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Evaluation of an antimicrobial reverse-thermal gel for use as a surgical drape

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Title:
Evaluation of an antimicrobial reverse-thermal gel for use as a surgical drape
Creator:
Stein, Madia Elizabeth
Place of Publication:
Denver, Colo.
Publisher:
University of Colorado Denver
Publication Date:
Language:
English

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Degree:
Master's ( Master of Science)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Bioengineering, CU Denver
Degree Disciplines:
Bioengineering

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University of Colorado Denver Collections
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Auraria Library
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Copyright MADIA ELIZABETH STEIN. Permission granted to University of Colorado Denver to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Full Text
EVALUATION OF AN ANTIMICROBIAL REVERSE-THERMAL GEL FOR USE AS A
SURGICAL DRAPE by
MADIA ELIZABETH STEIN B.S., University of New Mexico, 2014
A thesis submitted to the Faculty of the Graduate School of the University of the Colorado in partial fulfillment of the requirements for the degree of Master of Science Bioengineering Program
2016


This thesis for the Master of Science degree by Madia Elizabeth Stein has been approved for the Bioengineering Program by
Daewon Park, Chair Vikas Patel Danielle Soranno
Date: December 17, 2016


Stein, Madia Elizabeth (M.S., Bioengineering)
Evaluation of an Antimicrobial Reverse-Thermal Gel for Else as a Surgical Drape Thesis directed by Assistant Professor Daewon Park
ABSTRACT
Surgical site infections (SSIs) are a dangerous complication of surgical procedures.
SSIs develop in roughly 2% of surgical procedures in the U.S. Despite efforts to reduce infection rates, SSIs are extremely costly and they remain a significant cause of morbidity and mortality. Generally, antibiotic prophylaxis, preoperative skin preparation, surgical hand preparation, and intraoperative skin antisepsis are all standard practices used in hospitals to reduce SSI occurrences. Most SSIs are caused by endogenous bacteria from a patients skin, therefore preoperative skin preparation and intraoperative skin antisepsis are essential. Surgical incision drapes (SIDs) impede microbial recolonization and prevent contamination in the surgical incision site during surgery. However, due to drawbacks of current SIDs drawbacks, including time consuming placement, loss of adhesion, and loss of an epidermal cell layer during drape removal, an antimicrobial reverse-thermal gel was developed for potential use as a SID. Gelling properties and antimicrobial activity of the reverse-thermal gel surface were demonstrated to be ideal for the use of this polymer as a SID. Performance of the polymer as a SID was evaluated in a mouse skin incision model and compared with commonly used commercially available SID. The studied polymer was found to have comparable performance as a SID to the commercial alternative.
The form and content of this abstract are approved. I recommend its publication.
Approved: Daewon Park
m


ACKNOWLEDGEMENTS
I would like to acknowledge my professors, mentors, friends, and family and for all of their support throughout my Masters Project. Without their help this work would not have been possible.
First, I would like to thank my advisor, Dr. Daewon Park, for allowing me to join the Translational Biomaterial Research Laboratory (TBRL) and for his mentorship and patience. Through Dr. Parks guidance I have learned many valuable skills. I would also like to express my gratitude to Dr. Vikas Patel and Dr. Danielle Soranno for their time, insight, and advice throughout my research.
I would also like to thank Dr. Todd for his time and help with design of bacterial testing.
My time in this program would not have been the same without my fellow TBRL members. I appreciate all of our experiences together both in and out of the lab. I would particularly like to thank David Lee, James Bardill, and Melissa Laughter for their help and encouragement.
Finally, I would like to thank my family. I cannot thank my mother and father enough for their continual faith in my success and constant encouragement. I would also like to express my gratitude to my boyfriend, Jay McCabe, for his unbelievable support and patience.
Animal model studies were conducted under the University of Colorado at Denver Institutional Animal Care and Use Committee (IACUC) protocol number 102913(12)1D.
IV


TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION.............................................................1
Overview.................................................................1
Skin Anatomy and Physiology..............................................1
Skin Microbiota..........................................................4
SSI Microbiology.........................................................7
SSI Pathogenesis.........................................................9
SSI Prevention..........................................................10
Antibiotic Prophylaxis.............................................11
Preoperative Skin Preparation.....................................11
Preoperative Surgical Hand Scrub...................................13
Intraoperative Skin Antisepsis: Surgical Incision Drapes...........13
Study Objective.........................................................16
II. BACKGROUND..............................................................17
Antimicrobials and Antimicrobial Resistance.............................17
Mechanisms of Antimicrobial Activity....................................18
Antimicrobial Polymers..................................................21
Quaternary Ammonium Compounds......................................22
Polyethyleneimine..................................................23
v


Effect of Alkyl Chain and Molecular Weight on Antimicrobial Activity.24
Biocompatibility of Quaternary Ammonium Compounds..................26
Reverse-Thermal Gels...................................................27
III. PREVIOUS WORK...........................................................30
Q-PEI-PMPAAm LCST......................................................30
Antimicrobial Activity of Q-PEI-PNIPAAm................................30
Cytotoxicity...........................................................32
IV. HYPOTHESIS AM) SPECIFIC AIMS............................................33
Hypothesis.............................................................33
Specific Aims..........................................................33
V. MATERIALS AM) METHODS...................................................34
Materials..............................................................34
Polymer Synthesis......................................................35
PNIPAAm Synthesis..................................................35
PEI-PNIPAAm Synthesis..............................................35
Q-PEI-PNIPAAm Synthesis............................................36
Polymer Characterization...............................................37
Proton Nuclear Magnetic Resonance (*H NMR).........................37
LCST Determination.................................................37
Bacteria Preparation and Dilutions.....................................37
Antimicrobial Q-PEI-PNIPAAm Surface Tests..............................38
vi


Antimicrobial Activity of a 2D Q-PEI-PNIPAAm Surface in a 3D Bacterial Suspension...........................................................38
Antimicrobial Activity of a 2D Q-PEI-PNIPAAm Surface against 2D Bacterial Samples....................................................39
Skin Incision Animal Model................................................40
Skin Incision Surgery................................................40
Quantification of Bacterial Swabs....................................41
Q-PEI-PNIPAAm Immune Response In Vivo.....................................41
Subcutaneous Injections..............................................41
Immunohistochemistry.................................................42
Statistical Analysis......................................................43
VI. RESULTS AND DISCUSSION....................................................44
Q-PEI-PNIPAAm Reaction Mechanism..........................................44
Polymer Characterization..................................................45
Characterization of PEI, PEI-PNIPAAm, and Q-PEI-PNIPAAm using 'H NMR..................................................................45
Lower Critical Solution Temperature (LCST)...........................47
Antimicrobial Q-PEI-PNIPAAm Surface Tests.................................49
Antimicrobial Activity of Q-PEI-PNIPAAm Surface in a Bacterial Suspension...........................................................49
In vitro Q-PEI-PNIPAAm Activity as a Surgical Incision Drape.........55
Murine Skin Incision Model................................................57
Subcutaneous Injections...................................................63
vii


VII. CONCLUSION..........................................................66
VIII. FUTURE WORK........................................................68
Additional Polymer Optimization.....................................68
Increase Mouse Model Sample Size....................................68
In Vivo Cytoxicity Assay............................................69
Skin Irritation Test................................................69
REFERENCES...............................................................70
viii


LIST OF FIGURES
FIGURE
1. The anatomical structure of the human integumentary system [11]....................2
2. A: H&E stained thick skin showing the layers of the epidermis and collagen
fibers in the dermis. B: Illustration of the layers of the epidermis [12].........3
3. Factors influencing skin microbiota diversity [26].................................6
4. Skin microenvironments and associated distributions of bacteria [16]...............7
5. A: Epidermal cell removal during SID lift off, which exposes bacteria found beneath the skin. B: Surgical incision drape after being peeled back in preparation for would closure. C: Skin avulsion injury from the SID removal
on an elderly patient.............................................................15
6. Mechanisms of antibacterial resistance, la: Elimination of cellular antimicrobials using efflux pumps, lb: Elimination of cellular antimicrobials by decreasing/blocking cellular uptake. 2: Alteration of antimicrobial target.
3: Direct inactivation of antimicrobials by cellular enzymes. 4: Alteration of metabolic pathways [97].......................................................18
7. Antimicrobial mechanisms of various antimicrobial agents against bacterial spores, bacteria, viruses, and fungi. QACs and CRAs are quaternary
ammonium compounds and chlorine-releasing agents, respectively [101]..............19
8. Structure of gram-negative and gram-positive bacteria. Both types of bacteria
contain negatively charged structures on their surface. Gram-negative bacteria have negatively charged phospholipids and lipopolysaccharides on their surface. Gram-positive bacteria have negatively charged teichoic and lipoteichoic acids on their surface [102].........................................20
9. Quaternizatioin of tertiary amine with an alkyl halide yielding a quaternary ammonium cation. In this chemical equation, R represents alkyl or aryl
groups, and X represents a halogen, such as Cl", Br", FI", or I"..................22
10. A: Behavior of PNIPAAm chains below (left side) and above (right side)
LCST. B: PNIPAAm chemical structure with labeled hydrophilic and
hydrophobic regions...............................................................29
11. PEI-PNIPAAm and Q-PEI-PNIPAAm were added to stationary-phase
bacteria, and samples were taken at 0, 30, 60, and 120 minutes to determine bacterial concentrations. Kill-curves were constructed for the following bacteria: A: S. aureus subsp. aureus Mu3 (MRSA/hetero-VISA) B: Staph, epidermidis C: E. coli D: S. aureus...............................................31
IX


12. MTT assay assessing the cytotoxicity of Q-PEI-PNIPAAm (QPP). No statistical difference between the negative control and cells plated on PNIPAAm and PEI-PNIPAAm was observed. No statistical significance was found between cells plated on QPP and Chlorhexidine. Statistical significance was found between experimental groups and the positive control
(** denotes p < 0.05 and *** denotes p > 0.05)............................32
13. Reaction mechanism for the synthesis of Q-PEI-PNIPAAm.....................44
14. *11 NMR spectrum of branched PEI confirming its molecular structure.......45
15. *11 NMR spectrum of PEI-PNIPAAm confirming the addition of PNIPAAm
to PEI.....................................................................46
16. *11 NMR spectrum of Q-PEI-PNIPAAm confirmed the conjugation of alkyl
chains and the presence of a slight peak at 3.6 ppm indicated the presence of quaternary ammoniums....................................................46
17. LCST of polymer samples. A: PNIPAAm LCST. B: PEI-PNIPAAm LCST.
C: Q-PEI-PNIPAAm LCST. D: Gelled Q-PEI-PNIPAAm............................47
18. Appearance of 5 wt% solutions of PNIPAAm, PEI-PNIPAAm, and Q-PEI-
PNIPAAm above and below the LCST. Below the LCST, PNIPAAm and PEI-PNIPAAm are transparent solutions and Q-PEI-PNIPAAm is a yellowish opaque solution. Above the LCST, PNIPAAm and PEI-PNIPAAm form opaque unstable gels, which do not retain the shape of the vial, and Q-PEI-PNIPAAm forms a stable opaque gel......................................48
19. Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase Corynebacterium amycolatum. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error
bars represent the standard error of the mean..............................50
20. Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase Escherichia coli. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the
standard error of the mean.................................................51
21. Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase MRSA. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard
error of the mean..........................................................51
22. Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase Staphylococcus aureus. Glass slides were coated with 1.25,
2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent
the standard error of the mean............................................52
x


23. Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against
stationary-phase Staphylococcus epidermidis. Glass slides were coated with
1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars
represent the standard error of the mean.....................................53
24. Q-PEI-PNIPAAm accumulation following 120-minute surface test with shaking at 250 rpm. A: Slide coated with 1.25% QPP. B: Slide coated with
7.5% QPP.....................................................................54
25. Growth of bacteria in grid pattern after 48 hour incubation on agar with no
polymer......................................................................55
26. In vitro simulation of Q-PEI-PNIPAAm antimicrobial activity when used as a SID. Growth after 48 hours is shown. Bacteria were plated in the grid pattern below and above the polymers on separate plates. In this simulation agar represents the skin and the polymer represents the drape. Bacteria grew on both PNIPAAm plates, and PEI-PNIPAAm had some bacterial growth. Q-PEI-PNIPAAm inhibited all bacterial growth except for a small untreated
area.....................................................................56
27. Murine model treatment groups. A: Q-PEI-PNIPAAm applied as a surgical drape over bacteria. B: IOBAN 2 iodine-impregnated drape applied over bacteria. C: Positive control group with no drape or bacteria. D: Negative
control group with applied bacteria and no drape.............................................58
28. The number of C. amycolatum colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the
standard error of the mean. indicates statistical difference with p < 0.05.................59
29. The number of E. coli colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard
error of the mean. indicates statistical difference with p < 0.05..........................60
30. The number of MRSA colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard
error of the mean. indicates statistical difference with p < 0.05..........................61
31. The number S. aureus colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard
error of the mean. indicates statistical difference with p < 0.05..........................61
32. The number Staph, epidermidis colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the
standard error of the mean. indicates statistical difference with p < 0.05.................62
xi


33. Representative confocal images of immunostaining for macrophages after 1 and 7 days. Macrophages were stained with anti-CD68 antibody and Alexa Fluor 594, which appears in red. DAPI was used to stain cell nuclei, which appear in blue. Cells containing both colors were identified as macrophages.
Scale bars represent 100 pm.....................................................64
34. Macrophages per unit area following 1 and 7 day subcutaneous injections of saline and Q-PEI-PNIPAAm. Error bars represent the standard error of the
mean. indicates statistical difference with p < 0.05..........................65
Xll


LIST OF TABLES
TABLE
1. Rank and distribution of the most common SSI causing pathogens reported
for three time periods..........................................................8
2. Properties of various types of antiseptics and examples of commercially
available products. Table adapted from Reichman et al. 2009. [57]..............12
3. LCST values reported for various conjugations of PEI-PNIPAAm and Q-PEI-
PMPAAm.........................................................................30
4. Sample size (n) values calculated by the power analysis for each bacterial
strain.........................................................................63
xiii


LIST OF ABBREVIATIONS
'hnmr proton nuclear magnetic resonance
ACA 4,4-azobis(4-cyanovaleric acid)
AMPs antimicrobial peptides
ANOVA analysis of variance
APC antigen-presenting cell
BC benzyl chloride
BSA bovine serum albumin
CDC Centers for Disease Control and Prevention
CFUs colony forming units
CHG chlorhexidine gluconate
CT connective tissue
DCD dermcidin
DMAEMA dimethylaminoethyl methacrylate
DMF N,N-dimethylformamide
ECM extracellular matrix
EDC N-(3-dimethylamino-propyl)-N'-ethylcarbodiimide hydrochloride
GI gastrointestinal
H&E haematoxylin and eosin
HACC hydroxypropyltrimethyl ammonium chloride chitosan
HAI healthcare-associated infection
LCST lower critical solution temperature
LOS length of stay
MIC minimum inhibitory concentration
MRSA methicillin-resistant Staphylococcus aureus
MW molecular weight
n2 nitrogen
nh2 amine group
NHS N-hydroxysuccinimide
nm nanometers
NIPAAm N-i sopropyl acryl ami de
OCT optimal cutting temperature
OR operating room
PCMX para-chloro-meta-xylenol
PEI polyethyl enimine
PBS phosphate buffered saline
PNIPAAm poly (N-i sopropyl acryl ami de)
QACs quaternary ammonium compounds
QPP quatemized-PEI-PNIPAAm
rpm revolutions per minute
xiv


RTG reverse thermal gel
SID surgical incision drape
spp. species
SSI surgical site infection
uv Ultraviolet
ZPT zinc pyrithione
XV


CHAPTER I
INTRODUCTION
Overview
Surgical site infections (SSIs) are a dangerous complication of surgical procedures. When the skin is cut during surgery, microorganisms may invade the incision, which may cause infection. A SSI has been defined by the Centers for Disease Control and Prevention (CDC) as an infection related to a surgical procedure which occurs at or near a surgical incision within 30 days of the operation or 1 year for medical implants [1][3]. SSIs account for 14-16% of healthcare-associated infection (HAI) and develop in roughly 2% of surgical procedures in the U.S. [2][6]. There are roughly 27 million surgeries in the U.S. per year, which equates to approximately 540,000 SSIs [3], SSIs are associated with mortality, increased cost, and prolonged length of stay (LOS) in hospitals. It is estimated that among patients with SSIs there is a 3% mortality rate and a 7-10 day increase in LOS [3], [5], [7], SSIs cost between $11,000 and $35,000 per patient, and it is estimated that up to $10 billion is spent on SSIs annually in the U.S. [8][10], Despite efforts to reduce infection rates, SSIs are extremely costly and they remain a significant cause of morbidity and mortality.
Skin Anatomy and Physiology
The skin, which has a surface area of 1.2 to 2.2 m2, is the bodys largest organ [11], Importantly, the skin acts as a container for and a protector of the bodys contents. The skin consists of two layers; the superficial avascular epidermis and the underlying dermis. These layers are supported by a basal layer of subcutaneous tissue known as the hypodermis (Figure 1). Together the skin and its appendages form the integumentary system.
1


Hair shaft
Dermis-
Reticular -layer
Hypodermis
(subcutaneous tissue; not part of skin)
Nervous structures
Sensory nerve fiber with free nerve endings
Lamellar corpuscle-----
Hair follicle receptor (root hair plexus)
Appendages of skin
Eccrine sweat gland
Arrector pili muscle
Sebaceous (oil) gland
Hair follicle
Hair root
Cutaneous plexus
Adipose tissue
Figure 1 The anatomical structure of the human integumentary system. [11]
The epidermis is chiefly composed of keratinocytes, but it also contains melanocytes, dendritic cells, and tactile cells. These cells are organized into four to five distinct layers of cells. Starting from the base of the epidermis they are stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum (Figure 2). The stratum basale consists of a single layer of stem cells. These stem cells divide into keratinocytes to replenish cells shed from the epidermis. The stratum basale also contains a small number of melanocytes and infrequent tactile cells. The next layer up, the stratum spinosum, is several cell layers thick. This layer contains many keratinocytes along with melanin granules and dendritic cells. Melanin granules shield cells in the skin from ultraviolet (UV) damage. The stratum granulosum consists of four to six cell layers in which the keratinocytes begin to keratinize, flatten, and their nuclei and organelles begin to disintegrate. In this stratum,
Dermal papillae
Subpapillary
plexus
Sweat pore
Epidermis-
Papillary
layer
2


lamellar granules containing water-resistant glycolipids are released into the extracellular matrix (ECM). The stratum lucidum is present in areas of thick skin such as the hands and feet. It consists of two to three rows of flat, clear, dead keratinocytes. The superficial stratum corneum is the thickest layer of the epidermis accounting for up to 75% of its thickness and containing 20 to 30 cell layers. The dead, flattened, enucleated keratin in this layer protects the body from abrasion and infiltration, while glycolipids prevent water loss. [11], [12]
-Stratum corneum
lucidum^ granulosum
-Stratum spinosum-Stratum basale
-----Dermis------
Dead keratinocytes
Living keratinocyte
Melanocyte
Epidermal dendritic cell Basement membrane Tactile cell
nerve ending
A B
Figure 2 A: H&E stained thick skin showing the layers of the epidermis and collagen fibers in the dermis. B: Illustration of the layers of the epidermis. [12]
The dermis is composed of flexible connective tissue (CT). Although appendages, such
as hair follicles, sweat glands, and sebaceous (oil) glands, are derived from the epidermis,
they largely reside within the dermis. The dermis has a papillary and a reticular layer. The
superficial papillary layer contains a plethora of blood vessels that supply nutrients to the
epidermis. The CT in the papillary layer is loosely woven, which allows macrophages, mast
cells, and other immune cells to traverse freely. The reticular layer makes up about 80% of
the dermis and it is composed of dense fibrous CT. This layer is nourished by blood vessels
in the cutaneous plexus, which lies between the dermis and the hypodermis. [11]
3


Functions of the integumentary system include protection, regulation of body temperature, and secretion. First and foremost, the skin is a barrier that is essential to protecting the body from microorganisms, harmful substances, and other environmental factors. Dead keratinized cells in the stratum corneum strengthen the skin, while glycolipids seal the gaps between cells much like brick and mortar, respectively [13], [14], This physical barrier prevents most substances from penetrating the skin, however, a few substances, including water, organic solvents, and lipid-soluble substances pass through the skin in small amounts [11], The skin is also a chemical barrier whose secretions protect the body. The skin has two type of sweat glands: the eccrine and apocrine glands. The eccrine gland secretes water, electrolytes, and salt. Secretion of water is vital to regulation of the bodys temperature, and secreted salts and electrolytes create an acidic environment, which impedes bacterial growth [15], [16], Furthermore, eccrine glands have also been found to secrete antimicrobial peptides (AMPs) in the form of dermicidin (DCD) [17], [18], Sebaceous glands secrete sebum and several AMPs, including P-defensins, cathelicidins, and histones [19],
[20], The skin also contains active biological barriers that protect the body. The first line of defense are dendritic cells found in the epidermis. Dendritic cells engulf foreign substances and travel to lymphocytes to activate the adaptive immune response against foreign invaders. Macrophages in the dermis act as a second line of defense. They directly engulf any bacteria or viruses that make it through the epidermis, and they act as antigen presenting cells (APCs), which can activate adaptive immunity [11],
Skin Microbiota
Despite the skins numerous antimicrobial defenses, the skin is inhabited by a diverse array of microbes, including bacteria, viruses, fungi, and mites [16], [21], The skin contains
4


transient flora, which dont survive well on the skin and are transmitted by contact with the external environment, and resident flora, which colonize the skin and are not readily removed by hand washing [16], [22], Collectively, these microbes form the skin flora or the skin microbiota. There are three potential outcomes for each species in microorganism-host interactions: positive, negative, or no impact. Commensalism is an interaction where one species benefits from interactions while the other is not affected, and mutualism is a relationship where both the species benefit [23], [24], The relationship between most microbes on the skin and the skin itself is mutualistic, commensal, or neutral [25], [26], The bacteria on an individuals skin has been found to remain somewhat constant over time, allowing interactions to remain in the categories above [27], The vast majority of SSIs are caused by bacteria, so this report will focus on bacteria as the source of infection [3],
It is estimated that there are 10,000 bacteria/cm2 on the surface of the skin and about 1,000,000 bacteria/cm2 throughout the epidermis and the dermis [28], Although a study detected 19 phyla on the skin, nearly all bacteria belong to the Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%) phylas [29], Bacteria from the genera Corynebacteria, Propionibacteria, and Staphylococci accounted for 22.8%, 23.0%, and 16.8% of the bacteria on the skin, respectively [29], Corynebacteria and Propionibacteria belong to the Actinobacteria phylum, and Staphylococci belongs to the Firmicutes phylum. In addition, the genera Micrococci, Streptococci, and Brevibacteria are also frequently present on the skin [24], Despite the presence of conserved bacterial genera from individual to individual, specific inter-person skin microbiota is extremely diverse [27], [30]-[32], Skin microbiota diversity is influenced by a combination of environmental and host factors, including age, hygiene, and geographical location (Figure 3) [33],
5


Figure 3 Factors influencing skin microbiota diversity. [26]
The human skin can be divided into three distinct microenvironments: dry, moist, and sebaceous [29], [34], Each of these microenvironments tends to favor particular bacterial genera (Figure 4). Although dry habitats are associated with the most diverse population of bacteria, Proteobacteria are common in this microenvironment [16], [29], Despite previously being regarded as gastrointestinal (GI) organisms, gram-negative bacteria are also frequently found in this microenvironment [26], [35], Staphylococci from the phylum Firmicutes and Corynebacteria from the phylum Actinobacteria are abundant in moist microenvironments, which includes the groin, the sole of the foot, the axilla, the inner elbow, and the back of the knee [27], [29], Bacteria found in sebaceous areas of the skin, which include facial areas and the back, are the least diverse and consist mainly of bacteria of the Propionibacteria genus from the Actinobacteria phylum [16], [36], [37], Aside from the three listed microenvironments, glands and follicles are thought to have their own characteristic flora [28], [36],
6


