content of PNIPAAm affecting the quaternization process. Also, as ment ioned in the literature review, increasing the molecular weight will substantially affect the antibacterial properties; by conjugating 30% more primary amines to PNIPAAm (total of 50% conjug ation), the molecular weight would increase substantially since the PNIPAAm chains hav e a higher molecular weight than the 0 1 2 3 4 5 6 7 8 9 0306090120Log Bacterial Count (CFUs/mL) Bacteria Sample PEI-PNIPAAm (20%) Q-PEI-PNIPAAm (20%) Time (minutes)

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50 alkyl chains. At the same time, longer chains can a lso play a role in inhibiting the positive charges of the polymer to destabilize the negative charges associated with the bacterial cellÂ’s outer structure. Due to this, the polymer was modified once again, a nd PEI-PNIPAAm (30% conjugation) and Q-PEI-PNIPAAm (30% conjugation) were synthesize d. As shown in Figure 17, the first antimicrobial test against S. aureus showed promising results, over an 8-log10 CFU/ml bacterial reduction. This test was conducte d two more times to ensure the killing capacity of the polymer. The tests were carried out using different polymer samples and different bacteria cultures. St andard deviations were calculated, accounting for every sample. Figure 17. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) wer e added to stationary-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. 0 1 2 3 4 5 6 7 8 9 0306090120Log Bacterial Count (CFUs/mL)Time (minutes) Bacteria Sample PEI-PNIPAAm (30%) Q-PEI-PNIPAAm (30%)

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51 The results shown in Figure 17 also show that PEI-P NIPAAm (30% conjugation) itself possesses antibacterial activity. This is consisten t with the literature; PEI itself has been shown to kill S. aureus [48][27]. This is due to the presence of protonate d ammonium groups and non-protonated amine groups. The ethylene backb one serves as hydrophobic groups, which create repeating cationic, amphiphilic struct ures along the polymer backbone at a neutral pH without any further chemical modificatio n. At the same time, it can be observed that this polymer exhibited a 2-, 3-, and 4-log red uction after 30, 60, and 120 minutes, which is slightly lower than what the PEI-PNIPAAm (20% co njugation and 3-, 4-, and 5-log reduction after 30, 60, and 120 minutes), likely du e to having more PNIPAAm conjugated and less primary free amines. Tests with Staphylococcus aureus subsp. aureus Mu3 (MRSA/hete ro-VISA) Once the antibacterial properties against S. aureus were confirmed, a new strain was tested. Tests were conducted to measure the killing capacit y of the polymer against S. aureus subsp. aureus Mu3 (MRSA/hetero-VISA). Once again, the test was conducted three times usin g different polymer samples and different bacteria cu ltures. Standard deviations were calculated, accounting for every sample. In this case, PEI-PNIPAAm (30% conjugation) also ex hibited an 8-log10 bacterial reduction. Two tests showed a 6-log10 reduction in 30 minutes, while the third test show ed an 8-log reduction was achieved in the same period of time. This variability is normal, and it was concluded that in 1 hour, the polymer was able to a chieve an 8-log10 bacteria reduction in all cases. One of the controls previously used, PEI-PNI PAAm (30% conjugation) that had previously shown to possess antibacterial activity, did not show the same behavior in this case. The reason for this is because it appears tha t vancomycin resistance in S. aureus is

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52 conferred by alterations in the bacterial cell wall [68][69]. Specific to Mu3, Hanaki et Al. studied the activated cell-wall synthesis associate d with vancomycin resistance in methicillinresistant S. aureus clinical strains Mu3 and Mu50. They demonstrated that cell-wall synthesis and turnover are upregulated in VRSA isolates, lead ing to thicker and more disorganized cell walls [70]. Hence, since the cell wall is thicker, the long alkyl chains penetrate the cell wall to achieve cell death. The quaternization of PEI-PNIPA Am is strictly required to achieve cell death. Figure 18. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conju gation) were added to stationary-phase cultures of Staphylococcus aureus subsp. aureus Mu3 (MRSA/heter o-VISA). Samples were taken after 30, 60, and 120 minutes. N umbers indicate reductions in log10 CFU/ml. Tests with S. epidermidis S. epidermidis is a gram-positive organism and a major inhabitant of the skin, and in some areas, it makes up more than 90% of the resident ae robic flora [71]. Due to this, antibacterial tests were conducted to ensure that the polymer wou ld be able to kill this strain. The results, as shown in 0 1 2 3 4 5 6 7 8 9 0306090120Log Bacterial Count (CFUs/mL)Time (minutes) Bacteria Sample PEI-PNIPAAm (30%) Q-PEI-PNIPAAm (30%) PNIPAAm

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53 Figure 19, confirm that Q-PEI-PNIPAAm (30% conjugat ion) also exhibited an 8-log10 bacterial reduction. Figure 19. Q-PEI-PNIPAAm and PEI-PNIPAAm (20% and 3 0% conjugation) were added to stationary-phase cultures of Staphylococcus epidermidis. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. Tests with E. coli Staphylococcus aureus and Escherichia coli are among the most prevalent species of grampositive and gram-negative bacteria. Since the anti bacterial properties of Q-PEI-PNIPAAm (30% conjugation) were confirmed against gram-posit ive bacteria, the next step was to test it against gram-negative bacteria. As mentioned in the literature review, gram-positive bacteria have a thick cell wall, containing several layers o f peptidoglycan, while gram-negative bacteria contains a similar a cell wall, but is als o surrounded by an outer membrane. This outer membrane can be harder to destabilize at time s. Although gram-positive bacteria are 0 1 2 3 4 5 6 7 8 0306090120Log Bacterial Concentration (CFUs/ml)Time (minutes) Bacteria Sample Q-PEI-PNIPAAm (Conj 30%) Q-PEI-PNIPAAm (Conj 20%) PEI-PNIPAAm

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54 prevalent on the skin, gram-negative bacteria can a lso be present in a clinical setting, and due to this, the polymer antibacterial activity against E. coli was assessed. Q-PEI-PNIPAAm (30% conjugation) exhibited a 2-, 3-, and 8-log10 bacteria reduction after 30, 60, and 120 minutes, respectively. The lower ba ctericidal action, when compared to the previous tests, can be explained from the cell wall structural differences already mentioned between gram-positive and gram-negative bacteria. Q-PEI-PNIPAAm (20% conjugation) was also tested in this case but only exhibited sli ghtly higher bactericidal action. On the other hand, one of the controls, PEI-PNIPAAm (30% conjuga tion), had very little antibacterial activity, similar to the effect it had over the Mu3 strain of S. aureus Figure 20. Q-PEI-PNIPAAm and PEI-PNIPAAm (30 and 20 % conjugation) were added to stationary-phase cultures of E. coli. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. Test with S. aureus during Log Phase Finally, the last agar test conducted was to ensure the antibacterial properties of the polymer against bacteria in the log phase. As mentioned pre viously, some antibiotics require the cells 0 1 2 3 4 5 6 7 8 9 0306090120Log Bacterial Count (CFUs/mL) Bacteria Sample Q-PEI-PNIPAAm (Conj 30%) Q-PEI-PNIPAAm (Conj 20%) PEI-PNIPAAm Time (minutes)

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55 to be multiplying in order to kill the bacteria. Th e mechanism of action of this polymer does not require this. But it is important to ensure tha t the polymer can still be effective when subjected to continuously dividing cells. As can be observed in Figure 21, Q-PEI-PNIPAAm (30% conjugation) achieved a 6-log bacteria reducti on in 30 minutes. Figure 21. Q-PEI-PNIPAAm and PEI-PNIPAAm (30% conjugation) wer e added to log-phase cultures of Staphylococcus aureus. Samples were taken after 30, 60, and 120 minutes. Numbers indicate reductions in log10 CFU/ml. 5.3. Cytotoxicity Tests of Q-PEI-PNIPAAm An MTT assay was conducted to assess the biocompati bility of Q-PEI-PNIPAAm. Several methods were used to conduct this test. Previously, the polymer had been placed on top of the cells mixed with the media, however, due to the gel ling nature of the sample the polymer was not in contact with the cells for long period of ti me. Due to this, the methodology was modified and the cells were plated on top of the po lymer and then tripsinized and re-plated before conducting the test. This method also was pr oven faulty because tripsinizing 0 1 2 3 4 5 6 7 0306090120Log Bacterial Count (CFUs/mL) Bacteria Sample PEI-PNIPAAm Q-PEI-PNIPAAm Time (minutes)