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ty
#
q
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# Alar crease
External auditory canal
Nare
# Manubrium Axillary vault
t-
5
Antecubital fossa
Volar forearm
W k/
r/
x \
# Hypothcrvar
p/.lrri
Interdigital web space
I Inguinal crease
i Umbilicus
' IM
Reiroaur icular | crease
Occiput |
Back#
Buttock#
Gluteal# | crease
f\?pliteal fossa# Plantar heel#
#
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Key
Actinobacteria
| Corynebacteriaceae | Propionibacteriaceae | Micrococciaceae | Other Actinobacteria
Bacteroidetes j Cyanobacteria
Firmicutes
| Other Firmicutes | Staphylococcaceae
Proteo bacteria
Divisions contributing <1% Unclassified
Sebaceous
Moist
Dry
I Toe web space
Figure 4 Skin microenvironments and associated distributions of bacteria. [16]
SSI Microbiology
There are two main sources of pathogens for SSIs: endogenous and exogenous. Endogenous microbiota coming from the skin, the GI tract, the respiratory tract, and the genitourinary tract cause most SSIs [38], The most common endogenous pathogens are aerobic gram-positive cocci, which are associated with the skin microbiota [3], Exogenous sources include the air in the operating room (OR), the OR personnel, the OR equipment, surgical instruments, and anything brought into the surgical sterile field [2], [3], When outbreaks of SSIs occur that are associated with uncommon pathogens, they are typically caused by exogenous sources [3], For example, water contamination, elastic bandages, and an anesthesiologist were the cause of outbreaks of Pseudomonas multivorans, Clostridium perjr ingens, andNocardiafarcinica, respectively [39][41],
7


Although a variety of microbes are present on the skin, most SSIs are caused by bacteria. Table 1 lists the eight categories of pathogens that most frequently cause SSIs. Together, these pathogen categories account for 75-79% of SSIs, and the distribution of these pathogens has remained somewhat constant over time [42][44]. According to a recent report, Staphylococcus aureus, coagulase-negative staphylococci (e.g., Staphylococcus epidermidis), Enterococcus spp., and Escherichia colt are responsible for approximately 30.4%, 11.7%, 11.6%, and 9.4% of SSIs, respectively [44], Corynehacteria (e.g., C. amycolatum) have also been associated with SSI development [45], It has been found that an increasing amount of SSIs are caused by drug-resistant strains of many of these pathogens as well as other unlisted categories of pathogens [46], [47], As can be seen in Table 1, a small amount of SSIs are also consistently caused by the fungal pathogen Candida albicans, but this report will focus on bacteria.
Table 1 Rank and distribution of the most common SSI causing pathogens reported for three time periods.
Pathogen Percentage of Pathogens
Rank 1990-1996 (n=17,671)41 2006-2007 (n=7,025)42 2009-2010 (n=21,100)43
Staphylococcus aureus 1 20 30 30.4
Coagulase-negative staphylococci 2 14 13.7 11.7
Enterococcus spp. 3 12 11.2 11.6
Escherichia coli 4 8 9.6 9.4
Pseudomonas aeruginosa 5 8 5.6 5.5
Enterobacter spp. 6 7 4.2 4
Klebsiella pneuminiae 7 3 3 4
Candida albicans 8 3 1.6 1.3
Total 75 79 78
There are likely SSI pathogens associated with different types of surgeries. This is largely due to the fact that the distribution of endogenous microbiota varies by anatomical location. As discussed in the skin microbiota section, skin contains mostly gram-positive aerobes, while the GI tract has gram-negative bacteria and anaerobes. Therefore, a surgery
8


that only disrupts the skin exposes a patient to markedly different pathogens than a surgery where the integrity of the GI tract surface is broken. The likely SSI pathogens for neurosurgery, implants, graft placements, heart surgery, and vascular surgery are Staphylococcus aureus and coagulase-negative staphylococci. Appendectomies and colorectal surgeries are more likely to have SSIs caused by gram-negative bacilli or anaerobes. [3]
SSI Pathogenesis
Penetration of the skin during surgery exposes patients to microbial contamination.
One clinical trial found that out of 166 clean surgeries, 3 patients (1.8%) developed SSIs despite the fact that 101 (61%) of the incisions contained 1 to 200 colony forming units (CFUs) of bacteria during surgery [48], This indicates that while microbial contamination is a required precursor to SSIs, the presence of bacterial does not guarantee that a SSI will occur. It has been demonstrated that the presence of >105 bacteria/gram of tissue greatly increases the risk of a SSI [49], Using various strains of staphylococcus it was also demonstrated that while a subcutaneous injection of >106 bacteria is required for infection, 102 bacteria may cause infection in the presence of sutures composed of various materials [50][52],
Therefore, the presence of foreign material increases the risk of a SSI developing with markedly smaller numbers of bacterial contamination.
Development of SSIs also depends on the patients immune system as well as the virulence of the bacteria. Risk of surgical infection has been defined by the following equation [53]:
Dose of bacterial contamination x Virulence
Risk of SSI = -------------------- ---------------------- (1)
Resistance of the host patient
9


In this context, virulence is a measure of the severity of an infection caused by a particular species of bacteria. Many bacteria have specific defensive mechanisms that allow them to evade host immunity and survive within the host, these are termed virulence factors [3], For example, coagulase-negative staphylococci produce slime and glycocalyx, which both shields bacteria from phagocytes and protects bacteria from antimicrobial agents [54], The resistance of the host patient to infection is dependent both on their immune system and other patient risk factors, including age, obesity, nutrition, microbial colonization, and length of time in hospital prior to surgery [55], Aside from microbial and patient risk factors, operation risk factors also influence the risk of SSI [56], Some operational risk factors include sterile surgical technique, preoperative shaving, preparation of the surgical incision site, and antimicrobial prophylaxis [55],
SSI Prevention
Although standard practices in the OR vary slightly between hospitals, numerous guidelines for preventing and reducing the occurrence of SSIs have been published [3], [57], [58], Generally, antibiotic prophylaxis, preoperative skin preparation, surgical hand preparation, and intraoperative skin antisepsis are all standard practices used in hospitals to reduce SSI occurrences [58], [59], Since the skin is thought to be the main source of SSI pathogens, preoperative skin preparation and intraoperative skin antisepsis are essential [38], [60], Hair removal with a razor has been shown to increases SSI occurrence, so it is recommended that hair is removed only if it interferes with the procedure [61], [62], In cases where hair is removed, it is cut with clippers immediately prior to skin preparation as razors riddle the skin with microscopic lacerations [3], [62],
10


Antibiotic Prophylaxis
The goal of antibiotic prophylaxis is to prevent SSIs while having no adverse effects on the patient or the normal flora of the patient and the hospital [3], [63], This is achieved by decreasing microbial contamination during surgery to a quantity that the body can defend against with the use of antibiotics [3], In order for antibiotic prophylaxis to be effective, antibiotic levels should exceed the minimum inhibitory concentration (MIC) in the tissue and serum for the duration of surgery [64], [65], For this reason, antimicrobial agents are typically administered intravenously within 60 minutes before surgical incision [63], [64], [66], [67], In general, 1 dose of antibiotics is sufficient, but for longer surgeries the number of doses may vary [68], The exact antibiotics administered to a patient vary based on the type of surgery, and are selected to be active against the pathogens that are most likely to contaminate the surgical site [67],
Preoperative Skin Preparation
The goal of preoperative skin preparation is to significantly reduce the number of microorganisms on the skin surrounding the incision site [22], There are three main types of antiseptics used in preoperative skin preparation: alcohol-based, chlorhexidine gluconate (CHG)-based, and iodine-based [3], [69], Commercially available skin preparations use one or more of these types of antiseptics in their products (Table 2) [57],
Iodine-based antiseptics are effective against fungi, viruses, and gram-negative and gram-positive bacteria [22], These antiseptics function through penetration of the cell wall followed by the oxidation/substitution of microbe intracellular components with free iodine [3], [22], A drawback of iodine-based antimicrobial agent use is skin sensitivity exhibited by some patients. Povidone-iodine (PI) is a widely used iodophor, which causes less irritation
11


than iodine itself [22], [69], PI antiseptics are typically active for 2 hours, but they may become inactive upon contact with blood [57],
CHG-based solutions are active against gram-positive and gram-negative bacteria, yeasts, anaerobes, and some viruses, including human immunodeficiency virus (HIV) [3], [70], However, their activity against fungal microbes is lower than iodine-based and alcohol-based antiseptics [22], Due to the presence of quaternary ammoniums in its structure, chlorhexidine is a cationic compound that disrupts negatively charged bacterial cell wall leading to microbial death [71], Chlorhexidine gluconate exhibits antimicrobial activity for up to 6 hours [57],
Alcohol-based antiseptics are effective against gram-negative bacteria, gram-positive bacteria, fungi, and viruses [22], Alcohols denature proteins in the cell wall of microbes, which results in rapid lysis of the microbes [22], Despite the rapid action of alcohol-based antiseptics, antimicrobial properties of these antiseptics are short lived due to evaporation of alcohol. One approach to improving the longevity of alcohol-based antiseptics is to add zinc pyrithione (ZPT), which serves as a preservative [72],
Table 2 Properties of various types of antiseptics and examples of commercially available products. Table adapted from Reichman et al. 2009. [57]
Antimicrobial Coverage
Antiseptic Mechanism of Action Gram- positive Bacteria Gram- negative Bacteria Fungi Virus Onset Duration Examples
Aqueous- iodophor Oxidation/substitution by free iodine E G G G Moderate 2 h Betadine)
Aqueous-CEIG Disrupts cell membranes E G F G Moderate 6 h Hibiclens)
Alcohol Denatures proteins E E G G Most Rapid None
Alcohol-iodine Denatures proteins povacrylex Oxidation/substitution by free iodine E E ID G Rapid 48 h DuraPrep
Alcohol-CHG Denatures proteins Disrupts cell membranes E E ID G Rapid 48 h ChloraPrep)
CHG, chlorhexidine gluconate; E, excellent; F, fair; G, good; ID, insufficient data;
Betadine Microbicide, Purdue Products L.P, Stamford, CT; ChloraPrep, CareFusion, Inc., Feawood, KS; DuraPrep Surgical Solution, 3M Elealth Care, St. Paul, MN; Ehbiclens, Molnlycke Elealth Care Inc., Norcross, GA.
12


Preoperative Surgical Hand Scrub
To reduce risk of microbial contamination from the surgical team, individuals who have contact with anything in the sterile field must scrub their hands and forearms with an antiseptic [3], [57], [58], This is performed immediately before donning of sterile gloves and gowns. The three types of antiseptics used for skin preparation may also be used for scrubbing [3], [57], In addition to these, para-chloro-meta-xylenol-based (PCMX) and triclosan-based antiseptics may be used [3], Scrub duration varies based on the antiseptic used. Scrubbing for 3 to 5 minutes is typically required for CHG-based and Pi-based antiseptics, however, a study found hand-rubbing with an alcohol antiseptic for 1 minute to be equally effective [57], [73],
Intraoperative Skin Antisepsis: Surgical Incision Drapes
While preoperative skin preparation reduces the number of microbes on the skin, it is not possible to sterilize the skin. During surgery, bacteria residing deeper in the skin may recolonize the skin [74], The purpose of intraoperative skin antisepsis is to avoid infection by sequestering any remaining microbes on the skin to prevent their entry into the incision site [75], [76], Surgical incision drapes (SIDs) have been used for over 50 years to impede microbial recolonization and prevent contamination in the surgical incision site [77], [78], The first commercially available SIDs were simple plastic adhesive sheets, but over time SIDs have been impregnated with antimicrobials, such as iodophor, to increase their effectiveness against SSIs [79], [80], A microbial sealant has also been developed as an alternative to traditional SIDs [81], [82], IOBAN, Tiburon, and InteguSeal are iodine common SIDs manufactured by 3M, Cardinal Health, and Kimberly-Clark, respectively. IOBAN is an iodine-impregnated drape, while Tiburon is three-layer adhesive drape
13


with no antimicrobial agent. InteguSeal is a microbial sealant, which will be discussed later in this report. Even though iodine-impregnated drapes have been demonstrated to reduce bacterial contamination, a review of seven studies found that there is no evidence that SSI rates are reduced with the use of plastic adhesive SIDs [60], [80], In fact, the review found that in some cases use of plastic adhesive SIDs increases SSI rates [60],
There are numerous drawbacks to the use of current adhesive SIDs. First, the placement of SIDs is time consuming and requires good technique [77], Improper placement may result in wrinkles and air bubbles, which would allow bacteria to recolonize these areas of the skin. SIDs typically come in specific sizes and shapes, and in some cases they may not conform well to a patients shape, which may result in drape lift. Loss of adhesion, leading to drape lift, is a common problem with SIDs. A study found that loss of adhesion at the skin edges of the incision site resulted in a 6-fold increase in SSI rates [83], It has also been found that the type of skin preparation used affects the adhesion of SIDs [84],
Another problem is that the use of non-antimicrobial SIDs has been found to reduce the amount of time required for bacterial recolonization of the skin compared with no drape at all [76], [85], This indicated that SIDs create a warm moist environment at the skin surface, which is favorable to bacterial proliferation. For this reason, it is important that SIDs contain antimicrobial agents. A drawback of iodine-impregnated drapes is the existence of patient allergies to iodine. Another disadvantage is that antimicrobial SIDs typically only contain a certain amount of leachable antimicrobials, which limits the amount of time that they exhibit antimicrobial activity. In the case of iodine-impregnated drapes, all iodine is released after about 6 hours [86],
14


There are several other drawbacks of SIDs associated with incision closure and drape removal. First, in most surgical procedures the drape is peeled back from the incision before the incision site is closed with sutures. Lifting of the incision drape during closure has been found to cause bacterial contamination of the surgical site [87], If SIDs are peeled back for wound closure, it has recently been recommended that skin preparation be reapplied to the exposed area [87], Additionally, when SIDs are removed, a layer of epidermal cells adheres to the drape, and thus is removed as well (Figure 5). This exposes bacteria found in deeper layers of the skin. If the skin of the patient is fragile, such as in elderly patients, skin avulsion injuries may occur where large portions of epidermal cells are removed upon SID removal [88] (Figure 5). Once SIDs have been removed, bacteria are free to recolonize the areas around and incision.
Figure 5 A: Epidermal cell removal during SID lift off, which exposes bacteria found beneath the skin. B: Surgical incision drape after being peeled back in preparation for would closure. C: Skin avulsion injury from the SID removal on an elderly patient. [88][90]
15


Study Objective
Despite efforts to reduce SSI occurrences, rates remain unchanged. Due to drawbacks of current SIDs, including time consuming placement, loss of adhesion, and loss of an epidermal cell layer during drape removal, an antimicrobial reverse-thermal gel was developed for potential use as a SID. The main objective of this study is to evaluate a previously reported quaternized-polyethyleneimine-poly(N-isopropylacrylamide) (Q-PEI-PNIPAAm or QPP) copolymer [91], The described Q-PEI-PNIPAAm was designed as a broad spectrum antimicrobial non-leaching polymer that would have minimal toxicity and be easy to apply and remove as a surgical drape. Q-PEI-PNIPAAm has been characterized, demonstrated to have antimicrobial activity, and assessed for cell cytotoxicity [91], The aim of this study was to evaluate the performance of Q-PEI-PNIPAAm used as a surgical incision drape in a mouse skin incision surgery model.
16


CHAPTER II
BACKGROUND
Antimicrobials and Antimicrobial Resistance
Microbial contamination is of great concern in a number of industries, including food packaging, household sanitization, water treatment, and the medical industry [92], [93], The emergence of drug-resistant bacteria and the prevalence of healthcare-associated infections have further prompted the growth and development of the antimicrobial industry. Major classes of antimicrobial agents include disinfectants, antiseptics, and antibiotics, all of which typically use low molecular weight antimicrobial agents in their formulations. Hypochlorites, hydrogen peroxides, silver salts, alcohols, triclosans, and quaternary ammonium compounds (QACs) are common antimicrobial agents used in disinfectants [94], Unfortunately, disinfectants negatively impact the environment, and the use of antimicrobials contributes to the development of antimicrobial resistance, which presents a challenge to the treatment of microbial infections [94][97]. Another disadvantage of antimicrobial agents with low molecular weight is their short-term antimicrobial activity [93],
Antimicrobial resistance occurs when a microbe remains unaffected by an antimicrobial agent at a concentration that should be lethal [95], Some microbes have an inherent resistance to particular antimicrobials, but resistance can also be acquired through mutations or direct transfer of resistant genes by conjugation [95], [98], When antimicrobials are used, susceptible microbes are killed, but resistant strains may survive and proliferate further propagating the existence of populations of resistant strains. There are four main mechanisms by which bacteria become resistant: elimination of antimicrobials from the cell, alteration of antimicrobial targets, direct inactivation of the antimicrobial agent, and
17


alterations in metabolic pathways (Figure 6). Elimination of antimicrobials from the cell can occur through efflux pumps or decreased uptake of the antimicrobial. Understanding how bacteria acquire antimicrobial resistance is important for the development of new antimicrobials that minimize the risk of bacterial resistance. [98]


(lb) C# Decreased uptake
n
CZ' Target alterations
A X > B *
o
Alternative enzyme 4
Figure 6 Mechanisms of antibacterial resistance, la: Elimination of cellular antimicrobials using efflux pumps, lb: Elimination of cellular antimicrobials by decreasing/blocking cellular uptake. 2: Alteration of antimicrobial target. 3: Direct inactivation of antimicrobials by cellular enzymes. 4: Alteration of metabolic pathways. [99]
Mechanisms of Antimicrobial Activity
In general, antimicrobial agents inhibit cell wall synthesis, protein synthesis, or nucleic acid synthesis or they target the microbe cell membranes/wall (Figure 7) [98], Antibiotics, a class of antimicrobial agents, typically target a specific cellular process [100], To do this, most antibiotics pass through the bacterial cell envelope and impede biochemical pathways without affecting the structure of the cell envelope [101], [102], Mechanisms of antibacterial resistance allow bacteria to acquired resistance to antimicrobials that target specific intracellular processes. However, it is more difficult for bacteria to acquire resistance to
18


antimicrobials that disrupt the cell envelope. Resistance to disruption of the cell wall would
require the modification of many proteins to achieve complete alteration of the cell wall and membrane structure.
Spore core
Formaldehyde
Glutaraldehyde
Hydrogen peroxide
Spore coats Alkali f Bacterial Vegetative
If / spore bacterium Vgaf
Leakage of intracellular components
Chlorhexidine QAC's
Cytoplasm
Chlorhexidine
Copper (II) salts CRA's
Gluteraldehyde
. Hexachlorophene Hydrogen peroxide Mercury (II) salts
Organic mercurials
Phenols
QAC s
Silver salts
Cell wall
CRAs
Formaldehyde
. Mercury (II) salts
Organic mercurials
Phenols
Envelope
. Membrane-active agents, e.g. Alcohols, QAC's, chlorhexidine
Virus
Nucleic acid
* Peracetic acid
GRAs
Thiol groups
. Bronopol
Copper (I I) salts Ethylene oxide
Gluteraldehyde
. Hydrogen peroxide
CRA s
Iodine
. Mercuric (II) salts Organic mercurials Silver salts p -Propiolactone
Fungus
Capsid
Alcohols CRAs
Gluteraldehyde
Iodine
Peracetic acid
Phenols QACs
Amino groups
CRA s
Ethylene oxide Formaldehyde
Gluteraldehyde
. p -Propiolactone
Sulfur dioxides
Sulfites
Ribosomes
. Hydrogen peroxide Iodine
Plasma membrane
. Alcohols Chlorhexidine Esters Organic acids QAC s
Figure 7 Antimicrobial mechanisms of various antimicrobial agents against bacterial spores, bacteria, viruses, and fungi. QACs and CRAs are quaternary ammonium compounds and chlorine-releasing agents, respectively. [103]
To combat increasing antimicrobial resistance, new strategies for the development of
antimicrobials have focused on targeting common bacterial characteristics that are less likely
to contribute to the development of bacterial resistance. One such common characteristic is
the existence of negatively charged cell walls in all bacteria. There are two general types of
19


bacteria, and they are characterized by different cell wall structures: gram-positive and gramnegative bacteria (Figure 8). Gram-positive bacteria have one inner cell membrane and an outer cell wall. The outer wall contained teichoic and lipiteichoic acid, which are negatively charged. In contrast, gram-negative bacteria have an outer and inner cell membrane surrounding a peptidoglycan wall. Phospholipids and lipopolysaccharides of the outer membrane give gram-negative bacteria their negatively charged cell envelope [100],
a Gram-negative bacteria
b Gram-positive bacteria
Outer
membrane
Periplasmic
space
Periplasmic
space
Cell
membrane
Figure 8 Structure of gram-negative and gram-positive bacteria. Both types of bacteria contain negatively charged structures on their surface. Gram-negative bacteria have negatively charged phospholipids and lipopolysaccharides on their surface. Gram-positive bacteria have negatively charged teichoic and lipoteichoic acids on their surface. [104]
The common negative bacterial cell wall charge has led to the study and development of polycationic antimicrobials. Polycationic antimicrobials are able to disrupt the cell envelope of gram-positive and gram-negative bacterial cells as well as the membrane of fungal cells with attractive electrostatic forces [105], It should be noted that mammalian cell membranes have less negative surface charge than bacteria, so cationic antimicrobials selectively target bacteria cells over human cells [106], The surface of mammalian cells primarily contains sphingomyelin (SM) and phosphatidylcholine (PC), which both have no net charge [107], This makes polycations ideal for use as antimicrobials as they will target bacteria and fungi without harming mammalian cells.
20


Antimicrobial Polymers
The development of polymeric antimicrobials has gained great interest in the last couple of decades [93], [97], [100], Antimicrobial polymers have several advantages over conventional antimicrobial agents. First, they have been demonstrated to contain long-term activity and limited toxicity. Polymeric antimicrobials are also chemically stable, nonvolatile, and they do not pass through the skin [93], There are three general types of antimicrobial polymers: biocidal polymer, polymeric biocide, and biocide-releasing polymer. Biocidal polymers have intrinsic antimicrobial activity, while polymeric biocides contain a polymer backbone with attached biocides. These two types of antimicrobial polymers are nonleaching systems. Biocide-releasing polymers are systems in which biocidal agents are trapped within a polymeric matrix to be released at some time [105], These leaching antimicrobial polymers are necessary when a biocide must enter a microbial cell to exhibit antimicrobial activity. Leaching antimicrobial polymers have a limited lifespan, while nonleaching polymers with inherent antimicrobial activity exhibit long-term antimicrobial activity [97], This review will focus on biocidal polymers with inherent antimicrobial activity.
In general, ideal antimicrobial polymers should be active against a broad spectrum of microbes, have long lasting antimicrobial activity, not be toxic or irritating mammalian cells, be synthesized easily, and be associated with minimal antimicrobial resistance [93], Antimicrobial polymers are typically based on polycations, which meet these listed requirements [97], [105], Polycationic antimicrobial polymers cause microbial death by adsorption and penetration of the bacterial cell surface and wall, adsorption onto the bacterial cell membrane, disturbance of the cell membrane, leakage of cytoplasmic contents,
21


degradation of nucleic acids and cytoplasmic proteins, and lysis of the bacterial cell wall [93], [108], Commonly studied cationic antimicrobial polymers include quaternary ammonium compounds (QACs), antimicrobial peptides (AMPs), chitosan, polyguanidines, N-halamines, and phosphonium and sulfonium containing antimicrobials.
Quaternary Ammonium Compounds
Quaternary ammonium compounds (QACs) are the most frequently researched type of antimicrobial polymers [109], Use of polymers containing QACs is advantageous owing to their chemical stability and reduced toxicity to the environment [110], They are organic cationic compound having a general structure of NR4+, where N is nitrogen and R represents either an alkyl or an aryl group. QACs are commonly used in clinical disinfectants, domestic cleaning products, fabric softeners, and some products requiring antielectrostatic agents, such as shampoo [108], [111]. QACs may be synthesized by a quaternization reaction. Quaternization reactions involve an irreversible reaction between tertiary amines and alkyl halides (Figure 9) [108],
Rt
R2------N: + R4---------X
R3 Alkyl
Halide
Tertiary
Amine

Rt
R2 n r4 + x
R3 Halide
Anion
Quaternary
Ammonium
Cation
Figure 9 Quaternization of tertiary amine with an alkyl halide yielding a quaternary ammonium cation. In this chemical equation, R represents alkyl or aryl groups, and X represents a halogen, such as CT, Br", FT, or I".
22