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56 centrifuging and re-plating cells lead to higher va riability in number of cells per well and since the cells were not left to attached on the pl ates for long enough the results were inaccurate. Finally the polymer was dissolved in al cohol, left to evaporate and then the cells were plated on top. Since the MTT measures absorba nce, blanks were required for each sample. However, the layer of polymer on the plate needed to be very thin so the polymer concentration tested was1, 3 and 5%. Q-PEI-PNIPAAm (20% Conjugation) was chosen since it had the highest number of quaternized amin e groups and was assumed to be most toxic. At the same time, chlorhexidine 2%, a common skin preparation solution was tested as well.

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57 Figure 22. No statistical difference between negati ve control and cells exposed to PNIPAAm and PEI-PNIPAAm. Statistically significant differen ce was observed between the experimental samples, negative and positive control (cells cultured with 5% DMSO) (*** indicates p > 0.05, and ** indicates p < 0.05.). No statistical difference between cells exposed to Chlorhexidine (FDA approved) and Q-PEI-PNIPAAm 0 20 40 60 80 100 120 QPP (20% Conjugation) PEI-PNIPAAm (20% Conjugation) PNIPAAmChlorhexadineAbsorbance, % of ControlSamples Positive control Negative Control 1% 3% 5% ** ** *** *** ** **

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58 The cells exposed to Q-PEI-PNIPAAm showed statistic ally significant difference in metabolic activity when compared to cells cultured with complete growth medium alone and to the ones exposed to 5%DMSO. Hence, a reduction i n metabolic activity was observed but the same difference was obtained in the samples wit h cells subjected to an FDA approved skin preparation, chlorhexidine 2%. At the same ti me, it must be mentioned that the cells showed higher metabolic activity than the positive control as well. MTT-assays asses metabolic activity and live-dead staining would nee d to have more conclusive information regarding toxicity. Similarly, the toxicity of the cells was assessed w hen skin sensitization and irritation tests would probably be more appropriate when considering the polymer application. 5.4. Functional Tests Antimicrobial Activity of Commercially Available Su rgical Incision Drapes The preoperative skin preparations that are current ly used before surgeries disinfect the superficial layer of the skin. However, some remain ing bacteria may cause recolonization during surgery [11]. 3M, one of the major companies producing SIDs, states that surgeons use antimicrobial SIDs as an added protection to lo wer the risk of potential recolonization. However, the literature concerning risk of recoloni zation whilst using a drape is controversial. Many studies publish faster recolonization of skin with plastic drape than without plastic drape[54]. To evaluate whether bacteria does recolonize the sk in surface, agar plates were inoculated with a used surgical drape. Colonies were observed after a 24 hour period as can be observed in Figure 23.

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59 Figure 23. Bacterial growth after inoculation with used SIDs. For the test, pieces of SIDs were taken right after a surgery, inoculated on 5 s pots on an agar plate. Surprisingly, a formation of bacterial colony was observed on 2 spo ts, indicating bacterial recolonization Antimicrobial Activity of Polymer-Coated Glass Slid es Since the polymer will be used in a similar manner to a coating, it was tested after coating glass slides. All the different polymers were disso lved in water and placed in a spray bottle to assess the application method. Unfortunately, the v iscosity of the samples was too high, and the coating seemed uneven. Due to this, a new techn ique was developed. Instead of dissolving the polymer in dH2O, the new approach involved dissolving the polymer samples (1% w/v) in 70% ethanol and then spraying it and al lowing the ethanol to evaporate. This new approach also could potentially mean that the ‘ skin-prepping’ step using ethanol could be skipped and, hence, the steps before the surgery reduced. The glass slides coated with Q-PEI-PNIPAAm (20% and 30%) showed no bacterial growth, while the controls, did as can be seen in Figure 24 The coating with Q-PEI-PNIPAAm has an orange/yellowish color, but no colonies were seen, while PEI-PNIPAAm (20 and 30%) showed colonies. The bacterial dilutions performed showed that the initial bacterial concentration was 7.96 x 107CFU/ml.

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60 Figure 24. 1 x 1 cm glass slides coated with polyme r and then sprayed with bacteria. The half-coated/half-non-coated slide showed no bac terial growth on the coated region, as can be observed in Figure 25. Figure 25. Glass slide that was half coated with th e polymer and half uncoated. The left insert shows colonies that grew on the non-coated edges of the slide. Images were taken of the glass slide coated with th e polymer-ethanol solution before and after soaking in dH2O for 24 hours.

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61 Figure 26. Image of the glass slide sprayed with th e polymer-ethanol solution before soaking in water. Figure 27. A) Image of the glass slide after it was soaked for 24 hours in water. B) A razor blade was used to remove part of the polymer on the pre-soaked slide. Figure 26 and Figure 27 do not show any significant changes, and the coating is still present on the slide.

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62 6. Conclusions and Future Directions Conclusion of specific aim 1: Design and synthesize a reverse-thermal gel the quaternized to obtain antimicrobial properties An antimicrobial reverse-thermal gel was designed a nd characterized. Initially, PSHUPNIPAAm and Q-PSHU-PNIPAAm were developed and teste d. Although both polymers exhibited reverse-thermal gelling properties, they did not display good antimicrobial properties. Based on the knowledge gathered from th e initial development, a new polymer, PEI-PNIPAAm and its quaternized version were design ed and synthesized. First, PEIPNIPAAm (20% conjugation) and Q-PEI-PNIPAAm (20% co njugation) were obtained, and the zeta potential was measured. The results indica ted the presence of positive charges on the macromolecule. Further characterization was conduct ed through NMR that confirmed the conjugation of PEI to PNIPAAm and the quaternizatio n step. At the same time, UV-visible spectroscopy was used to verify the LCST. However, although this polymer exhibited a transition temperature, th e gel obtained was unstable. Finally, two variations of this polymer were obtained by varying the conjugation ratio to 30% and 50%. The detection of positive charges using zeta potent ial was impeded by the presence of PNIPAAm that also dominated the NMR spectra at 50% conjugation. In conclusion, three polymers were designed and cha racterized with one exhibiting properties suitable for antimicrobial applications. However, i n terms of future work, further analysis should be conducted to quantify the number of quate rnized amine groups and analyze the effect of these on the overall properties of the po lymer. A possible option would be to use confocal Raman spectroscopy, X-ray photoelectron sp ectroscopy (XPS), or a solution of uorescein (sodium salt) that can be used in a simi lar manner to a dye. Then, the conversion

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63 of the dye concentration to surface charge density can be determined, assuming that one surface quaternary ammonium group complexes with on e dye molecule. However, functional antibacterial tests were prioritized in this study, and a more thorough analysis is suggested as part of future work. Conclusion of specific aim 2: Characterize the anti microbial activity of the polymer The antimicrobial properties of the polymers synthe sized were assessed. As mentioned previously, initially, PSHU-PNIPAAm exhibited antim icrobial properties when evaluated using optical density tests. However, agar tests co nfirmed the opposite. The antibacterial test protocols and approach were modified, and a new set of polymer-quaternized PEI-PNIPAAm polymers of different conjugation ratios were teste d. All samples were exposed to bacterial suspensions of 108 CFU/ml. The results showed that although a conjuga tion ratio of 20% exhibits an 8-log10 bacteria reduction, the polymer did not satisfy other requirements. Later, the 50% conjugation ratio proved to have insufficie nt antibacterial activity, while the 30% conjugation ratio showed an 8-log10 bacterial reduction. At the same time, Q-PEI-PNIPAAm (30% conjugation) w as tested against different bacterial species. The polymer exhibited 8-log10 bacterial reductions when testing gram-positive an d gram-negative bacteria. The time-kill graphs showed differences in each case, and these were related to the outer cell wall structural differenc es of the strains tested. In conclusion, a polymer exhibiting excellent antib acterial properties and good gelling behavior was obtained and tested. The next step is to conduct in vivo test using Q-PEIPNIPAAm (30% conjugation) and compare it to SIDs. A n animal protocol has been submitted and accepted for this next test.