The antimicrobial activity of QACs depends on the composition of the four organic R groups and the number of cationic nitrogen atoms [97], Inclusion of at least one long alkyl chain as an R group provides a hydrophobic structure for the penetration of the bacterial cell envelope [110], This increases the antimicrobial activity of the polymer, and creates an amphiphilic QAC, which has the ability to attract bacteria with its cationic charge and penetrate the bacterial envelope with its hydrophobic structures. When the hydrophobic tail of a QAC penetrates into the hydrophobic core of a bacterial membrane, leakage of cell content occurs resulting in cell lysis [108],
Polyethyleneimine
Polyethyleneimine (PEI) is a cationic, nonbiodegradable, synthetic polymer, which has primary, secondary, and tertiary amines within its structure. Due to the large quantity of reactive amines in its structure, PEI has been studied for use as an antimicrobial polymer. Particular attention has been paid to branched PEI, which has the capacity for high charge density [97], Lin et al. covalently attached PEI to a glass surface, and found that PEI alone was not antibacterial. However, the study also found that alkylated PEIs conjugated to a glass surface exhibited bactericidal activity against both gram-positive and gram-negative bacteria
[112] , Kiss et al. also found that quaternized PEIs have significant antimicrobial activity
[113] , Park et. al developed a N-alkyl-PEI paint, which killed gram-positive S. aureus and gram-negative E. coli by rupturing the bacterial envelope upon contact [114], Alkyl PEI was also found to exhibit antifungal activity [115], Another study found that N-alkyl-PEI was both harmless to mammalian cells and no apparent bacterial resistance was formed in successive generations of E. coli and S. aureus [116], The alkylation of PEIs increased its hydrophobicity and cationic charge density by generating quaternary amines. This suggests
23


that PEI alone does not contain antimicrobial activity due to inadequate levels of hydrophobicity and/or cationic charge [112], This is supported by a study by Ilker et al. where a series of nonamphiphilic cationic polymers and amphiphilic cationic polymers were synthesized. In comparing the two types of polymers nonamphiphilic cationic polymers, which have no hydrophobic group, exhibited significantly less antibacterial activity [117], Thus antimicrobial activity of antimicrobial polymers is dependent on the presence of both cationic charge and hydrophobic groups.
Effect of Alkyl Chain and Molecular Weight on Antimicrobial Activity
The antimicrobial activity of a particular polymer depends on a number of factories including alkyl chain length, molecular weight, ratio of alkyl to cationic groups, charge density, and counter ions [92], [93], [118], This review will address two of these factors: alkyl chain length and molecular weight (MW).
As previously mentioned, the antimicrobial activity of QACs requires electrostatic forces between the antimicrobial and the bacterial cell surface followed by penetration of the bacteria envelope by a hydrophobic alkyl tail. The length of the hydrophobic tail, which penetrates the bacterial envelope, is important to the antimicrobial activity of the polymer. Generally, QACs with 8-18 carbon alkyl chains possess good antimicrobial activity [92], However, gram-positive bacteria are more susceptible to antimicrobial penetration due to loose packing of the peptidoglycan layer, and gram-negative are generally less sensitive due to the presence of an additional outer membrane [97], [119], Gilbert et al. found that polymers with alkyl chain lengths of 12-14 carbons provide optimum antibacterial activity against gram-positive bacteria, while alkyl groups with 14-16 carbons have better activity against gram-negative bacteria [120],
24


Lin et al. investigated the effect of alkyl chain length on the antimicrobial activity of alkylated PEI. PEI was covalently attached to a glass surface and alkylated with chains ranging from 2-18 carbons in length. Additionally, quaternized versions of these polymers were also synthesized in a reaction with iodomethane. As the alkyl chain length increased from 2 to 6 carbons, so did the antimicrobial activity of both the methylated and unmethylated polymer. However, when the alkyl chain length was increased above 6 carbons to a maximum of 18 carbon alkyl chains, the antibacterial activity plateaued for methylated-alkylated-PEI, which contained greater quantities of quaternary ammoniums, and decreased for alkylated-PEI [112], Alkylation with longer chains increases the hydrophobicity and penetrative ability of the polymer, but also decreases the accessibility of cationic amine groups, therefore a balance of charge and alkyl chain length is required for optimum antimicrobial activity. Longer alkyl chains may also result in aggregate clumps of hydrophobic tails, which would decrease the bactericidal activity.
Molecular weight also plays a role in determining the antimicrobial activity of polymers. The optimal range of molecular weights for antimicrobial activity is specific to the polymeric system, but general molecular weight ranges have been noted. Ikeda et al. examined the bactericidal activity of polymethacrylates and poly(vinylbenzyl ammonium chlorides) with various molecular weights against gram-positive S. aureus and they reported an optimal molecular weight range of 5xl04 tolxlO5 Da [121], Lin et al. functionalized textiles with N-hexylated and methylated PEI to examine its antibacterial and antifungal properties. This study also used PEI with molecular weights of 0.8, 2, 25, and 750 kDa to examine the influence of molecular weight on antimicrobial activity. It was found that antimicrobial activity against S. aureus increased with increasing molecular weight, with 750
25


kDa having the highest bacterial activity. It should be noted the alkylated polymers with low molecular weight PEI did contain some antimicrobial activity [115], With regard to molecular weight, high molecular weight alkylated PEIs appear to have better antimicrobial activity.
Biocompatibilitv of Quaternary Ammonium Compounds
As mentioned previously, polycationic polymers, such as QACs, selectively target bacteria cells over human cells due to the negative cell surface charge of bacteria [106], In contrast to bacteria cell envelopes, mammalian cell surfaces are composed primarily of components with no net charge [107], As a result, it is thought that QACs do not penetrate mammalian cells due to reduced attractive electrostatic forces when compared with electrostatic forces between QACs and bacterial cell envelopes. These attractive electrostatic forces are essential to the insertion of the hydrophobic tail into the cell. However, despite the fact that QACs should not directly penetrate mammalian cells, the presence of QACs may alter the cell microenvironment, which in turn may negatively impact the cell. As an example, one way that QACs may alter the microenvironment is through increasing the pH, which will affect both bacterial and mammalian cells.
A study by Peng et al. examined the cytotoxicity of a polymer, hydroxypropyltrimethyl ammonium chloride chitosan (HACC), with various degrees of substitution with quaternary ammoniums. This study found that HACC with 6% and 18% quaternary ammonium substitution did not reduce cell enzymatic activity, meaning they were not cytotoxic. In contrast, HACC with 44% substitution was cytotoxic. The trends of this study may demonstrate a positive correlation between the degree of quatemization and cytotoxicity.
This study also found that increasing quatemization leads to higher antimicrobial activity
26


[122], Therefore, an optimal polymer would be a balance of maximizing antimicrobial activity while minimizing cytotoxicity.
A second study by Lu et al. examined the skin irradiation and acute toxicity of dimethylaminoethyl methacrylate (DMAEMA) quaternized with benzyl chloride (BC). To examine skin irritation, 4 rabbits were inoculated with a DMAEMA-BC 5 times a day for 7 days, and no irritation was observed during the test. To examine the acute toxicity of DMAEMA-BC, 40 rats ingested 4 different doses of the polymer and it was found that the two lower doses did not visibly effect the rats, while rats in the two higher dose groups gradually died. Therefore, this study concluded that DMAEMA-BC was not a skin irritant, and not acutely toxic at lower doses [123], This study indicated that while QACs are not irritants on the skin surface, however, they may be acutely toxic at higher dosages. However, it should be noted that the dosage at which a QAC may be acutely toxic will vary based on the exact polymer composition, and typically the amount of polymer required to kill bacteria effectively exhibits low toxicity [123], [124],
Reverse-Thermal Gels
Increased bacterial contamination caused by improper placement is of concern when using traditional SIDs such as Ioban. Placement of traditional SIDs may be difficult if the drape does not conform well to the shape of the skin, and air bubbles and wrinkles may lead to bacterial recolonization under the drape. As such, there is a need for an effective antimicrobial system for intraoperative skin asepsis that is easy to apply and has minimal chance of misapplication.
Kimberly-Clark developed a cyanoacrylate microbial sealant named InteguSeal Microbial Sealant, which is an alternative to current SIDs. This microbial sealant is applied
27


to the skin as a liquid following skin preparation and prior to surgery. Upon application, exposure to moisture and proteins on the patients skin causes the cyanoacrylate to polymerize and create an adhesive barrier on the epidermal surface [125], [126], Similar to current SIDs, this adhesive barrier is intended to sequester bacteria on the surface of the epidermis [82], [127], [128], Although waterproof, the sealant is breathable, which prevents the development of a warm moist environment under the sealant [126], [129], It is important to note that InteguSeal itself is not antimicrobial [127], Several studies examined the efficacy of InteguSeal compared with the use of no intraoperative asepsis. Dromzee et al. found no evidence that InteguSeal reduces SSI rates, and Falk-Brynhildsen et al. found no difference in intraoperative bacterial growth with the use of InteguSeal [128], [130], However, Dohmen et al. reported reduced occurrence of SSIs with the use of InteguSeal in 676 CABG procedures [126], A review of seven studies involving cyanoacrylate microbial sealants found insufficient evidence that InteguSeal reduces SSI rates [131], Although InteguSeal is easy to apply, a polymer that acts as both a sealant and an antimicrobial agent may provide a better system.
Smart polymers are stimuli-responsive polymers, which change in response to external stimuli, such as temperature, light, or pH. Reverse thermal gels (RTGs) are stimuli responsive polymers that gel when heated above a particular temperature. RTGs are frequently used in the biomedical industry. Poly(N-isopropylacrylamide) (PNIPAAm) is one of the most frequently researched RTGs owing to its sudden gelation near body temperature. PNIPAAm dispersed in water undergoes a hydrophilic to hydrophobic phase transition resulting in gel formation at the lower critical solution temperature (LCST) (Figure 10). The LCST of PNIPAAm typically occurs at 32 C [132], [133],
28


Figure 10 A: Behavior of PNIPAAm chains below (left side) and above (right side) LCST. B: PNIPAAm chemical structure with labeled hydrophilic and hydrophobic regions.
Dincer et al. investigated NIPAAm-PEI block copolymers for use as a gene delivery vector. Addition of PEI to PNIPAAm was found to increase the LCST from approximately 29 C for PNIPAAm to 36-40 C, which is close to body temperature. The increase in LCST can be attributed to the addition of hydrophilic PEI chains. Results of this study also indicated that PNIPAAm-PEI had increased solubility in water when low molecular weight PNIPAAm was used [134],
In recent years, interest in antimicrobial reverse thermal gels has grown [132], [133], [135], [136], Dizman et al. developed aNIPAAM and quaternized methacrylamide derivative polymer, which contained antimicrobial and thermos-responsive characteristics [132], Mattheis et al. also developed an antibacterial RTG coating composed of a copolymer of NIPAAm and 2-aminnoethyl methacrylate [135], Most recently Yu at al. and Pappas et al. investigated p-phenylene ethynylene/PNIPAAm derived films for use as self-sterilizing surfaces [133], [136],
29


CHAPTER III
PREVIOUS WORK
Q-PEI-PNIPAAm has previously been designed and studied as a potential surgical incision drape. A synthesis method was established and variations of Q-PEI-PNIPAAm were characterized. Additionally, antimicrobial testing, and cytotoxicity assays were completed for Q-PEI-PNIPAAm [91],
Q-PEI-PNIPAAm LCST
LCST was determined for 20, 30, and 50% conjugations of PEI-PNIPAAm and Q-PEI-PNIPAAm with LCST being defined as the temperature where 50% reduction in UV transmittance occurs (Table 3). It was found that the addition of PEI increased the LCST, and alkylation and quaternization decreased the LCST. This can be explained by the chemical structure of both additions. Hydrophilic interactions dominate below LCST, so the addition of hydrophilic PEI increases the low temperature range where hydrogen bonding dominates, thus increasing the LCST. Likewise, hydrophobic alkyl chains added during quaternization extend the high temperature range at which the polymer will be a gel, thus decreasing the LCST. [91]
Table 3 LCST values reported for various conjugations of PEI-PNIPAAm and Q-PEI-PNIPAAm.
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
Antimicrobial Activity of Q-PEI-PNIPAAm
In vitro bacterial activity of Q-PEI-PNIPAAm was assessed using time-kill curves against four species of bacteria: S. aureus, MRS A, Staph, epidermidis, and if coli. Activity was assessed against stationary phase (non-multiplying) bacteria as bacteria rarely encounter
30


conditions that support log phase growth. For this test, Q-PEI-PNIPAAm with 30% conjugation of PEI amines with PNIPAAm was dissolved in a bacterial solution with a known concentration, and aliquots were taken at 30-minute time intervals for a duration of 2 hours. Q-PEI-PNIPAAm was found to achieve an 8-log MRSA (S. aureus subsp. aureus Mu3) reduction in 60 minutes, a 7- log Staph, epidermidis reduction in 30 minutes, an 8-log E. coli reduction in 120 minutes, and a 5-log S. aureus reduction in 30 minutes (Figure 11). PEI-PNIPAAm was also found to have some antibacterial activity against S. aureus, Staph, epidermis, and E. coli. It should be noted that since the polymer samples were placed directly in solution, the bacteria was surrounded with antimicrobial polymer in three-dimensions. However, only the surface of SIDs contacts bacteria during surgery, therefore, it is necessary
to assess the antimicrobial activity of a two-dimensional layer of Q-PEI-PNIPAAm.
A
C
Bacteria Sample -PEI-PNIPAAm (30%)
Time (minutes)
-Bacteria Sample -^PEI-PNIPAAm (30%)
-Q-PEI-PNIPAAm (30%) "-PNIPAAm
Time (minutes)
B -Bacteria Sample -^PEI-PNIPAAm (30%)
D
Bacteria Sample -"-PEI-PNIPAAm -^Q-PEI-PNIPAAm
7
Ei 6
0 ----
0 30 60 120
Time (minutes)
Figure 11 PEI-PNIPAAm and Q-PEI-PNIPAAm were added to stationary-phase bacteria, and samples were taken at 0, 30, 60, and 120 minutes to determine bacterial concentrations.
Kill-curves were constructed for the following bacteria: A: S. aureus subsp. aureus Mu3 (MRSA hetero-VISA) B: Staph, epidermidis C: E. coli D: S. aureus. [91 ]
31


Cytotoxicity
Cytotoxicity was assessed using fibroblasts. Films of Q-PEI-PNIPAAm (20% conjugation), PEI-PNIPAAm (20% conjugation), chlorhexidine 2%, plain media, and media containing 5% DMSO were coated with fibroblasts. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay then was used to determine cell viability. No statistical difference was found between Q-PEI-PNIPAAm (20% conjugation) and chlorhexidine 2%, which is used as a surgical skin preparation (Figure 12).
QPP (20% PEI-PNIPAAm PNIPAAm Chlorhexadine
Conjugation) (20% Conjugation)
Samples
Figure 12 MTT assay assessing the cytotoxicity of Q-PEI-PNIPAAm (QPP). No statistical difference between the negative control and cells plated on PNIPAAm and PEI-PNIPAAm was observed. No statistical significance was found between cells plated on QPP and Chlorhexidine. Statistical significance was found between experimental groups and the positive control (** denotes p < 0.05 and *** denotes p > 0.05). [91]
32


CHAPTER IV
HYPOTHESIS AND SPECIFIC AIMS Hypothesis
Based on previous research indicating that Q-PEI-PNIPAAm has antimicrobial activity and the ability to gel near body temperature, it was hypothesized that a two-dimensional Q-PEI-PNIPAAm surface would have antimicrobial activity and its effectiveness as a surgical drape would be comparable to commonly used SIDs, such as IOBAN.
Specific Aims
The first specific aim was to synthesize and characterize the Q-PEI-PNIPAAm formulation chosen for this study. It was particularly important to confirm conjugation of PEI and quatemization of the polymer as these steps give the polymer its antimicrobial properties. The second specific aim was to evaluate the antimicrobial killing capacity of the surface of Q-PEI-PNIPAAm against five relevant bacterial strains: Corynebacterium amycolatum, Escherichia coli, Staphylococcus aureus, MRSA, and Staphylococcus epidermidis. Evaluation included the development of time-kill curves for different weight percent solutions of Q-PEI-PNIPAAm coated on a glass surface, and an in vitro simulation of the polymers activity as a surgical drape. The third specific aim was to conduct an animal model to assess the performance of Q-PEI-PNIPAAm as a surgical drape and to determine if the polymer instigates an immune response. A murine incision model was completed to compare the effectiveness of Q-PEI-PNIPAAm and an IOBAN drape in killing and trapping different types of bacteria. Subcutaneous injections of Q-PEI-PNIPAAm were completed to examine the immune response prompted by the polymer. Tissue surrounding the injection site was harvested, cryosectioned, and stained for the presence of macrophages.
33


CHAPTER V
MATERIALS AND METHODS Materials
N-isopropylacrylamide (NIPAAm) was purchased from Tokyo Chemical Industry (Chuo-ku, Tokyo, Japan). N,N-dimethylformamide (DMF) was purchased from EMD Millipore (Billerica, MA, USA). 4,4'-azobis(4-cyanovaleric acid) (ACA), N-hydroxysuccinimide (NHS), sodium bicarbonate, 1-bromohexane, TWEEN 20, Triton X-100, y-globulins from bovine blood, and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, MO, USA). N-(3-dimethylamino-propyl)-N-ethylcarbodiimide hydrochloride (EDC), polyethyleneimine (PEI) molecular weight (MW) 10,000, anhydrous methanol, iodomethane, agar powder, lysogeny broth (LB) (Millers modification), and chloroform-d were purchased from Alfa Aesar (Ward Hill, MA, USA). Anhydrous diethyl ether was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ioban 2 was purchased from 3M (St. Paul, MN, USA). Ketoprofen, saline, and isoflurane were purchased from MWI Veterinary Supply (Boise, ID, USA). Coated Vicryl 4-0 sutures were purchased from Ethicon (Somerville, NJ, USA). Adult C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). 10 % formalin was purchased from JT Baker (Phillipsburg, NJ, USA). Sucrose (RNASE & DNASE free) was purchased from VWR Life Science (Radnor, PA, USA). Optimal cutting temperature (OCT) compound was purchased from Sakura (Torrance, CA, USA). Phosphate buffered saline (PBS) was purchased from HyClone Laboratories, Inc. (South Logan, Utah, USA). Anti-CD68 antibody (ab!25212) was purchased from Abeam (Cambridge, UK). Alexa Fluor 594 (goat anti-rabbit
34


IgG) was purchased from Life Technologies (Carlsbad, CA, USA). Dapi flouromount-G was purchased from Electron Microscope Sciences (Hartfield, PA, USA).
Polymer Synthesis
PNIPAAm Synthesis
PNIPAAm was conjugated using radical polymerization with an azobis initiator. NIPAAm (5.0 g, 44.2 mmol) and ACA (0.186 g, 0.664 mmol) were dissolved in anhydrous methanol (25 mL), and the mixture was bubbled with nitrogen gas for 30 minutes at room temperature. A reflux condenser apparatus was set up, and the solution was stirred for three hours at 68C. Next, the solution was precipitated into Milli-Q water at 60C in a dropwise manner. Following precipitation, the warm water was discarded and 40 mL of cold Milli-Q water was added to the precipitate. The precipitate was allowed to dissolve into the water in a cold room at 4C. The resulting solution was purified using 3500 kDa MWCO dialysis tubing, which was placed in Milli-Q water with stirring for 48 hours. The solution was dried utilizing lyophilization, which yielded purified PNIPAAm.
PEI-PNIPAAm Synthesis
PEI-PNIPAAm was synthesized using EDC/NHS chemistry. In preparation for completing this reaction under anhydrous conditions, PEI (500 mg) was lyophilized. After 24 hours of PEI lyophilization, PNIPAAm (700 mg) was dissolved in anhydrous DMF (5 mL).
A 1.2 molar excess of EDC (0.0161 g) and NHS (0.0097 g) to PNIPAAm carboxylic acid groups was then added to the mixture. The mixture was stirred in a dark environment at room temperature for 24 hours under nitrogen (N2). After 48 hours of lyophilization, PEI was dissolved in anhydrous DMF (5 mL) with stirring. The activated PNIPAAm was then aspirated into a syringe and slowly added to the PEEDMF solution. The reaction was left for
35


an additional 24 hours with stirring at room temperature. A rotary evaporator was then used to remove some of the DMF. Subsequently, the one half of the reaction solution was precipitated in cold ethyl ether to remove unused reactants and any remaining DMF. Excess ethyl ether was poured off, and this process was repeated for the remaining reaction solution. Following precipitation, a rotary evaporator was once again used to remove excess ethyl ether. Milli-Q water (10 mL) was added to the product, and the flask was placed in a cold room with a stir bar to allow the product to dissolve. After the polymer was dissolved, the solution was placed in 12-14 kDa dialysis tubing for 48 hours. The purified polymer was frozen and lyophilized to yield the final PEI-PNIPAAm product.
O-PEI-PNIPAAm Synthesis
PEI-PNIPAAm primary amines were converted to quaternary ammoniums by quaternization. In this process first primary amines were alkylated with 1-bromohexane, which converted primary amines to tertiary amines. Then, an alkyl halide was added to convert tertiary amines to quaternary ammonium. Molar excess calculations were based on the number of primary amines in PEI-PNIPAAm. Sodium bicarbonate (0.066 g, 3 molar excess) was added to anhydrous DMF (8 mL) with stirring. PEI-PNIPAAm (100 mg) was added then to the solution and allowed to dissolve. A 20 molar excess of 1-bromohexane (0.7386 mL) was added and the mixture was reacted at 95 C with stirring and with a cold water reflux condenser apparatus. After 48 hours, the temperature was reduced to 60 C and 20 molar excess iodomethane (0.3287 mL) was added to the reaction. The mixture was allowed to react for an additional 12 hours followed by cooling to room temperature. Subsequently, the solution was rotary evaporated to remove as much DMF as possible. A series of three precipitations in cold diethyl ether were used to remove DMF and unreacted
36


polymer. The rotary evaporator was then used to ensure removal of all diethyl ether, and 10 mL of Milli-Q water was added to the product. After the polymer was dissolved in water, the solution was added to 12-14 kDa MWCO dialysis tubing for 48 hours. Lyophilization was used to isolate the final Q-PEI-PNIPAAm product.
Polymer Characterization Proton Nuclear Magnetic Resonance QH NMR)
Proton nuclear magnetic resonance (*H NMR) was completed with a Varian Inova 500 mHz NMR Spectrometer. PEI, PNIPAAm, PEI-PNIPAAm, and QPP samples (3-5 mg) to be analyzed were dissolved in 600 pL of chloroform-d. Spectra were processed and analyzed using iNMR reader software. Online Advanced Chemistry Development i-Lab was used to predict *H NMR spectra peaks associated with each polymer at 5 MHz.
LCST Determination
The LCST was measured to assess the gelling properties of PNIPAAm, PEI-PNIPAAm, and Q-PEI-PNIPAAm. LCST data was acquired using a CARY 100 Bio UV-Visible Spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA). Polymer samples of 5 wt% were prepared by dissolving the polymers in Milli-Q water. Absorbance values were measured at a 500 nm wavelength for temperatures ranging from 25C to 45C.
Bacteria Preparation and Dilutions
Millers LB broth (5 mL) was inoculated with bacteria from a frozen monoclonal stock solution, and incubated at 37 C for 16 hours with shaking at 250 rpm. After 16 hours, the bacterial suspension was centrifuged for 5 minutes at 5,000 rpm and the LB broth was poured off. The bacterial pellet was then resuspended in sterile PBS, and a Genesys 10S UV-VIS (Thermo Scientific, Waltham, MA, USA) was used to measure the absorbance of the solution
37


at 600 nm. The absorbance was used to estimate the bacterial concentration of the suspension, and the solution was then diluted to the desired concentration. To determine the exact bacterial concentration, 20 pL aliquots were taken from the bacterial solution, and 10-fold serial dilutions were performed in triplicate in a 96-well plate. Aliquots (40 pL) from each well were plated on LB agar dishes and allowed to dry. Plates were incubated at 37 C overnight, and colonies were counted. The bacterial concentration was then calculated based on the following equation:
[Bacteria] =
(# of Colonies)x 10# fDllutlons
(2)
Aliquot Volume Antimicrobial Q-PEI-PNIPAAm Surface Tests
Antimicrobial Activity of a 2D O-PEI-PNIPAAm Surface in a 3D Bacterial Suspension Bacteria was proliferated, the concentration was estimated, and dilutions were completed using the previously listed method. Then 5 conical tubes (15 mL) were filled with 3 mL of a 108 CFU/mL bacterial solution. Spray bottles (3 mL) were filled with Q-PEI-PNIPAAm solutions with concentrations of 1.25, 2.5, 5, and 7.5 wt% dissolved in 70% ethanol. Glass slides were cut lengthwise into strips with a 1 cm width. In a 37 C warm room, both sides of 2 x 1 cm areas at the end of four slides were coated with one spray (-100 pL) each of 1.25, 2.5, 5, and 7.5 wt% Q-PEI-PNIPAAm solutions. As a control an additional slide was cleaned with 70% ethanol. Slides were placed in the conical tubes containing the bacterial solutions with the polymer coated side submerged in the solution. Therefore, a two-dimensional (2D) coated Q-PEI-PNIPAAm surface was submerged in a three-dimensional (3D) bacterial suspension. Each tube was then incubated at 37 C for 2 hours with shaking at 250 rpm. To quantify the antimicrobial capacity of the QPP surface, 20 pL aliquots were taken from each vial at 0, 30, 60, and 120 minutes. At each time point 3 aliquots were
38


removed from each tube to complete 10-fold serial dilutions in a 96-well plate. Aliquots from each vial and time point were plated on LB agar to determine the concentration of bacteria over time in each tube according to Equation 2. This experiment was completed for five bacterial strains that have all been associated with SSIs: Corynebacterium amycolatum (ATCC 49368), Escherichia coli C3000 (ATCC 15597), Staphylococcus aureus (ATCC 6538), Staphylococcus aureus subsp. aureus Mu3 (MRSA strain with vacomycin-intermediate resistance), and Staphylococcus epidermidis (Ron Gill Collection). Antimicrobial Activity of a 2D O-PEI-PNIPAAm Surface against 2D Bacterial Samples
C. amycolatum, E. coli, MRSA, S. aureus, and Staph, epidermidis were proliferated and the solution concentration with estimated and determined using the previously listed method. Stock bacterial solutions (0.5 mL) with concentrations of 109 CFU/mL in Lysogeny broth (LB) were made for each bacterial strain. A 200 pL volume of each 109 CFU/mL stock solution was plated in row A of a 96-well plate. Then, 10-fold dilutions were completed up to row G. This resulted in a plate of stock solutions of 109, 108, 107, 106, 105, 104, and 103 CFU/mL concentrations for each of the five bacterial strain. Samples (3 pL each) of all 35 stock solutions were then plated on a single LB agar plate in a grid pattern with 5 rows for each bacterial strain and 7 columns for each initial bacterial concentration. This plate served as a control plate and was incubated at 37C for 48 hours before being assessed for bacterial growth.
To simulate the antimicrobial activity of Q-PEI-PNIPAAm against bacteria on surface of the skin, three additional plates with the 35 sample grid (3 pL each) pattern were made and bacterial samples were allowed to completely dry. The plates were then placed on a 37C hot plate and 1 mL of 5 wt% PNIPAAm, 5 wt% PEI-PNIPAAm, or 5 wt% Q-PEI-PNIPAAm
39


was sprayed on each plate and allowed to gel. The plates were then incubated at 37C and examined for bacterial growth between the polymer and the LB agar after 48 hours.
To simulate antimicrobial activity of Q-PEI-PNIPAAm against bacteria that may fall onto the surface of a drape during surgery, three plates were then placed on a 37C hot plate and 1 mL of 5 wt% PNIPAAm, 5 wt% PEI-PNIPAAm, or 5 wt% Q-PEI-PNIPAAm was sprayed on each plate and allowed to gel. While the gelled plated were still on the hot plate, the 35 sample (3 pL each) grid pattern was plated on top of the gelled polymers. The plates were then incubated at 37C in a humid sealed container and examined for bacterial growth on top of the polymer after 48 hours.
Skin Incision Animal Model
Skin Incision Surgery
The skin incision surgery was approved by the Institutional Animal Care and Use Committee (IACUC). A total of 48 C57BL/6J mice were used in this portion of the study, and 3 mice were used per treatment group (Q-PEI-PNIPAAm/bacteria spray, IOBAN 2 drape/bacteria spray, no drape/bacteria spray, no drape/no bacteria spray). All groups involving bacterial spray were completed for C. amycolatum, E. coli, MRSA, S. aureus, and Staph, epidermidis. Adult C57BL/6J mice ranging from 12 to 24 weeks old were allowed 7 days to acclimate on a 14/10-hour light/dark cycle with access to water and food ad libitum.
Mice were anaesthetized with continuous inhalation of isoflurane and oxygen. Initially, 5% isoflurane in oxygen to induce loss of consciousness followed by reduction to 2% isoflurane in oxygen for the duration of the procedure. Preoperative subcutaneous injections of ketoprofen (5 mg/kg) were administered to minimize pain following the procedure. Fur below the base of the neck and 3 cm down the back of the mouse was removed with clippers.
40


The shaved area was then washed with warm water and disinfected with alcohol swabs. After
skin preparation, 0.5-1 mL of a 108 CFU/mL bacterial solution was sprayed onto the shaved area and allowed to dry. After inoculation of bacteria to simulate extreme operating room conditions, either 5 wt% Q-PEI-PNIPAAm in 70% ethanol (0.5-1 mL), an IOBAN 2 drape, or no drape was applied to the shaved area. As a positive control, a group was also completed with no drape and no bacterial inoculation. Next, a 1 cm skin incision was performed in the prepared area of the back, and the open incision was irrigated with 0.5 mL of sterile saline. Next, the incision was swabbed with cotton, which was used to inoculate an agar plate to assess bacterial presence in the incision site. The incision was then closed with 1-2 continuous sutures, and mice were monitored closely and allowed to recover. The incision was swabbed for bacterial growth again at times 0, 10, 20, 30, 40, 50, 60, 75, 90, 105, and 120 minutes following closure. After the 120 minute swab, mice were euthanized by CO2 inhalation followed by cervical dislocation.
Quantification of Bacterial Swabs
Agar plates with bacteria swabs from the skin incision surgery were placed in a 37C warm room for 24 hours or 48 hours in the case of C. amycolatum to allow bacteria to grow. Following bacterial growth, all LB agar plates were photographed for analysis of bacterial colony presence. Bacterial swabs were quantified by counting visible CFUs on each plate. This was completed using Image J software to process photos and detect particles.
Q-PEI-PNIPAAm Immune Response In Vivo Subcutaneous Injections
The subcutaneous injections were also approved by IACUC. A total of 12 C57BL/6J mice were used in this portion of the study, and 3 mice were used per injection group (5 wt%
41


Q-PEI-PNIPAAm in saline and sterile saline) for time points of 24 hours and 7 days. As with the skin incision study, adult C57BL/6J mice ranging from 12 to 24 weeks old were allowed 7 days to acclimate on a 14/10-hour light/dark cycle with access to water and food ad libitum. Mice were then administered 60 pL of 5 wt% Q-PEI-PNIPAAm in saline or 60 pL of sterile saline in the right dorsal flank. Euthanasia was carried out after 24 hours or 7 days by CO2 inhalation followed by cervical dislocation. After euthanasia, tissue surrounding the injection site was harvested.
Immunohi stochemi stry
After the skin incision procedure, harvested tissue was fixed overnight in 10% formalin. Tissue samples were then washed 3 times with IX PBS for 3 minutes each wash. Washed tissue was placed in 30% sucrose for 48 hours to cryoprotect the sample. Next, tissue was cut crosswise and embedded in OCT compound with the cut edge facing down in the mold. Embedded tissue was frozen at -80C. Tissue samples were then cryosectioned into 5 pm sections, which were placed on glass slides.
The following protocol was used to stain macrophages with CD68 antibody. First, slides with tissue sections were fixed in 10 % formalin for 10 min and washed 3 times for 5 min each in a washing buffer (IX PBS with 0.1% m/v Tween). Next, tissue sections were permeabilized for 10 minutes with a permeabilizing buffer (IX PBS with 0.5% m/v Triton X-100). Tissue sections were once again washed with the washing buffer 3 times for 5 minutes each followed by blocking with a blocking buffer (0.25% Triton X-100, 2% BSA, and 4% y-globulins in IX PBS) for 60 minutes to prevent non-specific binding. Next, tissue sections were stained with anti-CD68 antibody (1:500 in blocking buffer) overnight at 4C, washed 3 times with the washing buffer for 5 minutes each, and stained with Alexa Fluor 594 (1:500 in
42


blocking buffer) for 60 minutes. Following staining, tissue sections were washed 3 times with washing buffer and 3 times with DI water for 5 minutes each wash. Dapi flouromount-G and glass coverslips were then added to each slide.
Confocal images of stained slides were taken using a Zeiss LSM 780. Four to five images were taken for each mouse. Images where quantified using Zen 2.3 blue edition to select 250 x 250 pixel regions of interest (ROI) for each photo. The number of macrophages in each ROI was then counted and converted to counts per area of tissue.
Statistical Analysis
Results are presented as the mean the standard error of the mean. Statistical significance was determined through computation of the analysis of variance (ANOVA) followed by two-tailed t-tests when applicable. Results were considered statistically significant when p < 0.05.
Sample size estimates were calculated using a power analysis based on the significance level alpha, a target power, a desired sensitivity to detection of the true mean, an effect size, and the variance of the initial study. The first three variables were targeted to be 0.05, 0.8, and 25%, respectively. The effect size and variance were calculated for each sample size estimate.
43


CHAPTER VI
RESULTS AND DISCUSSION
Q-PEI-PNIPAAm Reaction Mechanism
The synthesized Q-PEI-PNIPAAm was designed to maximize antimicrobial activity and gel around body temperature. PNIPAAm gives the polymer reverse-thermal gelling properties and thus the ability to gel close to body temperature. Addition of PEI and quaternization of PEI-PNIPAAm gives the polymer antimicrobial activity due to the addition
of cationic quaternary ammoniums as well as hydrophobic alkyl chains (Figure 13).
NIPAAm
1) EDC 'TvHS Room Temperature. 24 h
2)m x PEI
Room Temperature. 24 h
15 1 brotiiohexane'NHlICO;
95 C, 24 h
2) jodomethane 60 "C.12h
Q-PEI-PNIPAAm
Figure 13 Reaction mechanism for the synthesis of Q-PEI-PNIPAAm.
44


Polymer Characterization
Characterization of PEL PEI-PNIPAAm. and Q-PEI-PNIPAAm using 'H NMR
Synthesis of Q-PEI-PNIPAAm was completed as described in the methods section.
As shown in Figure 13, branched PEI makes up the backbone of the developed antimicrobial reverse-thermal gel. It was important to confirm the conjugation of PNIPAAm to PEI and the quaternization of PEI-PNIPAAm, as both were vital to the desired characteristics of Q-PEI-PNIPAAm. Polymer synthesis was confirmed by NMR (Nuclear Magnetic Resonance) at 500 MHz in chloroform-d (CDCb). First, the structure of purchased branched PEI was confirmed (Figure 14). Peaks associated with primary, secondary, and tertiary amines were present around 2.8, 2.7, and 2.6 ppm, respectively. Peak assignments were supported by literature and NMR predictions from Advanced Chemistry Development [137],
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
ppm
Figure 14 NMR spectrum of branched PEI confirming its molecular structure.
Next, NMR was used to confirm the conjugation of PNIPAAm to PEI. In addition to PEI peaks, peaks at 1.14 and 4.0 ppm supported the presence of NH-CH2-(CH3)2.
45


8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
ppm
Figure 15 ^NMR spectrum of PEI-PNIPAAm confirming the addition of PNIPAAm to PEI.
Finally, ^ NMR was used to confirm the quatemization of PEI-PNIPAAm. Additional peaks observed at 0.9 and 1.3 ppm confirmed the presence of alkyl chains.
k
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
ppm
Figure 16 ^ NMR spectrum of Q-PEI-PNIPAAm confirmed the conjugation of alkyl chains and the presence of a slight peak at 3.6 ppm indicated the presence of quaternary ammoniums.
46


Lower Critical Solution Temperature (LCST)
LCST is the temperature at which a material with reverse-thermal gel properties undergoes a phase transition. At temperatures below the LCST, reverse-thermal polymers in aqueous solutions are dominated by hydrophilic interactions. Above the LCST, hydrophobic interactions dominate, which results in gel formation. The formation of gel alters the light transmittance of the polymer. Therefore, UV-visible spectroscopy can be used to determine the LCST of temperature responsive polymers.
As previously discussed, PNIPAAm has the ability to gel near body temperature. For this study it was important to confirm that Q-PEI-PNIPAAm also possessed this property. Use of Q-PEI-PNIPAAm as a surgical drape requires that the polymer quickly gels on the surface of the skin prior to surgery. The LCST of PNIPAAm, PEI-PNIPAAm, and Q-PEI-PNIPAAm were measures with UV-visible spectroscopy (Figure 17).
120
100
PNIPAAm
B
01 u
C 80 ra
4*
60 E
£ 40 ra
H 20 0
25
30 35 40
Temperature (C)
45
120
100
80
60
40
20
0
PEI-PNIPAAm
25
30 35 40
Temperature (C)
45
Figure 17 LCST of polymer samples. A: PNIPAAm LCST. B: PEI-PNIPAAm LCST. C: Q-PEI-PNIPAAm LCST. D: Gelled Q-PEI-PNIPAAm.
47


PNIPAAm and PEI-PNIPAAm in water are colorless clear solutions below their
LCSTs (Figure 18). When they gel, both polymers form white opaque gels. Therefore, light transmittance is high below the LCST and low above the LCST. LCST values are commonly calculated as the temperature at which a 50% change in transmittance occurs. The LCSTs for PNIPAAm and PEI-PNIPAAm were 35.4C and 35.5C, respectively. It should be noted that 5 wt% solutions of PNIPAAm and PEI-PNIPAAm did not form stable gels that conformed to the shape of the vial. The unstable gelling of PNIPAAm and PEI-PNIPAAm may be attributed to the reduced molecular weight of PNIPAAm used in this research.
PNIPAAm
PEI-PNIPAAm
Below
LCST
(21C)
Q-PEI-PNIPAAm

Above
LCST
(45C)

Figure 18 Appearance of 5 wt% solutions of PNIPAAm, PEI-PNIPAAm, and Q-PEI-PNIPAAm above and below the LCST. Below the LCST, PNIPAAm and PEI-PNIPAAm are transparent solutions and Q-PEI-PNIPAAm is a yellowish opaque solution. Above the LCST, PNIPAAm and PEI-PNIPAAm form opaque unstable gels, which do not retain the shape of the vial, and Q-PEI-PNIPAAm forms a stable opaque gel.
Q-PEI-PNIPAAm in water is a yellowish opaque solution below LSCT and its appearance is the same to the naked eye after gelling. However, it was found that there is a distinct change in the transmittance of Q-PEI-PNIPAAm when it gels. As the temperature
48


increases from 25C to 35C, the UV transmittance of Q-PEI-PNIPAAm also increases, but upon gelation of Q-PEI-PNIPAAm the transmittance remains constant with increasing temperatures. Using the above definition, the LCST of Q-PEI-PNIPAAm used in this research was calculated to be 29.5C. However, it should be noted that this method of LCST calculation assumes somewhat constant transmittance values above and below the LCST. In the case of Q-PEI-PNIPAAm, the transmittance below LCST is not constant with temperature changes, and as a result the LCST calculated for Q-PEI-PNIPAAm may not be accurate.
Despite the potential inaccuracy in the measurement of Q-PEI-PNIPAAm LCST, UV-visible spectroscopy demonstrates that this formulation of Q-PEI-PNIPAAm is a fully formed gel at and above 35C. The exact temperature at which this transition occurs is uncertain, but confirmation of stable gel formation was confirmed upon removal of the sample from the spectrometer (Figure 17). Normal body temperature ranges between 36.1C and 37.2C, so this formulation of Q-PEI-PNIPAAm is a gel at temperatures slightly lower than body temperature and therefore is ideal for use as a surgical drape [138],
Antimicrobial Q-PEI-PNIPAAm Surface Tests Antimicrobial Activity of Q-PEI-PNIPAAm Surface in a Bacterial Suspension
In vitro antimicrobial studies were conducted to quantify the antimicrobial killing capacity of the surface of Q-PEI-PNIPAAm and determine the optimal polymer solution concentration. Previous studies of Q-PEI-PNIPAAm investigated polymer activity below LCST by mixing the solution based polymer with bacteria and constructing time-kill curves [91], Before using Q-PEI-PNIPAAm as a surgical drape it was necessary to verify that the two-dimensional surface of the polymer also exhibited antimicrobial activity. In this study,
49


time-kill curves were constructed to assess the antimicrobial activity of Q-PEI-PNIPAAm against C. amycolatam, E. coli, MRSA, S. aureus, and S. epidermidis.
A time kill-curve for the activity of Q-PEI-PNIPAAm surfaces against C. amycolatam is shown in Figure 19. Glass slides coated with 1.25% QPP reduced bacterial concentrations by 2-, 4-, and 5-logs after 30, 60, and 120 minutes. The surface coated with 2.5% QPP showed a 5-log reduction in bacterial concentration after 120 minutes. Slides coated with 5% and 7.5% QPP exhibited a 6-log reduction in bacteria after 30 minutes.
E
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LL.
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c
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no
o
h No QPP *1.25% QPP -i 2.5% QPP -*5%QPP *7.5% QPP
Figure 19 Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase Corynebacterium amycolatam. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean.
Figure 20 shows a kill-curve for the antimicrobial activity of Q-PEI-PNIPAAm
against E. coli. Results showed that slides coated with 5% and 7.5% QPP reduced bacterial
concentrations by 8-logs after 30 minutes. Glass slides coated in 1.25% and 2.5% QPP only
exhibited 2- and 4-log reductions after 120 minutes.
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0 20 40 60 80 100 120
Time [min]
Figure 20 Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phast Escherichia coli. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean.
A time kill-curve for the activity of Q-PEI-PNIPAAm surfaces against MRSA is given
in Figure 21. Slides coated with 5% and 7.5% QPP exhibited an 8-log reduction in bacteria
after 30 minutes, while 1.25% and 2.5% exhibited 3- and 4-log reductions after 120 minutes.
0 20 40 60 80 100 120
Time [min]
Figure 21 Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase MRSA. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean.
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Figure 22 shows the constructed time-kill curve of Q-PEI-PNIPAAm surface antimicrobial activity against S. aureus. After a period of 120 minutes, 1.25%, 2.5%, and 5% QPP coated surfaces reduced bacterial concentrations by 3-, 5- and 8-log, respectively. An 8-log reduction in S. aureus bacterial concentration was exhibited by 7.5% QPP after 30 minutes.
z>
Ll_
u
c o 5 (0
+> c
u
c o u
tu>
20
40
60
Time [min]
80
100
120
Figure 22 Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase Staphylococcus aureus. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean.
Staph, epidermidis was the final bacterial strain, which Q-PEI-PNIPAAm was tested against. Glass slides coated with 1.25% QPP demonstrated a 1-log reduction in bacterial concentration after 120 minutes. In this test, 2.5% QPP demonstrated surprisingly high antimicrobial activity with 3-, 4-, and 7-log reductions after 30, 60, and 120 minutes, respectively. Reductions of 6- and 7-log in bacterial concentration were exhibited by 5% and 7.5% QPP, respectively.
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h No QPP -*1.25% QPP -i 2.5% QPP -* 5% QPP -*-7.5% QPP
Figure 23 Antimicrobial activity of a gelled Q-PEI-PNIPAAm surface against stationary-phase Staphylococcus epidermidis. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean.
Two-dimensional Q-PEI-PNIPAAm surfaces were demonstrated to have antimicrobial activity against all of the tested bacterial strains. These strains included both gram-negative and gram-positive bacteria. As expected, it was demonstrated that surfaces coated with increasing concentrations of Q-PEI-PNIPAAm solutions have increased antimicrobial activity. However, it was observed that gelled polymer surfaces coated with 1.25% and 7.5% QPP were not very durable. In this experiment glass slides coated with Q-PEI-PNIPAAm surfaces were places in conical tubes with 3 mL of bacterial solution. For the duration of the experiment, these tubes were incubated with shaking at 250 rpm. It was observed that over time a significant amount of Q-PEI-PNIPAAm fell off the slide coated with 1.25% and 7.5% QPP and gathered at the bottom of the conical tube (Figure 24). It is likely that 1.25% QPP did not adhere to the slide were well because there was not enough polymer on the surface to create a stable continuous gel. In the case of 7.5% QPP, too much polymer likely results in a pliable layer that can be peeled off more easily under mechanical strain.
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Figure 24 Q-PEI-PNIPAAm accumulation following 120-minute surface test with shaking at 250 rpm. A: Slide coated with 1.25% QPP. B: Slide coated with 7.5% QPP.
For this experiment, polymer solutions were sprayed onto the glass slides to ensure even coating of the polymer. Due to the viscosity of 7.5% Q-PEI-PNIPAAm, several experiments were delayed due to difficulty spraying the polymer. In those instances, the problem was frequently solved by changing spray bottles or priming the spray nozzle with a lower concentration polymer followed by the more viscous 7.5% Q-PEI-PNIPAAm. However, it was determined that due to difficulty with the application, 7.5% Q-PEI-PNIPAAm was not ideal for application at a surgical drape.
Based on the results of this study, it was decided that a 5% Q-PEI-PNIPAAm formulation was ideal for use as a surgical drape. This solution had very good surface antimicrobial activity, the polymer surface created at this concentration showed good durability, and this concentration was easily sprayable, Therefore, is was determined that approximately 100 pL of Q-PEI-PNIPAAm should be applied for every 4 cm2.
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In vitro O-PEI-PNIPAAm Activity as a Surgical Incision Drape
The previous experiment demonstrated that the surface of Q-PEI-PNIPAAm has antimicrobial activity. However, bacteria was readily able to move around in the bacterial solution, which maximized bacterial contact with the antimicrobial surface. It was therefore necessary to demonstrate the Q-PEI-PNIPAAm surfaces have the ability to kill stationary layers of bacteria, such as those on the skin. Thus, an in vitro study was developed to simulate Q-PEI-PNIPAAm activity when used as a surgical drape. In this study, LB agar was used to simulate the epidermis.
The same five relevant bacterial strains were used in this simulation: C. amycolatum,
E. coli, MRSA, S. aureus, and S. epidermidis. For the purpose of this test, a grid pattern was developed in which all five bacterial strains could be tested at once with seven initial stock bacterial concentrations for each (Figure 25). As one of the controls, bacterial with starting concentrations varying from 103 to 109 CFU/mL grown on agar with no polymer treatment.
Stock Solution Concentration [CFU/mL]
C. amycolatum E. Coli MRSA S. Aureus Staph, epidermidis
Figure 25 Growth of bacteria in grid pattern after 48 hour incubation on agar with no polymer.
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The goal of this surface test was to determine if Q-PEI-PNIPAAm demonstrated antimicrobial activity against bacteria trapped between the drape and the skin and against bacteria that fall on top of the drape. This was completed as described in the methods, and as expected Q-PEI-PNIPAAm killed bacteria trapped between itself and agar and bacteria on its surface (Figure 26).
Bacteria Between Polymer and Agar
Bacteria on Top of Polymer
PNIPAAm
PEI-
PNIPAAm
Q-PEI-
PNIPAAm
Figure 26 In vitro simulation of Q-PEI-PNIPAAm antimicrobial activity when used as a SID. Growth after 48 hours is shown. Bacteria were plated in the grid pattern below and above the polymers on separate plates. In this simulation agar represents the skin and the polymer represents the drape. Bacteria grew on both PNIPAAm plates, and PEI-PNIPAAm had some bacterial growth. Q-PEI-PNIPAAm inhibited all bacterial growth except for a small untreated area.
56


Bacteria plated on agar then covered with PNIPAAm, PEI-PNIPAAm, and Q-PEI-PNIPAAm was used to simulate the presence of bacteria under a surgical incision drape. To simulate bacterial contamination on top of a SID, the polymers were gelled on top of agar and bacteria was plated on top. PNIPAAm was used as a control, and as expected bacterial growth was observed on both plates. In the image in Figure 26, it is difficult to see bacterial growth between PNIPAAm and agar, but upon closer examination MRSA, S. aureus, and S. epidermidis colonies are present. As previously mentioned, PEI-PNIPAAm has some antimicrobial activity [91], In this in vitro simulation, PEI-PNIPAAm had some bacterial growth under the polymer where the polymer was poorly applied around the plate edges, and E. coli growth was observed on top of the polymer. In the case of Q-PEI-PNIPAAm, all bacterial growth was inhibited except for a small untreated area where E. coli growth was observed. Based on this in vitro simulation, Q-PEI-PNIPAAm shows promising potential for use as a surgical drape.
Murine Skin Incision Model
Skin incisions were completed with one of four different skin treatments (Figure 27). As a positive control, an incision was performed after skin cleansing with no drape. Inoculation of the incision site with bacteria and no surgical incision drape was used as a negative control. Q-PEI-PNIPAAm applied to the incision site after inoculation with bacteria was used as the experimental SID, and bacterial inoculation followed by placement of IOBAN 2 was used as a comparison SID. All groups except for the positive control were completed five times each for C. amycolatum, E. coli, MRSA, S. aureus, and S. epidermidis bacterial strains.
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Figure 27 Murine model treatment groups. A: Q-PEI-PNIPAAm applied as a surgical drape over bacteria. B: IOBAN 2 iodine-impregnated drape applied over bacteria. C: Positive control group with no drape or bacteria. D: Negative control group with applied bacteria and no drape.
The skin incision surgery was completed successfully for 48 mice with 3 mice in each group. For each mouse, swabs of bacterial growth were successfully taken and plated on LB agar after incision and at 11 additional time points up to 2 hours after incision closure. No complications arose for any of the mice during the skin incision surgery. Growth on LB agar was quantified as described in the methods section and data was compiled into plots of bacterial colony forming units (CFUs) as a function of time following the skin surgery.
Figure 28 reports the number of C. amycolatum CFUs detected by bacterial swabs following skin incision surgery. Trends show that the largest amount of bacterial colonies
58


were detected for the negative control with no drape. Use of Q-PEI-PNIPAAm and IOBAN 2 resulted in reduced detection if CFUs. However, due to large standard deviations there is no statistical difference between most groups. In the case of Q-PEI-PNIPAAm compared with I OBAN 2, no statistical difference was observed at most time points. However, at 75 minutes mice treated with Q-PEI-PNIPAAm had significantly fewer detected colonies compared with mice treated with IOBAN 2.
0 20 40 60 80 100 120
Time [min]
Figure 28 The number of C. amycolatam colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05.
The number of E. coli CFUs detected by bacterial swabs following skin incision
surgery is shown in Figure 29. Based on detected CFUs, E. coli survived poorly on the
mouse epidermidis. At 0 minutes there were statistically fewer CFUs positive control mice
compared with negative control mice. In the case of Q-PEI-PNIPAAm compared with
IOBAN 2, no statistical difference was observed.
59