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64 Conclusion of specific aim 3: Cytotoxicity test of Q-PSHU-PNIPAAm and Q-PEIPNIPAAm The cytotoxicity of Q-PSHU-PNIPAAm was assessed by means of an MTT assay using smooth muscle cells. The results showed no signific ant statistical difference with the negative control. The tests were then repeated using Q-PEI-PNIPAAm (2 0% conjugation) because this sample exhibited the highest antibacterial properties. Fib roblasts that are more sensitive were used in this case. At the same time, chlorhexidine was used because it is the golden standard. The results showed a decrease in metabolic activity whe n compared to the negative control. However, the metabolic activity of the fibroblasts subjected to Q-PEI-PNIPAAm was comparable to the cells that were exposed to chlorh exidine. Since MTT assays are indicative of the cellÂ’s metabolic activity, it can be conclud ed that the polymer does affect the cellÂ’s metabolic activity; however, the results observed s how no statistical difference to those obtained with Chlorhexidine, an FDA-approved produc t of clinical use. However, as part of future work, skin sensitivity/irritation and sensit ization tests, such as the Bowman Berger tests recommended by the FDA, should be conducted in vivo Future work Finally, although further characterization would be interesting in order to correlate the number of quaternized amine groups to antibacterial properties, it can be concluded that a reverse-thermal gel with excellent antimicrobial pr operties was developed in this study. However, a major point that was not targeted in thi s study involves the analysis of adhesive and mechanical properties of the polymer. During su rgery, this material has to be able to withstand water and tough pulling of the skin. Alth ough preliminary tests showed that the

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65 polymer can remain attached to a surface during a p eriod of 24 hours in water, further tests need to be conducted. Research was conducted on possible methods to asses s the adhesive properties of the polymer and an interesting option was explored by Vernengo et al. [72]. They tested the adhesive properties of a PNIPAAm-PEG/PEI polymer blend using an Instron mechanical testing system. They placed the polymer on fresh porcine sk in and then removed it at a rate of 2 mm min-1 whilst obtaining load-displacement data that they then used to obtain stress-strain values. Using this method they then calculated the maximum force required to detach the gel form a skin surface. An assay similar to the one pr eviously described would be of interest to gain insight regarding the adhesive properties of t he polymer. Finally, future work concerns in vivo studies initially on mice (protocol and method approved) and comparing the polymer to current SIDs

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73 APPENDIX 1. H1NMR of PNIPAAm Figure A 1. 1H NMR (500 MHz, CDCl3) analysis of the PEI. Peak assignments were corroborated by NMR modeling (Advanced Chemistry De velopment) 2. Agar test example images Images Figure B 1 Agar plates showing bacterial colonies. Dilutions w ere performed and each samples triplicated. Data was collected from counti ng colonies in E and extrapolating to obtain the initial bacterial concentration

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74 Figure B 2. Agar plates showing bacterial colonies. Time points at t= 60 and 120 minutes. Dilutions were performed and each samples triplicat ed. Data was collected from counting colonies in E and extrapolating to obtain the initi al bacterial concentration Figure B 3. Agar plates showing bacterial colonies subjected to PEI-PNIPAAm (30% conjugation). Dilutions were performed and each sam ples triplicated. Data was collected from counting colonies in E and extrapolating to ob tain the initial bacterial concentration

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75 Figure B 4. Agar plates showing no bacterial growth after 30 mi nute antibacterial test with QPEI-PNIPAAm (30% conjugation) Figure B 5. Agar plates showing no bacterial growth. The bacter ia had been subjected to QPEI-PNIPAAm (30% conjugation) for 60 minutes Figure B 6. Agar plates showing no bacterial growth. The bacter ia had been subjected to QPEI-PNIPAAm (30% conjugation) for 120 minutes