Figure 29 The number of E. coli colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean.
* indicates statistical difference with p < 0.05.
Figure 30 shows the number of MRSA CFUs detected following skin incision surgeries. It should be noted that the low number of CFUs reported at time 0 for the negative control was due to the presence of large fused MRSA colonies. These large fused colonies indicated the presence of large amounts of bacteria, and therefore CFUs at this time point should be higher. Trends once again indicated that the negative control group had the highest CFUs.
The polymer mice had consistently fewer MRSA colonies when compared with IOBAN 2 mice. At times 0, 10, 20, and 75 minutes, Q-PEI-PNIPAAm drape mice had statistically significant lower numbers of CFUs compared with the IOBAN 2 drape group, and at all other times there was no statistical difference between the two groups. This data suggests that the use of Q-PEI-PNIPAAm as a SID is comparable to IOBAN 2 use, and that Q-PEI-PNIPAAm may even be a more effective SID than IOBAN 2.
60


900
-* + Bacteria, SID
Figure 30 The number of MRSA colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean.
* indicates statistical difference with p < 0.05.
The number of S. aureus CFUs following murine skin incision surgeries is shown in Figure 31. Significantly fewer CFUs were detected for the polymer mice at time 0 compared with the IOBAN 2 drape, but there was no statistical difference at other time points.
Time [min]
Figure 31 The number S. aureus colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean.
* indicates statistical difference with p < 0.05.
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Figure 32 shows the number of Staph, epidermidis CFUs detected by bacterial swabs following skin incision surgery. Trends show that the highest amount of bacterial colonies was observed for the negative control with no drape. There was no statistical difference between the use of Q-PEI-PNIPAAm or IOBAN 2 drapes.
swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05.
Results from this animal model consistently show lower numbers of detected CFUs with the use of Q-PEI-PNIPAAm as a surgical drape compared with IOBAN 2. However, in most of these cases the difference in CFUs detected with the use of these two drapes is not statistically different. Data from all five bacterial strains indicated that the performance of Q-PEI-PNIPAAm as a surgical drape is comparable with the use of IOBAN 2. It should be noted that while Q-PEI-PNIPAAm remained on the mouse skin following surgery, the IOBAN 2 drapes were removed, thus Q-PEI-PNIPAAm had more time to kill bacteria. However, Q-PEI-PNIPAAm is intended to remain on the skin following surgery, so this study tests this polymer as it is intended to be used.
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A power analysis to estimate the sample size required for the detection of the mean within 25% with a power of 0.80 was completed for each bacterial strain. A sample size (n) required to meet the above parameters was calculated at each time point based on the negative control data as this data frequently exhibited the highest variance. A minimum, maximum, and mean n value were calculated and those values are reported for each bacterial strain in Table 4. Based on these calculations if it is desired to determine the mean within 25% of its value with a power of 0.80 at every time point, then the study would need to be completed for the maximum n value. However, the maximum n value is quite high for some of these bacterial strains and it would be costly to complete such a study.
Table 4 Sample size (n) values calculated by the power analysis for each bacterial strain.
C. amycolatum E. coli MRS A S. aureus S. epidermidis
Minimum 21 14 1 16 3
Maximum 165 223 70 198 76
Mean 65 79 35 118 30
Subcutaneous Injections
Although Q-PEI-PNIPAAm was designed for topical use as a surgical drape, trace amounts of the polymer are likely to enter the body cavity during surgery. To assess the immune response instigated by the polymer, subcutaneous injections were successfully conducted for 12 mice. Samples of sterile saline and Q-PEI-PNIPAAm were injected into mice for time points of 1 and 7 days with 3 mice in each experimental group. Tissue harvested from the area surrounding the injection site was then stained for macrophage presence with an anti-CD68 antibody. Stained tissue samples were imaged with confocal microscopy (Figure 33).
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7 Day
1 Day
Saline
QPP
Figure 33 Representative confocal images of immunostaining for macrophages after 1 and 7 days. Macrophages were stained with anti-CD68 antibody and Alexa Fluor 594, which appears in red. DAPI was used to stain cell nuclei, which appear in blue. Cells containing both colors were identified as macrophages. Scale bars represent 100 pm.
Macrophages were observed in all subcutaneous injection groups. Immune response was quantified by determining the number of macrophages per unit area. Figure 34 shows a graph of macrophages per mm2 for the polymer and saline at both time points. Q-PEI-PNIPAAm injections at both time points had higher macrophage counts per area than saline. There was no statistical difference between Q-PEI-PNIPAAm and saline macrophages per area for 1 day subcutaneous injections. For 7 day subcutaneous injections, Q-PEI-PNIPAAm
64


had statistically higher counts of macrophages per area. While macrophages per area increased for Q-PEI-PNIPAAm and saline injections between 1 and 7 days, the increases were not statistically significant.
Time [day]
Figure 34 Macrophages per unit area following 1 and 7 day subcutaneous injections of saline and Q-PEI-PNIPAAm. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05.
Results from subcutaneous injections indicate that both saline and Q-PEI-PNIPAAm injections prompt an immune response. In the case of biocompatible materials, any initial immune response should dissipate over time. Saline is commonly used as a sham injection in research, and it has been shown that any immune response to saline disappears with time [139], In this study, the immune response to saline injections increased from 1 to 7 days. Therefore, there was no control group in which immune response returned to baseline. This means that although it can be concluded the Q-PEI-PNIPAAm prompts on immune response, no conclusions can be made about the biocompatibility or toxicity of the polymer.
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CHAPTER VII
CONCLUSION
The first specific aim of this study was to characterize the formulation of Q-PEI-PNIPAAm intended for use as a surgical drape. Successful synthesis of the desired structure of Q-PEI-PNIPAAm was confirmed with 'H NMR. Analysis of the polymers LCST also verified that Q-PEI-PNIPAAm forms a stable gel at a temperature slightly below body temperature.
The antimicrobial activity of the two-dimensional surface of Q-PEI-PNIPAAm was investigated with two studies. The first study demonstrated that the Q-PEI-PNIPAAm surface has antimicrobial activity against all five bacterial strains used in this research. From this study, it was also concluded that a 5 wt% solution of Q-PEI-PNIPAAm in 70% ethanol was ideal for use as a sprayable gelling surgical drape.
The second surface test was an in vitro simulation of Q-PEI-PNIPAAm antimicrobial activity as a surgical drape. This study demonstrated Q-PEI-PNIPAAm has the potential to kill bacterial trapped between the polymer and the skin during surgery as well as any bacterial contamination that may fall onto the drape surface.
The effectiveness of Q-PEI-PNIPAAm used as a surgical drape was compared to the use of a commercially available widely used iodine-impregnated SID in a murine skin incision model. In this model, the incision site was cleansed, inoculated with a particular strain of bacteria, and an IOBAN 2 drape or Q-PEI-PNIPAAm was applied to the area. Drape performance was evaluated based on the relative differences in detectable CFUs between the tested groups. Although at most time points there was no statistical difference between the number of CFUs detected in mice where Q-PEI-PNIPAAm or IOBAN 2 were
66


applied as a drape, overall data trends and a small number of statistical points indicated that Q-PEI-PNIPAAm may be more effective as a SID than IOBAN 2. Data from this murine model supports that hypothesis that Q-PEI-PNIPAAm performance as a surgical drape is comparable to a commercially available drape.
Subcutaneous injections of sterile saline and Q-PEI-PNIPAAm were administered for 1 and 7 days to determine if Q-PEI-PNIPAAm instigates an in vivo immune response. It was found the between 1 and 7 days, both saline and Q-PEI-PNIPAAm injections result in an increase in macrophages per area. This indicates that both prompt and immune response. However, no conclusions can be made about the biocompatibility of the polymer without a full cytotoxicity assay where the control group displays a dissipation of the initial immune response.
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CHAPTER VIII
FUTURE WORK Additional Polymer Optimization
Although during the animal model it was observed that Q-PEI-PNIPAAm gelled on the mouse skin surface, the gelling temperature of the polymer should be further tailored. The Q-PEI-PNIPAAm formulation used in this study was reported to form a stable gel by 35C. However, the skin surface temperature varies between approximately 33-37C [140], [141], Therefore, to ensure that the polymer remains gelled on the skin during surgery, the polymer should be designed to form a stable gel around 32C. This may be achieved by increasing the length of the alkyl chains conjugated the PEI-PNIPAAm, which would increase the amount of hydrophobic groups on the polymer. Increasing the hydrophobic groups will result in gelation at a lower temperature.
Increase Mouse Model Sample Size
Although data trends of the current study suggest that Q-PEI-PNIPAAm has the potential to outperform IOBAN 2, high variability in reported CFUs rendered many of the trends statistically insignificant. In many cases, there was no statistical differences even between the negative and positive control. An increased sample size is thus necessary and should reduce some of the variability. It is expected that with an increased sample size, more statistical differences will be seen between the groups. A power analysis was completed to calculated the required sample size to detect the number of CFUs within 25% of its true mean with an 0.8 power, and results of the analysis were reported in Table 4. The analysis indicated that increased sample size would improve the significance of the data.
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In Vivo Cytoxicity Assay
Q-PEI-PNIPAAm is not intended for injection into the body cavity. However, trace amounts of the polymer are likely to enter the body cavity through the incision site during surgery. For this reason, it is important to conduct an in vivo cytotoxicity where it can be determined if Q-PEI-PNIPAAm is biocompatible. Biocompatibility can be assessed with the addition of longer duration subcutaneous injection groups.
Skin Irritation Test
Surgical drapes may remain on the skin surrounding the incision site for long periods of time during surgery. For this reason, it would be useful to assess whether Q-PEI-PNIPAAm causes skin irritation after prolonged contact with the epidermis. As mentioned in the background previous skin irritation tests of a quaternized polymer showed no skin irritation for a 7 day duration. However, a similar test should be completed with the polymer formulation used in this study.
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Full Text

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EVALUATION OF AN ANTIMICROBIAL REVERSE THERMAL GEL FOR USE AS A SURGICAL DRAPE by MADIA ELIZABETH STEIN B.S., University of New Mexico, 2014 A thesis submitted to the Faculty of the Graduate School of the University of the Colorado in partial fulfillment of the requirements for the degree of Master of Science Bioengineering Program 2016

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ii This thesis for the Master of Science degree by Madia Elizabeth Ste in has been approved for the Bioengineering Program by Daewon Park, Chair Vikas Patel Danielle Soranno Date: December 17, 2016

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iii Stein, Madia Elizabeth (M.S. Bioengineering) Evaluation of an Antimicrobial Reverse Thermal Gel for Use as a Surgical Drape Thesis directed by Assistant Professor Dae w on Park ABSTRACT Surgical site infections (SSIs) are a dangerous complication of surgical procedures. SSIs develop in roughly 2% of surgical procedures in the U.S. Despite efforts to reduce infection rates, SSIs are extremely costly and they remain a significant cause of morbidity and mortality. Generally antibiotic prophylaxis, preoperative skin preparation, surgical hand preparation and intraoperative skin antise psis are all standard practices used in hospitals to reduce SSI occurrences therefore preoperative skin preparation and intraoperative skin antisepsis are essential S urgical incision drap es (SIDs) impede microbial recolonization and prevent contamination in the surgical incision site during surgery. However, due to drawbacks of current SIDs drawbacks including time consuming placement, loss of adhesion, and loss of an epidermal cell layer during drape removal, an antimicrobial reverse thermal gel was developed for potential use as a SID. Gelling properties and antimicrobial activity of the reverse thermal gel surface were demonstrated to be ideal for the use of this polymer as a SID. Perfo rmance of the polymer as a SID was evaluated in a mouse skin incision model and compared with commonly used commercially available SID. The studied polymer was found to have comparable performance as a SID to the commercial alternative. The form and conte nt of this abstract are approved. I recommend its publication. Approved: Daewon Park

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iv ACKNOWLEDGEMENTS I would like to acknowledge my professors, mentors, friends, and family and for all of their supp ort heir help this work would not have been possible. First, I would like to thank my advisor, Dr. Daewon Park, for allowing me to join the Translational Biomaterial Research Laboratory (TBRL) and for his mentorship and patience. I have learned many valuable skills I would also like to express my gratitude to Dr. Vikas Patel and Dr. Danielle Soranno fo r their time, insight, and advice throughout my research. I would also like to thank Dr. Todd for hi s time and help with design of ba cterial testing. My time in this program would not have been the same without m y fellow TBRL members. I appreciate all of our experiences together both in and out of the lab. I would particularly like to thank David Lee, James Bardill, and Melissa Laughter for their help and encouragement. Finally, I would like to thank my family. I can no t thank m y mother and father enough for their continual faith in my success and constant encouragement I would also like to express my gratitude to my boyfrien d, Jay McCabe, for his unbelievable support and patience Animal model studies were conducted under the University of Colorado at Denver Institutional Animal Care and Use Committee ( IACUC ) protocol number 102913(12)1D

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v TABLE OF CONTENTS CHAPTER I. IN TRODUCTION ................................ ................................ ................................ ........ 1 Overview ................................ ................................ ................................ ....................... 1 Skin Anatomy and Physiology ................................ ................................ ...................... 1 Skin Microbiota ................................ ................................ ................................ ............ 4 SSI Microbiolo gy ................................ ................................ ................................ .......... 7 SSI Pathogenesis ................................ ................................ ................................ ........... 9 SSI Prevention ................................ ................................ ................................ ............ 10 Antibiotic Prophylaxis ................................ ................................ ....................... 11 Preoperative Skin Preparation ................................ ................................ ............ 11 Preoperative Surgical Hand Scrub ................................ ................................ ..... 13 Intraoperative Skin Antisepsis: Surgical Incision Drapes ................................ 13 Study Objective ................................ ................................ ................................ ........... 16 II. BACKGROUND ................................ ................................ ................................ ......... 17 Antimicrobials and Antimicrobial Resistance ................................ ............................ 17 Mechanisms of Antimicrobial Activity ................................ ................................ ...... 18 Antimicrobial Polymers ................................ ................................ .............................. 21 Quaternary Ammonium Compounds ................................ ................................ 22 Polyethyleneimine ................................ ................................ .............................. 23

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vi Effect of Alkyl Chain and Molecular Weight on Antimicrobial Activity ......... 24 Biocompatibility of Quaternary Ammonium Compounds ................................ 26 Reverse Thermal Gels ................................ ................................ ................................ 27 III. PREVIOUS WORK ................................ ................................ ................................ .... 30 Q PEI PNIPAAm LCST ................................ ................................ ............................. 30 Antimicrobial Activity of Q PEI PNIPAAm ................................ .............................. 30 Cytotoxicity ................................ ................................ ................................ ................. 32 IV HYPOTHESIS AND SPECIFIC AIMS ................................ ................................ ..... 33 Hypothesis ................................ ................................ ................................ ................... 33 Specific Aims ................................ ................................ ................................ .............. 33 V MATERIALS AND METHODS ................................ ................................ ................ 34 Materials ................................ ................................ ................................ ..................... 34 Polymer Synthesis ................................ ................................ ................................ ....... 35 PNIPAAm Sy nthesis ................................ ................................ .......................... 35 PEI PNIPAAm Synthesis ................................ ................................ .................. 35 Q PEI PNIPAAm Synthesis ................................ ................................ .............. 36 Polymer Characterization ................................ ................................ ............................ 37 Proton Nuclear Magnetic Resonance ( 1 H NMR) ................................ ............... 37 LCST Determination ................................ ................................ .......................... 37 Bacteria Preparation and Dilutions ................................ ................................ ............. 37 Antimicrobial Q PEI PNIPAAm Surface Tests ................................ ......................... 38

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vii Antimicrobial Activity of a 2D Q PEI PNIPAAm Surface in a 3D Bacterial Suspension ................................ ................................ ................................ ......... 38 Antimicrobial Activity of a 2D Q PEI PNIPAAm Surface against 2D Bacterial Samples ................................ ................................ ............................... 39 Skin Incision Animal Model ................................ ................................ ....................... 40 Skin Incision Surgery ................................ ................................ ......................... 40 Quantification of Bacterial Swabs ................................ ................................ ..... 41 Q PEI PNIPAAm Immune Response In Vivo ................................ ........................... 41 Subcutaneous Injections ................................ ................................ ..................... 41 Immunohistochemistry ................................ ................................ ...................... 42 Statistical Analysis ................................ ................................ ................................ ...... 43 V I RESULTS AND DISCUSSION ................................ ................................ ................. 44 Q PEI PNIPAAm Reaction Mechanism ................................ ................................ ..... 44 Polymer Characterization ................................ ................................ ............................ 45 Characterization of PEI, PEI PNIPAAm, and Q PEI PNIPAAm using 1 H NMR ................................ ................................ ................................ .................. 45 Lower Critical Solution Temperature (LCST) ................................ ................... 47 Antimicrobial Q PEI PNIPAAm Surface Tests ................................ ......................... 49 Antimicrobial Activity of Q PEI PNIPAAm Surface in a Bacterial Suspension ................................ ................................ ................................ ......... 49 In vitro Q PEI PNIPAAm Activity as a Surgical Incision Drape ..................... 55 Murine Skin Incision Model ................................ ................................ ....................... 57 Subcutaneous Injections ................................ ................................ .............................. 63

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viii VII. CONCLUSION ................................ ................................ ................................ ........... 66 VIII. FUTURE WORK ................................ ................................ ................................ ........ 68 Additional Polymer Optimization ................................ ................................ ............... 68 Increase Mouse Model Sample Size ................................ ................................ ........... 68 In Vivo Cytoxicity Assay ................................ ................................ ............................ 69 Skin Irritation Test ................................ ................................ ................................ ...... 69 REFERENC ES ................................ ................................ ................................ ....................... 70

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ix L IST OF FIGURES FIGURE 1 The anatomical structure of the human integumentary system [11]. ................................ .. 2 2 A: H&E stained thick skin showing the layers of the epidermis and collagen fibers in the dermis B: Illustration of the layers of the epidermis [12]. ............................. 3 3 Factors influencing skin microbiota diversity [26]. ................................ ............................ 6 4 Skin microenvironments and associated distributions of bacteria [16]. ............................. 7 5 A: Epidermal cell removal during SID lift off, which exposes bacteria found beneath the skin. B: Surgical incision drape after being peeled back in prepa ration for would closure. C: Skin avulsion injury from the SID removal on an elderly patient. ................................ ................................ ................................ ......... 15 6 Mechanisms of antibacterial resistance. 1a: Elimination of cellular antimicrobials using efflux pumps. 1b: Elimination of cellular antimicrobials by decreasing/blocking cellular uptake. 2: Alteration of antimicrobial target. 3: Direct inactivation of antimicrobials by cellular enzym es. 4: Alteration of metabolic pathways [97]. ................................ ................................ ................................ .. 18 7 Antimicrobial mechanisms of various antimicrobial agents against bacterial ammonium compounds and chlorine releasing agents, respectively [101]. ..................... 19 8 Structure of gram negative and gram positive bacteria. Both types of bacteria contain negatively charged structures on their surface. Gram negative bacteria have negatively charged phospholipids and lipopolysaccharides on their surface. Gram positive bacteria have negatively charged teichoic and lipoteichoic acids on their surface [102]. ................................ ................................ .......... 20 9 Quaternizatioin of tertiary amine with an alkyl halide yielding a quaternary ammonium cation. In this chemical equation, R represents alkyl or aryl groups, and X represents a halogen, such as Cl Br Fl or I ................................ ....... 22 10 A: Behavior of PNIPAAm chains below (left side) and above (right side) LCST. B: PNIPAAm chemical structure with labeled hydrophilic and hydrophobic regions. ................................ ................................ ................................ ......... 29 11 PEI PNIPAAm and Q PEI PNIPAAm were added to stationary phase bacteria, and samples were taken at 0, 30, 60, and 120 minutes to determine bacterial c oncentrations. Kill curves were constructed for the following bacteria: A: S. aureus subsp. aureus Mu3 (MRSA/hetero VISA) B: Staph. epidermidis C: E. coli D: S. aureus. ................................ ................................ .................. 31

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x 12 MTT assay assessing the cytotoxicity of Q PEI PNIPAAm (QPP). No statistical difference between the negative control and cells plated on PNIPAAm and PEI PNIPAAm was observed. No statistical significance was found between cells plated on QPP and Chlorhexidi ne. Statistical significance was found between experimental groups and the positive control (** denotes p < 0.05 and *** denotes p > 0.05). ................................ .............................. 32 13 Reaction mechanism for the synthesis of Q PEI PNIPAAm. ................................ .......... 44 14 1 H NMR spectrum of branched PEI confirming its mol ecular structure. ......................... 45 15 1 H NMR spectrum of PEI PNIPAAm confirming the addition of PNIPAAm to PEI. ................................ ................................ ................................ ............................... 46 16 1 H NMR spectrum of Q PEI PNIPAAm confirmed the conjugation of alkyl chains and the presence of a slight peak at 3.6 ppm indicated the presence of quater nary ammoniums. ................................ ................................ ................................ .... 46 17 LCST of polymer samples. A: PNIPAAm LCST. B: PEI PNIPAAm LCST. C: Q PEI PNIPAAm LCST. D: Gelled Q PEI PNIPAAm. ................................ ............. 47 18 Appearance of 5 wt% solutions of PNIPAAm, PEI PNIPAAm, and Q PEI PNIPAAm above and below the LCST. Below the LCST, PNIPAAm and PEI PNIPAAm are transparent solutions a nd Q PEI PNIPAAm is a yellowish opaque solution. Above the LCST, PNIPAAm and PEI PNIPAAm form opaque unstable gels, which do not retain the shape of the vial, and Q PEI PNIPAAm forms a stable opaque gel. ................................ ................................ ....... 48 19 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Corynebacterium amycolatum. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solution s, which were allowed to gel. Error bars represent the standard error of the mean. ................................ ................................ .. 50 20 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Escherichia coli. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean. ................................ ................................ ............................... 51 21 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase MRSA. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean. ................................ ................................ ................................ .............. 51 22 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Staphylococcus aureus. Glass slides were coated with 1.25, 2 .5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean. ................................ ................................ ......................... 52

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xi 23 Antimicrobia l activity of a gelled Q PEI PNIPAAm surface against stationary phase Staphylococcus epidermidis. Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean. ................................ ................................ .......... 53 24 Q PEI PNIPAAm accumulation following 120 minute surface test with shaking at 250 rpm. A: Slide coated with 1.25% QPP. B: Slide coated with 7.5% QPP. ................................ ................................ ................................ ......................... 54 25 Growth of bacteria in grid pattern after 48 hour incubation on agar with no polymer. ................................ ................................ ................................ ............................ 55 26 In vitro simulation of Q PEI PNIPAAm antimicrobial activity when used as a SID. Growth after 48 hours is shown. Bacteria were plated in the grid pattern below and above the polymers on separate plates. In this simulation agar represents the skin and the p olymer represents the drape. Bacteria grew on both PNIPAAm plates, and PEI PNIPAAm had some bacterial growth. Q PEI PNIPAAm inhibited all bacterial growth except for a small untreated area. ................................ ................................ ................................ ................................ ... 56 27 Murine model treatment groups. A: Q PEI PNIPAAm applied as a surgic al drape over bacteria. B: IOBAN TM 2 iodine impregnated drape applied over bacteria. C: Positive control group with no dr ape or bacteria. D: Negative control group with applied bacteria and no drape. ................................ ............................ 58 28 The number of C. amycolatum colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. .................... 59 29 The number of E. coli colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard er ror of the mean. *_indicates statistical difference with p < 0.05. ................................ .. 60 30 The number of MRSA colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. *_indicates statistical difference with p < 0.05. ................................ .. 61 31 The number S. aureus colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. *_indicates statistical difference with p < 0.05. ................................ .. 61 32 The number Staph. epidermidis colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. .................... 62

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xii 33 Representative confocal images of immunostaining for macrophages after 1 and 7 days. Macrophages were stained with anti CD68 antibody and Alexa Fluor 594, which appears in red. DAPI was used to stain cell nucl ei, which appear in blue. Cells containing both colors were identified as macrophages. Scale bars represent 100 m. ................................ ................................ ............................ 64 34 Macrop hages per unit area following 1 and 7 day subcutaneous injections of saline and Q PEI PNIPAAm. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. ................................ ...................... 65

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x iii L IST OF TABLES TABLE 1. Rank and distribution of the most common SSI causing pathogens reported for three time periods. ................................ ................................ ................................ ......... 8 2 Properties of various types of antiseptics and examples of commercially available products. Table adapted from Reichman et al. 2009. [57] ................................ 12 3 LCST values reported for various conjugations of PEI PNIPAAm and Q PEI PNIPAAm. ................................ ................................ ................................ ........................ 30 4 Sample size (n) values calculated by the power analysis for each bacterial strain. ................................ ................................ ................................ ................................ 63

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xiv LIST OF ABBREVIATION S 1 H NMR proton nuclear magnetic resonance ACA a zobis(4 cyanovaleric acid) AMPs antimicrobial peptides ANOVA analysis of variance APC antigen presenting cell BC benzyl chloride BSA bovine serum albumin CDC Centers for Disease Control and Prevention CFUs colony forming units CHG chlorhexidine gluconate CT connective tissue DCD dermcidin DMAEMA dimethylaminoethyl methacrylate DMF N,N dimethylformamide ECM extracellular matrix EDC N (3 dimethylamino propyl) ethylcarbodiimide hydrochloride GI gastrointestinal H&E haematoxylin and eosin HACC hydroxypropyltrimethyl ammonium chloride chitosan HAI healthcare associated infection LCST lower critical solution temperature LOS length of stay MIC minimum inhibitory concentration MRSA methicillin resistant Staphylococcus aureus MW molecular weight N 2 nitrogen NH 2 amine group NHS N hydroxysuccinimide nm nanometers NIPAAm N i sopropylacrylamide OCT optimal cutting temperature OR operating room PCMX para chloro meta xylenol PEI polyethylenimine PBS phosphate buffered saline PNI PAAm poly(N i sopropylacrylamide) QAC s quaternary ammonium compounds QPP quaternized PEI PNIPAAm rpm revolutions per minute

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xv RTG reverse thermal gel SID surgical incision drape spp. species SSI surgical site infection UV U ltraviolet ZPT zinc pyrithione

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1 CHAPTER I INTRODUCTION Overview Surgical site infections (SSIs) are a dangerous complication of surgical procedures When the skin is cut during surgery, microorganisms may invade the incision which may cause infection A SSI has been defined by the Centers for Disease Control and Prevention (CDC) as an infection related to a surgical procedure which occurs at or near a surgical incision within 30 days of the operation or 1 year for medical implants [1] [3] SSIs account for 14 16% of healthcare associated infection (HAI) and develop in roughly 2% of surgical procedures in the U.S. [2] [6] T here are roughly 27 million surgeries in the U S per year which eq u ates to approximately 540,000 SSIs [3] SSIs are associated with mortality, increased cost, and prolon ged length of stay (LOS) in hospitals It is estimated that a mong patients with SS Is there is a 3% mortality rate and a 7 10 day increase in LOS [3], [5], [7] SSI s co st between $11,000 and $35,000 per patient and i t is estimated that up to $10 billion is spent on SSIs annually in the U S [8] [10] Despite efforts to reduce infection rates, SSIs are extremely costly and they remain a significant c ause of morbidity and mortality. Skin Anatomy and Physiology The skin which has a surface area of 1.2 to 2.2 m 2 is the largest organ [11] I mportantly, the skin The skin consists of two layers; the superficial avascul ar epidermis and the underlying dermis. The se layers are supported by a basal layer of subcutaneous tissue known as the hypodermis (Figure 1) Tog ether the skin and its appendages form the inte g u mentary system.

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2 Figure 1 The anatomical structure of the human integumentary system [11] The epidermis is chiefly composed of keratinocytes, but it also contains melanocytes, dendritic cells, and tactile cells. The se cells are organized into four to five distinct layers of cells S tarting from the base of the epidermis they are stratum basale stratum spinosum stratum granulosum, stratum lucidum and stra tum corneum (Figure 2) The stratum basale consists of a single layer of stem cells These stem cells divide into keratinocytes to replenish cells shed from the epider mis. The stratum basale also contains a small number of melanocytes and infrequent tactile cells. The next layer up, the stratum spinosum, is several cell layers thick. T his layer contains many keratinocytes along with melanin granules and dendritic cells Melanin granules shield cells in the skin from ultraviolet (UV) damage. The st r atum granulosum consists of four to six cell layers in which the keratinocytes begin to keratinize, flatten, and their nuclei and organelles begin to disintegrate. In this stra tum

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3 lamellar granules containing water resistant glycolipids are released into the extracellular matrix (ECM). The stratum lucidum is present in areas of thick skin such as the hands and feet. It consists of two to three rows of flat, clear, dead keratino cytes. The superficial stratum corneum is the thickest layer of the epidermis accounting for up to 75% of its thickness and containing 20 to 30 cell layers. The dead, flattened, enucleated keratin in this layer protects the body from abrasion and infiltrat ion while glycolipids prevent water loss. [11], [12] Figure 2 A: H&E stained thick skin showing the layers of the epidermis and collagen fibers in the dermis. B: Illustration of t he layers of the epidermis [12] The dermis is composed of flexible connective tissue (CT) Although appendages such as hair follicles, sweat glands, and sebaceous (oil) glands are derived from the epidermis, they largely reside within the de rmis The dermis has a papillary and a reticular layer. The superficial papillary layer contains a plethora of blood vessels that supply nutrients to the epidermis. The CT in the papillary lay er is loosely woven, which allows macrophages, mast cells and other imm une cells to traverse freely. The reticular layer makes up about 80% of the dermis and it is composed of dense fibrous CT This layer is nourished by blood vessels in the cutaneous plexus which lies between the dermis and the hypodermis. [11]

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4 F unctions of t he integumentary system include protection regul ation of body temperature, and secretion. First and foremost, t he skin is a barrier that is essen tial to protecting the body from microorganisms, harmful substances, and other environmental factors. Dead k eratinized cells in the stratum corneum strengthen the skin, while glycolipids seal the gaps between c ells much like brick and mortar, respectively [13], [14] This physical barrier prevents most substances from penetrating the skin, however, a few substances including water, organic solvents, and lipid soluble substances pass through the skin in small amounts [11] The skin is also a chemical barrier whose secretions protect the body. The skin has two type of sweat glands: the eccrine and apocrine glands. The eccrine gland secretes water, electrolytes temperature, and secreted s alt s and electrolytes create an acid ic environment, which impedes bacterial growth [15], [16] Furthermore, eccrine glands have also been found to secrete antimicrobial peptides (AMPs) in the form of dermicidin (DCD ) [17], [18] Sebaceous glands secrete sebum and several AMPs defensins, cathelicidins, and histones [19], [20] The skin also contains active biological barriers that protect the body. The first line of defense are dendritic cells found in the epidermis. Dendritic cells engulf foreign substances and travel to lymphocytes to activate the adaptive immune response agai nst foreign invaders. Macrophages in the dermis act as a second line of defense. They directly engulf any bacteria or viruses tha t make it through the epidermis and they act as antigen presenting cells (APCs), which can activate adaptive immunity [11] Skin Microbiota Desp numerous antimicrobial defenses t he skin is inhabited by a diverse array of microbes including bacteria, viruses, fungi, and mi tes [16], [21] The skin contains

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5 external environment, and resident flora, which colonize the skin and are not rea dily removed by hand washing [16], [22] There are three potential outcomes for each species in microorganism host interactions: positive, negative, or no impact. C ommensalism is an interaction where one species benefits from interactions while the other is not affected, and mutualism is a relationship where both the species benefit [23], [24] The relationship between m ost microbes on the skin and the skin itself is mutualistic, commensal, or neutral [25], [26] The s skin has been found to remai n somewhat constant over time allowing interactions to remain in the categories above [27] The vast majority of SSIs are caused by bacteria, so this report will focus on bacteria as the source of inf ection [3] It is estimated that there are 10,000 bacteria/cm 2 on the surface of the skin and about 1,000,000 bacteria/cm 2 throughout the epidermis and the dermis [ 28] Although a study detected 19 phyla on the skin, n early all bact e ria belong to the Actinobacteria (51.8%) Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%) phylas [29] B acteria from the genera Corynebacteria Propionibacteria and Staphylococci accounted for 22.8%, 23.0%, and 16.8% of the bacteria on the skin, respectively [29] Coryneb acteria and Propionibacteria belong to the Actinobacteria phylum, and Staphylococci belongs to the Firmicutes phylum. In addition t he genera Micrococci Streptococci and Brevibacteria are also frequently present on the skin [24] Despite the presence of conserved bacterial genera from individual to individual, specific inter person skin microbiota is extremely diverse [27], [30] [32] Skin microbiota diversity is influenced by a combination of environmental and host factors including age, hygiene, and geographical location (Figure 3) [33]

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6 Figure 3 Factors influencing skin microbiota diversity [26] The human skin can be divided into three distinct microenvironments : dry, moist, and sebaceous [29], [34] Each of these microenvironments tends to favor particular bacterial genera (Figure 4 ) Although dry habitats are associated with the most diverse population of bacteria, Proteobacteri a are common in this microenvironment [16], [29] Despite previously being regarded as gastrointestinal (GI) organisms, g ram negative bacteria are also frequently found in this microenvironment [26], [35] Staphyloco cci from the phyl um Firmicutes and Corynebacter ia from the phylum Actinobacteria are abundant in m oist microenvironments, which include s the groin, the sole of the foot, the axilla, the inner elbow, and the back o f the knee [27], [29] Bacteria found in sebaceous areas of the skin which include facial areas and the back, are the least diverse and consist mainly of bac teria of the Propionibacteria genus from the Actinobacteria phylum [16], [36], [37] Aside from the three listed microe nviro nments, glands and follicles are thought to have their own characteristic flora [28], [36]

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7 F igure 4 Skin microenvironments and associated distributions of bacteria [16] SSI Microbiology There are two main sources of pathogens for SSIs: endogenous and exogenous. Endogenous microbiota coming from the skin, the GI tract, the respiratory tract, and the genitourinary tract cause most SSIs [38] The most common endogenous pathogens are aerobic gram positive cocci which are associated with the skin microbiota [3] Exogenous sources include the air in the operating room (OR), the OR personnel, the OR equipment, surgical instruments, and anything brought into the surgical sterile field [2], [3] When outbreaks of SSIs occur that are associated with uncommon pathogens, they are typic ally caused by exogenous sources [3] For example, water contamination, elastic bandages, and an anesthesiologist were the cause of outbreaks of Pseudomonas multivorans, Clostridium perfringens and Nocardia farcinica respectively [39] [41]

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8 Although a variety of microbes are present on the skin, most SSIs are caused by bacteria. Table 1 lists the eight categories of pathogens that most frequently cau se SSIs. Together, these pathogen categories account for 75 79% of SSIs and the distribution of these pathogens has remained somewhat constant over time [42] [44] According to a recent report, Staphylococc us aureus coagulase negative staphylococci ( e. g. Staphylococcus epidermidis ) Enterococcus spp. and Escherichia coli are responsible for approximately 30.4%, 11.7%, 11.6%, and 9.4% of SSIs, respectively [44] Corynebacteria (e.g., C. amycolatum ) h ave also been associated with SSI development [45] It has been found that an increasing amount of SSIs are caused by drug resistant strains of many of these pathogens as well as other unlisted categories of pathogens [46], [47] As can be seen in Table 1, a small amount of SSIs are also consistently caused by the fungal pathogen Candida albicans but this report will focus on bacteria. Table 1 Rank and distribution of the most common SSI causing pathogens reported for three time periods. There are likely SSI pathogens associated with different types of surgeries. This is largely due to the fact that the distribution of endogenous microbiota varies by anatomical location. As discussed in the skin microbiota section, skin contains mostly gram positive aerobes, while the GI tract has gram negative bacteria and anaerobes. Therefore, a surgery

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9 that only disrupts the skin expo ses a patient to mark edly different pathogens than a surgery where the integrity of the GI tract surface is broken. The likely SSI pathogens for neurosurgery, implants, graft placements, heart surgery, and vascular surgery are Staphylococcus aureus and coagulase negative staph ylococci. Appendectomies and colorectal surgeries are more likely to have SSIs caused by gram negative bacilli or anaerobes. [3] SSI Pathogenesis Penetration of the skin during surgery exposes patients to microbial contamination. One clin ical trial found that out of 166 clean surgeries, 3 patients (1.8%) developed SSIs despite the fact that 101 (61%) of the incisions contained 1 to 200 colony forming units (CFUs) of bacteria during surgery [48] This indicates that while microbial contamination is a required precursor to SSIs, the presence of bacterial does not guarantee that a SSI will occur. It has been demonstrated that the presence of >10 5 bacteria/gram of tissue greatly increases the risk of a SSI [49] Using various strains of staphylococcus it was also demonstrated that while a subcutaneous injection of >10 6 bacteria is required for infection, 10 2 bacteria may cause infection i n the presence of sutures composed of various materials [50] [52] Therefore, the presence of foreign material increases the risk of a SSI developing with markedly smaller numbers of bacterial contamination. as well as the virulence of the bacteria. Risk of surgical infection has been defined by the following equation [53] :

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10 In this context, virulence is a measure of the severity of an infection caused by a particular species of bacteria. Many bacteria have specific defensive mechanisms that allow them to evade ho st immunity and survive within the host these are termed virulence factors [3] For example, coagulase shields bacteria from phagocytes and protects bacteria from antimicro bial agents [54] The resistance of the host patient to infe ction is dependent both on their immune system and other patient risk factors including age, obesity, nutrition, microbial colonization, and length of time in hospital prior to surgery [55] Aside from microbial and patient risk factor s, operation risk factors also influence the risk of SSI [56] Some operational risk factors include sterile surgical technique, preoperative shaving, preparation of the surgical incision site, and antimicrobial prophylaxis [55] SSI Prevention Alth ough standard p ractic es in the OR vary slightly between hospitals n umerous guidelines for preventing and reducing the occurrence of SSIs have been published [3], [57], [58] Generally antibiotic prophylaxis, p reoperative skin preparation, surg ical hand preparation and intraoperative skin antisepsis are all standard practices used in hospitals to reduce SSI occurrences [58], [59] Since the skin is thought to be the main source of SSI pathogens, preoperative skin preparation and intraoperative skin antisepsis are essential [38], [60] Hair removal with a razor has been shown to increases SSI occurrence, so it is recom mended that hair is r emoved only if it interferes with the procedure [61], [62] In cases where hair is removed, it is cut with clippers immediately prior to skin preparation as razors riddle the skin with microscopic lacerations [3], [62]

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11 Antibiotic Prophylaxis The goal of antibiotic prophylaxis is to prevent SSIs while hav ing n o adverse effects on the patient or the normal flora of the patient and the hospital [3], [63] This is achieved by decreasing microbial contamination during surgery to a quantit y that the body can defend against with the use of antibiotics [3] In order for antibiotic prophylaxis to be effective, antibiotic levels should exceed the minimum inhibitory concentration (MIC) in the tissue and serum for the duration of surgery [64], [65] For this reason, a ntimicrobial agents are typically administered int ravenously within 60 minutes before surgical incision [63], [64], [66], [67] In general, 1 dose of antibiotics is sufficient, but for longer surgeries the number of doses may vary [68] The exact a ntibiotics administered to a patient vary based on the type of surgery, and are selected to be active against the pathog ens that are most lik ely to contaminate the surgical site [67] Preoperative Skin Preparation The goal of preoperative skin preparation is to significantly reduce the number of microorganisms on the skin surrounding t he incision site [22] There are three main types of antiseptics used in pre ope rative skin prepar a tion: alcohol based, chlorhexidine gluconate (CHG) based, and i odine based [3], [69] C ommercially available skin preparations use one or mo re of these types of antiseptics in their products (Table 2) [57] Iodine based antiseptics are effective agai nst fungi, viruses, and gram negative and gram positive bacteria [22] These antiseptics function through penetration of the cell wall followed by the oxidation/substitution of microbe intracellular components with free iodine [3], [22] A drawback of iodine based antimicrobial agent use is skin sensitivity exhibited by some patients. Povidone iodine (PI) is a widely used iodophor, which causes less irritation

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12 than iodine itself [22], [69] PI antiseptics are typically active for 2 hours, but they may become inactiv e upon contact with blood [57] CHG based solutions are active again st gram positive and gram negative bacteria, yeasts, anaerobes, and some viruses including human immunodeficiency virus (HIV) [3], [70] However, their activity against fungal microbes is lower than iodine based and alcohol based antiseptics [22] Due to the presence of quaternary ammoniums in its structure c hlorhexidine is a cationic compound that disrupts negatively charged bacterial cell wall leading to microbial death [71] Chlorhexidine gluconate exhibits antimicrobial activity for up to 6 hours [57] Alcohol based antiseptics are effective against gram negative bacteria, gram positive bacteria, fungi, and virus es [22] Alcohols denature proteins in the cell wall of microbes, which results in rapid lysis of the microbes [22] Despite the rapid action of alcohol based antiseptics, antimicrobial properties of these antisept ics are short lived due to evaporation of alcohol. One approach to improving the longevity of alcohol based antiseptics is to add zinc pyrithione (ZPT), which serves as a preservative [72] Table 2 Properties of various types of antiseptics and examples of commercially available products. Table adapted from Reichman et al. 2009 [57] Antimicrobial Coverage Antiseptic Mechanism of A ction G ram positive Bacteria G ram negative Bacteria Fungi Vi rus Onset Duration Examples A queous iodophor Oxidation/substitution b y fre e iodine E G G G Moderate 2 h Betadine A queous CHG Disrupt s cel l membranes E G F G Moderate 6 h Hibiclens Alcohol Denature s proteins E E G G Mos t Rapid None Alcohol iodine povacrylex Denature s proteins Oxidation/substitution b y fre e iodine E E ID G R apid 4 8 h Alcohol CHG Denature s proteins Disrupt s cel l membranes E E ID G R apid 4 8 h ChloraPrep CHG chlorhexidin e gluconate ; E e x cellent ; F fair ; G good ; ID insufficien t data; Betadine Microbicide Purdu e Product s L. P Stamford CT ; ChloraPrep CareFusion Inc. Leawood KS ; DuraPrep Surgica l Solution 3 M Healt h Care, St P aul MN ; Hibiclens Mlnlyc k e Healt h Car e Inc. Norcross GA

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13 Preoperative Surgical Hand Scrub To reduce risk of microbial contamination from the surgical team, individuals who have contact with anything in the sterile field must scrub their hands and forearms with an antiseptic [3], [57], [58] This is pe rformed immediately before donning of sterile gloves and gowns. The three types of antiseptics used for skin preparation may also be used for scrubbing [3], [57] In addition to these, para chloro meta xylenol based (PCMX) and triclosan based antiseptics may be used [3] Scrub duration varies based on the antiseptic used. Scrubbing for 3 to 5 minutes is ty pically required for CHG based and PI based antiseptics however, a study found hand rubbing with an alcohol antiseptic for 1 minute to be equally effective [57], [73] Intraoperative Skin Antisepsis : Surgical Incision Drapes While preoperative skin preparation reduces the number of microbes on the skin, it is not possible to sterilize the skin During surgery, bacteria residing deeper in the skin may recolonize the skin [74] T he purpose of intraopera tive skin antisepsis is to avoid infection by sequestering any remaining microbes on the skin to prevent their entry into the incision site [75], [76] S urgical incision drapes (SIDs) have been used for over 50 years to impede microbial recolonization and prevent contamination in the surgical incision site [77], [78] The first commercially available SIDs were simple plastic adhesive sheet s but over time SIDs have b een impregnated with antimicrobials, such as iodophor to increase their effectiveness against SSIs [79], [ 80] A m icrobial sealant has also been develop ed as an alternative to traditional SIDs [81], [82] IOBAN TM Tiburon, and InteguSeal are iodine common SIDs manufactured by 3M, Cardinal Health, and Kimberly Clark, respectively. IOBAN TM is an iodine impregnated drape, while Tiburon is three layer adhesive drape

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14 with no antimicrobial agent. InteguSeal is a microbial s ealant, which will be discussed later in this report. Even though iodine impregnated drapes have been demonstrated to reduce bacteria l contamination a review of seven s tudies found that there is no evidence that SSI rates are reduced with the use of plastic adhesive SIDs [60], [80] In fact, the review found that in some cases use of plastic adhesive SIDs increases SSI rates [60] There are numerous drawbacks to the use of c urrent adhesive SIDs. First, the placement of SIDs is time consuming and requires good tech nique [77] Improper placement may result in wrinkles and air bubbles, which would allow bacteria to recolonize these areas of the skin. SIDs typically c ome in specific size s and shapes, and in some cases they may not e, which may result in drape lift. Loss of adhesion leading to drape lift, is a common proble m with SIDs. A s tudy found that loss of adhesion at the skin edges of the incision site resulted in a 6 fold increase in SSI rates [83] It has al so been found that the type of skin preparation used affects the adhesion of SIDs [84] Another problem is that the use of non antimicrobial SIDs has been found to reduce the amount of time required for bacterial recolonization of the skin compared with no drape at all [76], [85] This indicated that SIDs create a warm moist environment at the skin surface which is favorable to bacterial proliferation. For this reason, it is important that SID s contain antimicrobial agents. A drawback of iodine impregnated drapes is the existence of patient allergies to iodine. Another disadvantage is that a ntimicrobial SIDs typic ally only contain a certain amount of leachable antimicrobial s which limits the a mount of time that they exhibit antimicrobial activity. In the case of i odine impregnated drapes all iodine is released after about 6 hours [86]

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15 There are several other drawbacks of SIDs associated with incision closure and drape removal. First, in most surgical procedures the drape is peeled back from the incision before the incision site is closed with sutures. Lifting of the incision drape during closure has been found to cause bacterial contamination of the surgical site [87] If SIDs are peeled back for wound c losure, it has recently been recommended that skin preparation be reapplied to the exposed area [87] Additionally, w hen SIDs are remo ved, a layer of epidermal cells adhere s to the drape and thus is removed as well (Figure 5 ). This exposes bacteria found in deeper layers of the skin. If the skin of the patient is fragile, such as in elderly patients skin avulsion inj uries may occur where large portions of epidermal cells are re moved upon SID removal [88] (Figure 5) Once SIDs have been removed, bacteria are free to recolonize the areas around and incision. Figure 5 A : Epidermal cell removal during SID lift off, which exposes bacteria found beneath the skin. B : Surgical incision drape after being peeled back in preparation for would closure. C : Skin avulsion injury from the SID removal on an elderly patient. [88] [90]

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16 Study Objective Despite efforts to reduce SSI occurrenc es, rates remain unchanged. Due to drawbacks of c urrent SIDs including time consuming placement, loss of adhesion, and loss of an epidermal cell layer during drape removal an antimicrobial reverse thermal gel was developed f or potential use as a SID. The main objective of this study is to evaluate a previously reported quaternized polyethyleneimine poly(N isopropylacrylamide) (Q PEI PNIPAAm or QPP ) copolymer [91] T he described Q PEI PNIPAAm was designed as a broad spectrum antimicrobial non leaching polymer that would have minimal toxicity and be easy to apply and remove as a surgical drape. Q PEI PNIPAAm has been characterized, demonstrated to have antimicrobial activity, and assessed for cell cytotoxicity [91] The aim of this study was to evaluate the performance of Q PEI PNIPAAm used as a surgical incision drape in a mouse skin incision surgery model.

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17 CHAPTER II BACKGROUND An timicrobials and Antimicrobial Resistance Microbial contamination is of great concern in a number of industries, including food packaging, household sanitization, water treatment, and the medical industry [92], [93] The emergence of drug resistant bacteria and the prevalence of healthcare associated infection s have further prompted the growth and development of the antimicrobial i ndustry. Major classes of antimicrobial agents include disinfectants, antiseptics, and antibiotics all of which typically use low molecular weight antimicrobial agents in their formulations Hypochlorites, hydrogen peroxides, silver salts, alcohols, tricl osans, and quaternary ammonium compounds (QACs) are common antimicrobial agents used in disinfectants [94] Unfortunately, disinfectant s negatively impact the environment, and t he use of antimicrobials contributes to the development of antimicrobial resistance which present s a challenge to the treatment of microbial infection s [94] [97] Another disadvantage o f antimicrobial agents with low molecular weight is their short term antimicrobial activity [93] Antimicrobial resistance occurs when a microbe remains unaffected by an antimicrobial agent at a concentrat ion that should be lethal [95] Some microbes have an inherent resistance to particular antimicrobials, but resistance can also be acquired through mutations or direct transfer of resistant genes by conjugation [95], [98] When antimicrobial s are used, susceptible microbes are killed, but resistant strains m ay survive and proliferate further propagating the existence of populations of resistant strains There are four main mechanisms by which bacteria become resistant: elimination of antimicrobials from the cell alteration of antimicrobial target s direct inactivation of the antimicrobial agent and

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18 alterations in metabolic pathways (Figure 6) Elimination of antimicrobials from the cell can occur through efflux pumps or decreased uptake of the antimicrobial. Und erstanding how bacteria acquire antim icrobial resistance is important for the development of new antimicrobials that minimize the risk of bacterial resistance [98] Figure 6 Mechanisms of antibacterial resistance. 1 a: Elimination of cellular antimicrobials using efflux pumps. 1b: Elimination of cellular antimicrobials by decreasing/blocking cellular uptake. 2: Alteration of antimicrobial target. 3: Direct inactivation of antimicrobials by cellular enzymes. 4: Alteration of metabolic pathways [99] Mechanisms of Antimicrobial Activity In general, antimicrobial agents inhibit cell wall synthesis, p rotein synth esis, or nucleic acid synthesis or they target the microbe cell membranes/ wall (Figure 7) [98] Antibiotics a class of antimicrobial agents, typically target a specific cellular process [100] To do this, m ost antibiotics pass through the bacterial cell envelope and impede biochemical pathways without affecting the structure of the cell envelope [101], [102] Mechanisms of antibacterial resistance allow bacteria to acquired resistance to antimicrobials that target specific intracellular processes. H owever, it is more difficult for bacteria to acquire resistance to

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19 antimicrobials tha t disrupt the cell envelope Resistance to disruption of the cell wall would require the modification of many proteins to achieve complete altera tion of the cell wall and membrane structure. Figure 7 Antimicrobial mechanisms of various antimicrobial agents against bacterial spores, chlorine releasing agents, respe ctively [103] To combat increasing antimicrobial resistance, new strategies for the development of antimicrobials have focused on targeting common bacterial characteristics that are less likely to contribute to the development of bacterial resistance. One such common characteristic is the existence of negatively charged cell walls in all bacteria There are two general types of

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20 bacteria and they are characterized by different cell wall structures : gram positive and gram negative bacteria (Figure 8) Gram positive bacteria have one inner cell membrane and an outer cell wall. The outer wall contained teichoic and lipiteichoic acid, which are negatively charged. In contrast, gram negative bacteria have an outer and inner cell membrane surrounding a peptidoglycan wall. Phospholipids and lipopolysaccharides of the outer membrane give gram negative bacteria their negatively charged cell envelope [100] Figure 8 Structure of gram nega tive and gram positive bacteria. Both types of bacteria contain negatively charged structures on their surface. Gram negative bacteria have negatively charged phospholipids and lipopolysaccharides on their surface. Gram positive bacteria have negatively ch arged teichoic and lipoteichoic acids on their surface [104] The common negative bacteria l cell wall charge has led to the st udy and development of polycationic antimicrobials. Polycationic antimicrobials are able to disrupt the cell envelope of gram positive and gram negative bacterial cells as well as the membrane of fungal cells with attrac tive electrostatic forces [105] It should be noted that mammalian cell membranes have less negative surface charge than bacteria, so cationic antimicrobials selectively target bacteria cells over human cells [106] The surface of mammalian cells primarily contains sphingomyelin (SM) and phosphatidylcholine (PC), which both have no net charge [107] This makes polycations ideal for use as antimicrobials as they will target bacteria and fungi without harming mammalian cells.

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21 Antimicrobial Polymers The development of polymeric antimicrobials has gained great interest in the last couple of decades [93], [97], [100] Antimicrobial polymers have several advantages over conventional antimicrobial agents. First, they have been demonstrated to contain long term activity and limited toxicity. Polymeric antimicrobials are also chemically stable, nonvolatile, and they do not pass through the skin [93] There are three general types of an timicrobial polymers: biocidal polymer, polymeric biocide, and biocide releasing polymer. Biocidal polymers have intrinsic antimicrobial activity, while polymeric biocides contain a polymer backbone with attached biocides. These two types of antimicrobial polymers are non leaching systems. Biocide releasing polymers are systems in which biocidal agents are trapped within a polymeric matrix to be released at some time [105] These leaching antimicrobial polymers are necessary when a biocide must enter a microbial cell to exhibit antimicrobial activity. Leaching antimicrobial polymers have a limited lifespan, while non leaching polymers with inherent antimicrobial activity exhi bit long term antimicrobial activity [97] This review will focus on biocidal polymers with inherent antimicrobial ac tivity. In general, ideal antimicrobial polymers should be active a gainst a broad spectrum of microbes, have long lasting antimicrobial activity, not be toxic or irritating mammalian cells, be synthesized easily and be associated with minimal antimicrobia l resistance [93] Antimicrobial polymers are typ ically based on polycations, which meet these listed requirements [97], [105] Polycatio n ic antimicrobial polymers cause microbial death by adsorption and penetration of the bacterial cell surface and wall adsorption onto the b acterial cell membrane, disturbance of the cell membrane, leakage of cytoplasmic contents,

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22 degradation of nucleic acids and cytoplasmic proteins, and lysis of the bacterial cell wall [93], [108] Commonly studied cationic antimicrobial polymers include quater nary ammonium compounds (QACs) an timicrobial pept ides (AMPs), chitosan, polyguanidines N halamines, and phosphonium and sulfo nium containing antimicrobials Quaternary Ammonium Compounds Quaternary ammonium compounds (QACs) are the most fre quently researched type of antimicrobial polymer s [109] Use of polymers containing QACs is advantageous owing to their chemical stability and reduced toxicity to the environment [110] They are organic cationic compound having a general structure of NR 4 + where N is nitrogen and R represents either an alkyl or an aryl group QACs are commonly used in clinical disinfectants, domestic cleaning products fabric softeners, and some products requiring antielectrostatic agent s such as shampoo [108], [111] QACs may be synthesized by a quaternization reaction. Quaternization reactions involve an irreversible reaction between tertiary amines and alkyl halid es (Figure 9) [108] Figure 9 Quaternizatio n of tertiary amine with an alkyl halide yielding a quaternary ammonium cation. In this chemical equation, R rep resents alkyl or aryl groups and X represents a halogen, such as Cl Br Fl or I

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23 The antimicrobial activity of QACs depends on the composition of the four organic R groups and the number of cationic nitrogen atoms [97] Inclusion of at least one long alkyl chain as an R group provides a hydrophobic structure for the penetr ation of the bacterial cell envelope [110] This increases the antimicrobial activity of the polymer and creates an amphiphilic QAC, which has the ability to attract bacteria with its cationic charge and penetrat e the bacterial envelope with its hydrophobic structures. When the hydrophobic tail of a QAC penetrates into the hydrophobic core of a bacterial membrane leakage of cell content occurs resulting in cell lysis [108] Polyethyleneimine Polyethyleneimine (PEI) is a cationic, nonbiodegradable, synthetic polymer, which has primary, secondary, and tertiary amines within its structure. Due to the large quantity of reactive amines in its structure PEI has been studied for use as an antimicrobial polymer. Particular attention has been paid to branched PEI, which has the capacity for high charge density [97] Lin et al. covalently attached PEI to a glass surface, and found that PEI alone was not antibacterial. However, t he study also fo und that alkylated PEIs conjugated to a glass surface exhibited bactericidal activity against both gram positive and g ram negative bacteria [112] Kiss et al. a lso found that quaternized PEIs have signific ant antimicrobial activity [113] Park et. al developed a N alkyl PEI paint, which killed gram positive S. aureus an d gram negative E. coli by rupturing the bacterial envelope upon contact [114] Alkyl PEI was also found to exhibit antifungal activity [115] A nother study found that N alkyl PEI was both harmless to mammalian cells and no apparent bacterial resistance was formed in successive generations of E coli and S aureus [116] The alkylation of PEIs increased its hydrophobicity and cationic charge density by generating quaternary amines This suggests

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24 that PEI alone does not contain antimicrobial activity due to inadequate levels of hydrophobicity and/o r c ationic charge [112] This is supported by a study by Ilker et al. where a series of nonamphiphilic cationic polymers and amphiphilic cationic polyme rs were synthesized In comp aring the two types of polymers nonamphiphilic cationic polymers which have no hydrophobic group, exhibited significantly less antibacterial activity [117] Thus antimicrobial activity of antimicrobial polymers is de pendent on the presence of both cationic charge and hydrophobic groups. Effect of Alkyl Chain and Molecular Weight on Antimicrobial Activity The a ntimicrobial activity of a particular polymer depends on a number of factories including alkyl chain length, molecular weight, ratio of alkyl to cationic groups, c harge density and counter ions [92], [93], [118] This review will address two o f these factors: alkyl chain length and molecular weight (MW) As previously mentioned, the antimicrobial activity of QACs requires electrostatic forces between the antimicrobial and the bacterial cell surface followed by penetration of the bacteria envelo pe by a hydrophobic alkyl tail. The length of the hydrophobic tail, which penetrates the bacterial envelo pe, is important to the antimicrobial activity of the polymer Generally, QACs with 8 18 carbon alkyl chains possess good antimicrobial activity [92] However g ram positive bacteria are mo re susceptible to antimicrobial penetration due to lo ose packing of the peptidoglycan layer and g ram negative a re gener ally less sensitive due to the presence of an additional outer membrane [97], [119] Gilbert et al. found that polymers with alkyl chain lengths of 12 14 carb ons provide optimum antibacterial activity against gram pos itive bacteria, while alkyl group s with 14 16 carbon s have better activity against gram negative bacteria [120]

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25 Lin et al. investigated the effect of alkyl chain length on the antimicrobial activity of alkylated PEI. PEI was covalently attached to a glass surface and alkylated with chains ranging from 2 18 carbons in length. Additionally, quaternized versions of these polymers were also synthesized in a reaction with iodomethane As the alkyl chain length increased from 2 to 6 carbons, so did the antimicrobial activity of both the methylated and unmethylated polymer. However, when the alkyl chain length was increased above 6 carbons to a maximum of 18 carbon alkyl chains, the antibacterial activity plateaued for methylated alkylated PEI, which contained greater quantities of quaternary ammoniums, and decreased for alkylated PEI [112] Alkylation with longer chains increases the hydrophobicity and penetrative ability of the polymer, but also decreases the accessibility of cationic amine groups, therefore a balance of charge and alkyl chain length is required for optim um antimicrobial activity. Longer alkyl chains may also result in aggregate clumps of hydrophobic tails, which would decrease the bacteri cidal activity. Molecular weight also plays a role in determining the antimicrobial activity of polymers. The optimal r ange of molecular weights for antimicrobial activity is specific to the polymeric system, but general molecular weight ranges have been noted. Ikeda et al. examined the bactericidal activity of polymethacrylates and poly(vinylbenzyl ammonium chlorides) wit h various molecular weights against gram positive S. aureus and t hey reported an optimal molecular weight range of 5x10 4 to 1 x10 5 Da [121] Lin et al. functionalized textiles with N hexylated and methylated PEI to examine its antibacterial and antifungal properties. This study also used PEI with molecular weights of 0.8, 2, 25, and 7 50 kDa to examine the influence of molecular weight on antimicrobial activity. It was found that antimicrobial activity against S. aureus increased with increasing molecular weight, with 750

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26 kDa having the highest bacterial activity. It should be noted the alkylated polymers with low molecular weight PEI did contain some antimicrobial activity [115] With regard to molecular weight, high molecular weight alkylated PEIs appear to have better antimicrobial activity. Biocompatibility of Quaternary Ammonium Compounds As mentioned previously, polycationic polymers, such as QACs, selectively target bacter ia cells over human cells due to the negative cell surface charge of bacteria [106] In contrast to bacteria cell envelopes, mammalian cell surfaces are composed primarily of components with no net c harge [107] As a result, it is thought that QACs do not penetrate mammalian cells due to reduced attractive electrostatic forces when compared with electrostatic forces between QACs and bacterial cell envelopes. These attractive electrostatic forces are essential to the insertion of the hydrophobic tail into the cell. However, despite the fact that QACs should not direc tly penetrate mammalian cells, the presence of QACs may alter the cell microenvironment, which in turn may negatively impact the cell. As an example, one way that QACs may alter the microenvironment is through increasing the pH, which will affect both bacterial and mammalian cells. A study by Peng et al. examined th e cytotoxicity of a polymer, hydroxypropyltrimethyl ammonium chloride chitosan (HACC), with various degrees of substitution with quaternary ammoniums. This study found that HACC with 6% and 18% quaternary ammonium substitution did not reduce cell enzymatic activity, meaning they were not cytotoxic. In contrast, HACC with 44% substitution was cytotoxic. The trends of this study may demonstrate a positive correlation between the degree of quaternization and cytotoxicity. This study also found that increasing quaternization leads to higher antimicrobial activity

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27 [122] Theref ore, an optimal polymer would be a balance of maximizing antimicrobial activity while minimizing cytotoxicity. A second study by Lu et al. examined the skin irradiation and acute toxicity of dimethylaminoethyl methacrylate (DMAEMA) quaternized with benzyl chloride (BC). To examine skin irritation, 4 rabbits were inoculated with a DMAEMA BC 5 times a day for 7 days, and no irritation was observed during the test. To examine the acute toxicity of DMAEMA BC, 40 rats ingested 4 different doses of the polymer an d it was found that the two lower doses did not visibly effect the rats, while rats in the two higher dose groups gradually died. Therefore, this study concluded that DMAEMA BC was not a skin irritant, and not acutely toxic at lower doses [123] This study indic ated that while QACs are not irritants on the skin surface, however, they may be acutely toxic at higher dosages. However, it should be noted that the dosage at which a QAC may be acutely toxic will vary based on the exact polymer comp osition, and typicall y the amount of polymer required to kill bacteria effectively exhibits low toxicity [123], [124] Reverse Thermal Gels Increased bacterial contamination caused by im proper placement is of concern when using traditional SIDs such as Ioban TM Placement of traditional SIDs may be difficult if the drape does not conform well to the shape of the skin and air bubbles and wrinkles may lead to bacterial recolonization under the drape As su ch, there is a need for an effective antimicrobial system for intraoperative skin asepsis that is eas y to apply and has minimal chance of misapplication Kimberly Clark developed a cyanoacrylate microbial sealant named InteguSeal Microbia l Sealant which is an alternative to current SIDs. This microbial sealant is applied

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28 to the skin as a liquid following skin preparation and prior to surgery. Upon application, e xposure to moisture and proteins on the patient s skin causes the cyanoacrylat e to polymerize and create a n adhesive barrier on the epidermal surface [125], [126] Similar to current SIDs, this adhesive barrier is intended to sequester bacteria on the surface of the epidermis [82], [127], [128] Although waterproof, the sealant is breathable, which prevents the development of a warm moist environment under the sealant [126], [129] It is important to note that InteguSeal itself is not antimicrobial [127] Several studies examined the efficacy of InteguSeal compared with the use of no intraoperative asepsis Dromzee et al. found no evidence that InteguSeal reduces SSI rates and Falk Brynhildsen et al. found no difference in i ntraoperative bacterial growth with the use of InteguSeal [128], [130] However, Dohmen et al. reported reduced occurrence of SSIs with the use of InteguSeal in 676 CABG procedures [126] A review of seven studies i nvolving cyanoacrylate microbial sealants found insufficient evidence that InteguSeal reduces SSI rates [131] Although InteguSeal is easy to apply, a polymer that acts as both a sealant and an antimicrobial agent may provide a better system Smart polymers are stimuli responsive polymers, which change in respons e to external s timuli, such as temperature light, or pH. Reverse thermal gels (RTGs) are stimuli responsive po lymers that gel when heated above a particular temperature. RTGs are frequently used in the biomedical industry. Poly(N isopropylacrylamide) (PNIPAAm) is one of the most frequently researched RTGs owing to its sudden gelation near body temperature. PNIPAAm dispersed in water undergoes a hydrophilic to hydrophobic phase transition resulting in gel formation at the lower critical solution temperature (LCST) (Figure 10) The LCST of PNIPAAm typically occurs at 32 C [132], [133]

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29 Figure 10 A: Behavior of PNIPAAm chains below (left side) and above (right side) LCST. B: PNIPAAm chemical structure with labeled hydrophilic and hydrophobic regions. Dincer et al. investigated NIPAAm PEI block copolymers for use as a gene delivery vector. Addition of PEI to PNIPAAm was found to increase the LCST from approximately 29 C for PNIPAAm to 36 40 C, which is close to body temperature. The increase in LCST can be attribut ed to the addition of hydrophilic PEI chains. Results of this study also indicated that PNIPAAm PEI had increased solubility in water when low molecular weight PNIPAAm was used [134] In recent years, interest in antimicrobial reverse thermal gels has grown [132], [133], [135], [136] Dizman et al. developed a NIPAAM and quaternized methacrylamide derivative polymer, which contained antimicrobial and thermos responsive ch aracteristics [132] Mattheis et al. also developed an antibacterial RTG coating comp osed of a copolymer of NIPAAm and 2 aminnoethyl methacrylate [135] Most recently Yu at al. and Pappa s et al. investigated p phenylene ethynylene/PNIPAAm derived films for use as self sterilizing surfaces [133], [136]

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30 CHAPTER III PREVIOUS WORK Q PEI PNIPAAm has previously been designed and studied as a potential surgical incision drape. A synthesis method was established and variations of Q PEI PNIPAAm were characterized. Additionally, antimicrobi al testing, and cytotoxicity assays were completed for Q PEI PNIPAAm [91] Q PEI PNIPAAm LCST LCST was determined for 20, 30, and 50% conjugations of PEI P NIPAAm and Q P EI P NIPAAm with LCST being defined as the temperature where 50% reduction in UV transmittance occurs (Table 3 ). It was found that the addition of PEI increased the LCST, and alkylat ion and quaternization decreased the LCST. This can be explained by the chemical structure of bo th additions. Hydrophilic interactions dominate below LCST, so the addition of hydrophilic PEI i ncreases the low temperature range where hydrogen bonding domina tes thus increasing the LCST. Likewise, hydrophobic alkyl chains added during quaternization extend the high temperature range at which the polymer will be a gel, thus decreasing the LCST. [91] Table 3 LCST values reported for various co njugations of PEI PNIPAAm and Q PEI PNIPAAm Antimicrobial Activity of Q PEI PNIPAAm In vitro bacterial activity of Q PEI PNIPAAm was assessed using time kill curves against four species of bacteria: S. aureus MRSA Staph. epidermidis and E. coli Activity was assessed against stationary phase (non multiplying) bacteria as bacteria rarely encounter

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31 conditions that support log phase growth. For this test Q PEI PNIPAAm with 30% conjugation of PEI amines with PNIPAAm was dissolved in a bacterial solut ion with a known concentration, and aliquots were taken at 30 minute time intervals for a duration of 2 hours. Q PEI PNIPAAm was found to achieve an 8 log MRSA ( S. aureus subsp. aureus Mu3 ) reduction in 60 minutes, a 7 log Staph. epidermidis reduction in 30 minutes, an 8 log E. coli reduction in 120 minutes, and a 5 log S. aureus reduction in 30 minutes (Figure 11). PEI PNIPAAm was also found to have some antibacterial activity against S. aureus, Staph. epidermis, and E. coli It should be noted that since the polymer samples were placed directly in solution, the bacteria was surrounded with antimicrobial polymer in three dimensions. However, only the surface of SIDs contacts bacteria during surgery, therefore, it is necessary to assess the antimicrob ial activity of a two dimensional layer of Q PEI PNIPAAm. Figure 11 PEI PNIPAAm and Q PEI PNIPAAm were added to stationary phase bacteria, and samples were taken at 0, 30, 60, and 120 minutes to det ermine bacterial concentration s Kill curves were constr ucted for the following bacteria: A: S aureus subsp. a ureus Mu3 (MRSA/hetero VISA) B: Staph. epidermidis C: E. coli D: S aureus [91]

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32 Cytotoxicity Cytotoxicity was assessed using fibroblasts. Films of Q PEI PNIPAAm (20% conjugation), PEI PNIPAAm (20% conjugation), chlorhexidine 2%, plain media, and media containing 5% DMSO were coated with fibroblasts. A 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide (MTT) assay then was used to determine cell viability. No statistical di fference was found between Q PEI PNIPAAm (20% conjugation) and chlorhexidine 2 %, which is used as a surgical skin preparation (Figure 12 ). Figure 12 MTT assay assessing the cytotoxicity of Q PEI PNIPAAm (QPP) No statistical difference between the negative control and cells plated on PNIPAAm and PEI PNIPAAm was observed. No statistical significance was found between cells plated on QPP and Chlorhexidine. Statistical significance was found between experimental groups and the p ositive control (** denotes p < 0.05 and *** denotes p > 0.05) [91]

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33 CHAPTER IV HYPOTHESIS AND SPECI FIC AIMS Hypothesis Based on previous research indicating that Q PEI PNIPAAm has antimicrobial activity and the ability to gel near body temperature, it was hypothesized that a two dimensional Q PEI PNIPAAm surface would have antimicrobial activity and its effectiveness as a surgical drape would be comparable to commonly used SIDs, such as IOBAN TM Specific Aims The first specific aim was to synthesize and character ize the Q PEI PNIPAAm formulation chosen for this study It was particular ly important to confirm conjugation of PEI and quaternization of the polymer as these steps give the polymer its antimicrobial properties The second specific aim was to evaluate the antimicrobial killing capacity of the surface of Q PEI PNIPAAm a gainst five relevant bacterial strains : Corynebacterium amycolatum Escherichia coli, Staphylococcus aureus MRSA and Staphylococcus epidermidis Evaluation included the development of time kill curves for different weight percent solutions of Q PEI PNIPAAm coated on a glass surface, and an in vitro simulatio surgical drape. The third specific aim was to conduct an animal model to assess the performance of Q PEI PNIPAAm as a surgical drape and to deter mine if the polymer instigates an immune response A murine incision model was completed to compare the effectiveness of Q PEI PNIPAAm and an IOBA N TM drape in killing and trapping different types of bacteria. Subcutaneous injections of Q PEI PNIPAAm were completed to examine the immune response prompted by the polymer. Tissue surrounding the injection site was harvested, cry o sectioned, and stained for the presence of macrophages.

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34 CHAPTER V MATERIALS AND METHOD S Materials N isopropylacrylamide (NIPAAm) was purchased from Toky o Chemical Industry (Chuo ku, Tokyo, Japan). N ,N dimethylformamide (DMF) was purchased from EMD Millipore (Billerica MA, USA ). 4,4' a zobis(4 cyanovaleric acid) (ACA) N hydroxysuccinimide (NHS), sodium bicarbonate, 1 bromohexane, TWEEN 20 Triton TM X 100 globulins from bovine blood and bovine serum albumin (BSA) were purchased from Sigma Aldrich (St. Louis, MO, USA) N (3 dimethylamino propyl) ethylcarbodiimide hydrochloride ( EDC), polyethyleneimi ne (PEI) molecular weight (MW) 10,000 anhydrous methanol iodomethane, agar powder, lysogeny broth (LB) modification) and chloroform d were purchased from Alfa Aesar (Ward Hill, MA, USA ). Anhydrous diethyl ether was purchased from Fisher Scientific (Pittsburgh, PA, USA). Ioban TM 2 was purchas ed from 3M ( St. Paul, MN, USA). Ketoprofen saline, and isoflurane were purchased from MWI Veterinary Supply (Boise, ID, USA). Coated Vicryl 4 0 sutures were purchased from Ethicon (Somerville, NJ, USA). Adult C57BL/6J mice were purchased from The Jackson Laboratory ( Bar Harbor, Maine, USA ). 10 % formalin was purchased from JT Baker (Phillipsburg, NJ, USA). Sucrose (RNASE & DNASE free) was purchased from VWR Life Science (Radnor, PA, USA ). Optimal cutting temperature (OCT) compound was purchased from Sakura (Torrance, CA, USA). Phosphate buffered s aline (PBS) was purchased from HyC lone Laboratories, Inc. ( South Logan Utah, USA ). Anti CD68 antibody (ab125212 ) was purchased from Abcam (Cambridge, UK ). Alexa Fluor 594 (goat anti rabbit

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35 IgG) was purchased from Life Technologies (Carlsbad, CA, USA). Dapi flouro mount G was purchased from Electron Microscope Sciences (Hartfield, PA USA ). Polymer Synthesis PNIPA A m Synthesis PNIPAAm was conjugated using radical polymerization with an azo bis initiato r NIPAAm (5.0 g, 44.2 m mol) and ACA (0.186 g, 0.664 m mol) were dissolved in anhydrous methanol ( 25 mL) and the mixture was bubbled with nitrogen gas for 30 minutes at room temperature A reflux condenser apparatus was set up a nd the solution was stirred for three hours at 68 C Next the solution was precipitated into Milli Q water at 60 C in a dropwise manner Following precipitation, the warm water was discarded and 40 mL of cold Milli Q water was added to the precipitate. The precipitate was allowed t o dissolve into the water in a cold room at 4 C. The r esulting solution was purified using 3500 kDa MWCO dialysis tubing, which was placed in Milli Q water with stirring for 48 hours. The solution was dried utilizing lyophilization, which yielded purified PNIPAAm. P EI PNIP A Am Synthesis PEI PNIPAAm was synthesized using EDC/NHS chemistry In preparation for completing this reaction under anhydrous conditions PEI (500 mg) was lyophilized. After 24 hours of PEI lyophilization, PNIPAAm (700 mg) was dissolved i n anhydrous DMF (5 mL) A 1.2 molar excess of EDC (0.0161 g) and NHS (0.0097 g) to PNIPAAm carboxylic acid groups was then added to the mix ture. The mixture was stirred in a dark environment at room temperature for 24 hours under nitrogen (N 2 ) After 48 hours of lyophilization, PEI was dissolved in anhydrous DMF (5 mL) with stirring. The activated PNIPAAm was then aspirated into a syringe and slo wly added to the PEI/DMF solution The reaction was left for

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36 an additional 24 hours with stirring at room tempe rature A rotary evaporator was then used to remove some of the DMF. Subsequently, the one half of the reaction solution was precipitated in cold ethyl ether to remove unused reactants and any remaining DMF Excess ethyl ether was poured off and this proc ess was repeated for the remaining reaction so lution Following precipitation, a rotary evaporator was once again used to remove excess ethyl ether. Milli Q w ater (10 mL) was added to the product and the flask was placed in a cold room with a stir bar to allow the product to dissolve. After the polymer was dissolved, the solution was placed in 12 14 kDa dialysis tubing for 48 hours. The purified polymer was frozen and lyophilized to yield the final PEI PNIPAAm product. Q PEI PNIPA A m Synthesis PEI PNIPAAm p rimary amines were converted to quaternary ammoniums by quaternization In this process first primary amines were alkylated with 1 bromohexane, which converted primary amines to tertiary amines. Then, an alkyl halide was added to convert tertiary amines to quaternary ammonium. Molar excess calculations were based on the number of primary amines in PEI PNIPAAm. Sodium bicarbonate (0.066 g, 3 molar excess) was added to anhydrous DMF (8 mL) with stirring. PEI PNIPAAm (100 mg) was added then to the solution and allowed to dissolve. A 20 molar excess of 1 bromohexane (0.7386 mL) was added and the mixture was reacted at 95 C with stirring and with a cold water reflux condenser apparatus. After 48 hours, the temperature was reduced to 60 C and 20 molar excess iodomethane (0.3287 mL) was added to the reaction. The mixture was allowed to react for an additional 12 ho urs followed by cooling to room temperature Subsequently, the solution was rotary evaporated to remove as much DMF as possible. A se ries of three precipitations in cold diethyl ether were used to remove DMF and unreacted

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37 polymer. The rotary evaporator was then used to ensure removal of all diethyl ether, and 10 mL of Milli Q water was added to the product. After the polymer was dissolv ed in water, the solution was added to 12 14 kDa MWCO dialysis tubing for 48 hours. Lyophilization was used to isolate the final Q P EI P NIPAAm product. Polymer Characterization Proton Nuclear Magnetic Resonance ( 1 H NMR) Proton nuclear magnetic resonance ( 1 H NMR) was completed with a Varian Inova 500 mHz NMR Spectrometer. PEI, PNIPAAm, PEI PNIPAAm, and QPP samples (3 5 mg) to be analyzed were dissolved in 600 L of chloroform d. Spectra were proc essed and analyzed using iNMR reader software Online Advanced Chemistry Development i Lab was used to predict 1 H NMR spectra pea ks associated with each polymer at 5 MHz LCST Determination The LCST was measured to assess the gelling properties of PNIPAAm, PEI PNIPAAm, and Q PEI PNIPAAm LCST data was acquired using a CARY 100 Bio UV Visible Spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA) Polymer samples of 5 wt% were prepared by dissolving the polymers in Milli Q water Absorbance values were measured at a 500 nm wavelength for tem peratures ranging from 25 45 Bacteria Preparation and Dilutions (5 mL) was inoculated with bacteria from a frozen monoclonal st ock solution and incubated at 37 C for 16 hours with shaking at 250 rpm. A fter 16 hours, the bacteri al suspension was centrifuged for 5 minutes at 5,000 rpm and the LB broth was poured off. The bacterial pellet was then res uspended in sterile PBS and a Genesys 10S UV VIS (Thermo Scientific, Waltham, MA, USA) was used to measure the absorbance of the sol ution

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38 at 600 nm. The absorbance was used to estimate the bacterial concentration of the suspension and the solution was then diluted to the desired concentration. To determine the exact bacterial concentration 20 L aliquots were taken from the bacterial solution and 10 fold serial dilutions were performed in triplicate in a 96 well plate. Aliquots (40 L) from each well were plated on LB agar dishes and allowed to dry. Plates were incubated at 37 C overnight, and colonies were counted. The bacterial co ncentration was then calculated based on the following equation: Antimicrobial Q P EI P NIPAAm Surface Test s Anti microbial A ctivity of a 2D Q P EI P NIPAAm Surface in a 3D Bacterial Suspension Bacteria was proliferated, the concentration was estimated, and dilutions were completed using the previously listed method. Then 5 conical tubes (15 mL) were filled with 3 mL of a 10 8 CFU/mL bacterial solution. Spray bottles (3 mL) were filled with Q PEI PNIPAAm solutions with concentrations of 1.25, 2.5, 5, and 7.5 wt% dissolved in 70% ethanol G lass slides were cut lengthwise into strips with a 1 cm width. In a 37 C warm room, both sides of 2 x 1 cm area s at the end of four s li de s were coate d with one spray (~100 L) each of 1.25, 2.5, 5, and 7.5 wt% Q PEI PNIPAAm solution s As a control an additional slide was cleaned with 70% ethanol. Slides were placed in the co nical tubes containing the bacterial solutions w ith the polymer coated side submerged in the solution Therefore, a two dimensional (2 D ) coated Q P EI P NIPAAm surface was submerged in a three dimensional ( 3D ) bacterial suspension. Each tube was then incubat ed at 37 C for 2 hours with shaking at 250 rpm. To quantify the antimicrobial capacity of the QPP surface, 20 L aliquots were taken from each vial at 0, 30, 60, and 120 minutes. At each time point 3 aliquots were

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39 removed from each tube to complete 10 fol d serial dilutions in a 96 well plate. Aliquots from each vi al and time point were plated on LB agar to determine the concentration of bacteria over time in each tube according to Equation 2 This experiment was completed for five bacteria l strains that ha ve all been associated with SSIs : Corynebacterium amycolatum (ATCC 49368 ), Escherichia coli C3000 (ATCC 15597) Staphylococcus aureus (ATCC 6538), Staphylococcus aureus subsp. aureus Mu 3 ( MRSA strain with vacomycin intermediate resistance ), and Staphylococcus epidermidis (Ron Gill Collection). Antimicrobial Activity of a 2D Q P EI P NIPAAm Surface against 2D Bacterial Samples C. amycolatum E. coli, MRSA, S. aureus, and Staph. epidermidis were proliferated and the solution concentration with estim ated and determined using the previo usly listed method. Stock bacterial solutions (0.5 mL) with concentrations of 10 9 CFU/mL in Lysogeny broth (LB) were made for each bacteria l strain A 200 L v olume of each 10 9 CFU/mL stock solution was plated in row A of a 96 well plate. Then, 10 fold dilutions were completed up to row G. This resulted in a plate of stock solutions of 10 9 10 8 10 7 10 6 10 5 10 4 and 10 3 CFU/mL concentrations for each of the five bacterial strain. S am ple s (3 L each ) of a ll 35 stock solutions were then plated on a single LB agar plate in a grid pattern with 5 rows for each bacterial strain and 7 columns for each initial bacterial concentration This plate served as a control plate and was incubated at 37 C for 48 hours before being assessed for bacterial growth To simulate the antimicrobial activity of Q PEI PNIPAAm against bacteria on surface of the ski n, three additional plates with the 35 sample grid (3 L each) pattern were made and bacterial samples were allowed to completely dry The plates were then placed on a 37 C hot plate and 1 mL of 5 wt% PNIPAAm, 5 wt% PEI PNIPAAm, or 5 wt% Q PEI PNIPAAm

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40 was sprayed on each plate and allowed to gel. The plates were then incubated at 37 C and examined f or bacterial growth between the polymer and the LB agar after 48 hours. To simulate antimicrobial activity of Q PEI PNIPAAm against bacteria that may fall onto the surface of a drape during surger y, three plates were then placed on a 37 C hot plate and 1 mL of 5 wt% PNIPAAm, 5 wt% PEI PNIPAAm, or 5 wt% Q PEI PNIPAAm was sprayed on each plate and allowed to gel. While the gelled plated were still on the hot plate, the 35 sample (3 L each) grid pattern was plated on top of the gelled polymers. The p lates we re then incubated at 37 C in a humid sealed container and examined for bacterial growth on top of the polymer after 48 hours. Skin Incision Animal Model Skin Incision Surgery The skin incision surgery was approved by the Institutional Animal Care and Use Committee (IACUC). A total of 48 C5 7BL/6J mice were used in this portion of the study and 3 mice were used per treatment group (Q PEI PNIPAAm /b acteria spray IOBAN TM 2 drape/ b acteria spray n o drape /b acteria spray, no drape/no bacteria spray ) All groups involving bacterial spray were completed for C. amycolatum E. coli MRSA S. aureus and Staph. epidermidis Adult C5 7BL/6J mice ranging from 12 to 24 weeks old were allowed 7 days to acclimate on a 14/10 hour light/dark cycle with access to water and food ad libitum. Mice were anaesthetized with continuous inhalation of isoflurane and oxygen. Initially, 5% isoflurane in oxygen to induce loss of consciousness followed by reduction to 2% isoflurane in oxygen for the duration of the procedure Preoperative s ubcutaneous injections of ketoprofen (5 mg/kg) were administered to minimize pain following the procedure. Fur below the base of the neck and 3 cm down the back of the mo use was removed with clippers.

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41 The shaved area was then washed with warm water and disinfected with alcohol swabs. After skin preparation, 0.5 1 mL of a 10 8 CFU /mL bacterial solution was sprayed onto the shaved area and allowed to dry. After inoculation of bacteria to simulate extreme operating room conditions, either 5 wt % Q PEI PNIPAAm in 70% ethanol (0.5 1 mL), an IOBAN TM 2 drape, or no drape was applied to the shaved area. As a positive control, a group was also completed with no drape and no bacterial inoculation. Next, a 1 cm skin incision was performed in the prepared area of the back, and the open incision was irrigated with 0.5 mL of sterile saline Next, the incision was swabbed with cotton, which was used to inoculate an agar plate to assess bacte rial presence in the incision site. T he incision was then closed with 1 2 continuous sutures, and m ice were monitored closely and allowed to recover. The incision was swabbed for bacterial growth again at times 0, 10, 20, 30, 40, 50, 60, 75, 90, 105, and 1 20 minutes following closure. After the 120 minute swab, mice were euthanized by CO 2 inhalation followed by cervical dislocation. Quantification of Bacterial Swabs Agar plates with bacteria swabs from the skin incision surgery were placed in a 37 C warm ro om for 24 hours or 48 hours in the case of C. amycolatum to allow bacteria to grow. Following bacterial growth, all LB agar plates were photographed for analysis of bacterial colony presence Bacterial swabs were quantified by counting visible CFUs on each plate This was completed using Image J software to process photos and detect particles Q PEI PNIPAAm Immune Response In Vivo Subcutaneous Injections The subcutaneous injections were also approved by IACUC. A total of 12 C5 7BL/6J mice were used in this portion of the study, and 3 mice were used per injection group (5 wt%

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42 Q PEI PNIPAAm in saline and sterile saline) for time points of 24 hours and 7 days. As with the skin incision study, adult C5 7BL/6J mice ranging from 12 to 24 weeks old were allowed 7 days to acclimate on a 14/10 hour light/dark cycle with access to water and food ad libitum. Mice were then administered 60 L of 5 wt % Q PEI PNIPAAm in saline or 60 L of sterile saline in the right dorsal flank. Euthanasia was carried out after 24 hours or 7 days by CO 2 inhalation followed by cervical dislocation. After euthanasia, t issue surrounding t he injection site was harvested Immunohistochemistry After the skin incision procedure, harvested tissue was fixed overnight in 10% formalin. T issue samples were then washed 3 time s with 1X PBS for 3 minutes each wash Washed tissue was placed in 30% sucrose for 48 hours to cryoprotect the sample Next tissue was cut crosswise and embedded in OCT compound with the cut edge facing down in the mold Embedded tissue was frozen at 80 C Tissue samples were then cryosectioned into 5 m sections, whi ch were placed on glass slides. The following protocol was used to stain macrophages with CD68 antibody. First, slides with tissue s ection s were fixed in 10 % formalin for 10 min and washed 3 times for 5 min each in a washing buffer (1X PB S with 0.1 % m/v Tween ) Next, tissue sections were permeabilized for 10 minutes with a permeabilizing buffer (1X PBS with 0.5% m/v Triton X 100). Tissue sections were once again washed with the washing buffer 3 times for 5 minutes each followed by blocking with a blocking buffer (0.25% Triton X globulins in 1X PBS) for 60 minutes to prevent non specific binding Next, tissue s ections were stained with anti CD68 antibody (1:500 in blocking buffer) overnight at 4 C washed 3 times with the washing buffer for 5 minutes each, and stained with Alexa Fluor 594 (1:500 in

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43 blocking buffer) for 60 minutes Following staining, tissue sect ions were washed 3 times with washing buffer and 3 times with DI water for 5 minutes each wash. Dapi flouromount G and glass coverslips were then added to each slide. Confocal images of stained slides were taken using a Zeiss LSM 780. Four to five images were taken for each mouse. Images where quantified using Zen 2.3 blue edition to select 250 x 250 pixel re gions of interest (ROI) for each photo. The number of macrophages in each ROI was then counted and converted to counts per area of tissue. Statistica l Analysis Resu lts are presented as the mean the standard error of the mean. Statistical significance was determined through computation of the analysis of variance (ANOVA) followed by two t ailed t test s when applicable. Results were considered statistically significant when p < 0.05. Sample size estimates were calculated using a power analysis based on the significance level alpha a target power, a desired sensitivity to detection o f the true mean an effect size, and the variance of the initial study. The first three variables were targeted to be 0.05, 0.8, and 25%, respectively. The effect size and variance were calculated for each sample size estimate.

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44 CHAPTER VI RESULTS AND DISCUSSI ON Q P E I P NIPAAm Reaction Mechanism The synthesized Q PEI PNIPAAm was designed to maximize antimicrobial activity and gel around body temperature PNIPAAm gives the polymer reverse thermal gelling properties and thus the ability to gel close to body temperature. Addition of PEI and q uaternization of PEI PNIPAAm gives the polymer antimicrobial activity due to the addition of cationic quaternary ammoniums as well as hydrophobic alkyl chains (Figure 13) Figure 13 Reaction mechanism for the synthesis of Q PEI PNIPAAm.

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45 Polymer Characterization Characterization of PEI, PEI PNIPAAm, and Q PEI PNIPAAm using 1 H NMR Synthesis of Q PEI PNIPAAm was completed as described in the methods section. As shown in Figure 13, branched PEI makes up the backbone of the dev eloped antimicrobial reverse thermal gel. It was important to confirm the conjugation of PNIPAAm to PEI and the quaternization of PEI PNIPAAm as both were vital to the desired characteristics of Q PEI PNIPAAm Polymer synthesis was confirmed by 1 H NMR (Nu clear Magnetic Resonance) at 500 MHz in chloroform d (CDCl 3 ). First, t he structure of purchased branched P EI was con firmed (Figure 14). Peaks associated with primary, secondary, and tertiary amines were present around 2.8, 2.7, and 2.6 ppm, respectively. P eak assignments were supported by literature and NMR predictions from Advan ced Chemistry Development [137] Figure 14 1 H NMR spectrum of branched PEI confirming its molecular structure. Next 1 H NMR was used to confirm the conjugation of PNIPAAm to PEI. In addition to PEI peaks, peaks at 1.14 and 4.0 ppm supported the presence of NH CH 2 ( CH 3 ) 2

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46 Figure 15 1 H N MR spectrum of PEI PNIPAAm confirming the addition of PNIPAAm to PEI. Finally 1 H NMR was used to confirm the quaternization of PEI PNIPAAm. Additional peaks observed at 0.9 and 1.3 ppm confirmed the presence of al kyl chains. Figure 16 1 H NMR spectrum of Q PEI PNIPAAm con firmed th e conjugation of alkyl chains and the presence of a slight peak at 3.6 ppm indicated the presence of quaternary ammoniums

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47 Lower Critical Solution Temperature (LCST) LCST is the temperature at which a material with reverse thermal gel properties undergoes a phase transition. At temperatures below the LCST, reverse thermal polymers in aqueous solutions are dominated by hydrophilic interactions. Above the LCST, hydrophob ic interactions dominate, which results in gel formation. The formation of gel alters the light transmittance of the polymer. Therefore, UV visible spectroscopy can be used to determine the LCST of temperature responsive polymers As previously discussed, PNIPAAm has the ability to gel near body temperature For this study it was important to confirm that Q PEI PNIPAAm also possessed this property Use of Q PEI PNIPAAm as a surgical drape requires that the polymer quickly gels on the surface of the skin pr ior to surgery. The LCST of PNIPAAm, PEI PNIPAAm, and Q PEI PNIPAAm were measures with UV visible spectroscopy (Figure 17) Figure 17 LCST of polymer samples. A: PNIPAAm LCST. B: PEI PNIPAAm LCST. C: Q PEI PNIPAAm LCST. D: Gelled Q PEI PNIPAAm.

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48 PNIPAAm and PEI PNIPAAm in water are colorless clear solutions below the ir LCST s (Figure 18) When the y gel, both polymers form white opaque gels. Therefore, light transmittance is high below the LCST and low above the LCST. LCST valu es are commonly calculated as the temperature at which a 50% change in transmittance occurs The LCSTs for PNIPAAm and PEI PNIPAAm were 35.4 C and 35.5 C, respectively. It should be noted that 5 wt% solutions of PNIPAAm and PEI PNIPAAm did not form stable gels that conformed to the shape of the vial. The unstable gelling of PNIPAAm and PEI PNIPA Am may be attributed to the reduced molecular weight of PNIPAAm used in this research. Figure 18 Appear ance of 5 wt% solutions of PNIPAAm, PEI PNIPAAm, and Q PEI PNIPAAm above and below the LCST. Below the LCST, PNIPAAm and PEI PNIPAAm are transparent solutions and Q PEI PNIPAAm is a yellowish opaque solution. Above the LCST, PNIPAAm and PEI PNIPAAm form opaque unstable gels, which d o n ot retain the shape of the vial, and Q PEI PNIPAAm forms a stable opaque gel. Q PEI PNIPAAm in water is a yellowish opaque solution below LSCT and its appearance is the same to the naked eye after gelling However, it was found that there is a distinct change in the transmittance of Q PEI PNIPAAm when it gels. As the temperature

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49 increases from 25 C to 35 C the UV transmittance o f Q PEI PNIPAAm also increases, but upon gelation of Q PEI PNIPAAm the transmittance remains constant with increasing temperatu res Using the above definition the LCST of Q PEI PNIPAAm used in this research was calculated to be 29.5 C. However, it should be noted that this method of LCST calculation assumes somewhat constant transmittance values above and below the LCST. In the c ase of Q PEI PNIPAAm, the transmittance below LCST is not constant with temperature changes and as a result the LCST c alculated for Q PEI PNIPAAm may not be accurate Despite the potential inaccuracy in the measurement of Q PEI PNIPAAm LCST, UV visible spectroscopy demonstrates that this formulation of Q PEI PNIPAAm is a fully formed gel at and above 35 C The exact temperature at which this transition occurs is uncerta in, but confirmation of stable gel formation was confirmed upon removal of the sample from the spectrometer (Figure 17 ) Normal body temperature ranges between 36.1 C and 37.2 C so this formulation of Q PEI PNIPAAm is a gel at temperatures slightly lower than body temperature and therefore is ideal for use as a surgical drape [138] Antimicrobial Q PEI PNIPAAm Surface Test s Antimicrobial Activity of Q PEI PNIPAAm Surface in a Bacterial Suspension In vitro antimicrobial studies were conducted to quantify the antimicrobial killing capacity of the surface of Q PEI PNIPAAm and determine the optimal polymer solution concentration Previous studies of Q PEI PNIPAAm investi gated polymer activity below LCST by mixing the solution based polymer with bacteria and constructing time kill curves [91] Before using Q PEI PNIPAAm as a surgical drape it was necessary to verify that the two dimensional surface of the polymer also exhibited antimicrobial activity. In this study,

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50 time kill curves were constructed to assess the antimicrobial activity of Q PEI PNIPAAm again st C. amycolatum E. coli MRSA S. aureus and S. epidermidis. A time k ill curve for the a ctivity of Q PEI PNIPAAm surfaces against C. amycolatum is shown in Figure 19. Glass slides coated with 1.25% QPP reduced bacterial concentrations by 2 4 and 5 l ogs after 30, 60, and 120 minutes. The surface coated with 2.5% QPP showed a 5 log reduction in bacterial concentration after 120 minutes. Slides coated with 5% and 7.5% QPP exhibited a 6 log reduction in bacteria after 30 minutes. Figure 1 9 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Corynebacterium amycolatum Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean Figure 20 shows a kill curve for the antimicrobial activity of Q PEI PNIPAAm against E. coli Results showed that slides coated with 5% and 7.5% QPP reduced bacterial concentrations by 8 logs after 30 minutes. Gl ass slides coated in 1.25% and 2.5% QPP only exhibited 2 and 4 log reductions after 120 minutes.

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51 Figur e 20 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Escherichia coli Glass slides we re coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean. A time kill curve for the activity of Q PEI PNIPAAm surfaces against MRSA is given in Figure 21. Slides coated with 5% and 7.5% QPP exhibited a n 8 log reduction in bacteria after 30 min utes, while 1.25% and 2.5% exhibited 3 and 4 log reductions after 120 minutes. Figur e 2 1 Antimicrobial activity of a gel led Q PEI PNIPAAm surface against stationary phase MRSA Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean.

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52 Figure 22 shows the constructed time kill curve of Q PEI PNIPAAm surface antimicrobial activity against S. aureus After a period of 120 minutes, 1.25%, 2.5%, and 5% QPP coated surfaces reduced bacteria l concentrations by 3 5 and 8 log, respectively. An 8 log reduction in S. aureus bacterial concentrati on was exhibited by 7.5% QPP after 30 minutes Figure 2 2 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Staphylococcus aureus Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% solutions, which were allowed to gel. Error bars represent the standard error of the mean. Staph. epidermidis was the final bacterial st rain, which Q PEI PNIPAAm was tested against. Glass slides coated with 1.25% QPP demonstrated a 1 log reduction in bacte rial concentration after 120 minutes. In this test, 2.5% QPP demonstrated surprisingly high antimicrobial activity with 3 4 and 7 log reductions after 30, 60, and 120 minutes, respectively. Reductions of 6 and 7 log in bacterial concentration were exh ibited by 5% and 7.5% QPP, respectively.

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53 Figur e 2 3 Antimicrobial activity of a gelled Q PEI PNIPAAm surface against stationary phase Staphylococcus epidermidis Glass slides were coated with 1.25, 2.5, 5, and 7.5 wt% soluti ons, which were allowed to gel. Error bars represent the standard error of the mean. Two dimensional Q PEI PNIPAAm surfaces were demonstrated to have antimicrobial activity against all of the tested bacterial strains. These strains included both gram nega tive and gram positive bacteria. As expected, it was demonstrated that surfaces coated with increasing concentrations of Q PEI PNIPAAm solutions have increased antimicrobial activity. However, it was observed that gelled polymer surfaces coated with 1.25% and 7 .5% QPP were not very durable. In this experiment glass slides coated with Q PEI PNIPAAm surfaces were places in conical tubes with 3 mL of bacterial solution. For the duration of the experiment these tubes were incubated with shaking at 250 rpm. It was observed that over time a significant amount of Q PEI PNIPAAm fell off the slide coated with 1.25% and 7.5% QPP and gathered at the bottom of the conical tube (Figure 24) It is likely that 1.25% QPP did not adhere to the slide were well because there was not enough polymer on the surface to create a stable continuous gel. In the case of 7.5% QPP, too much polymer likely results in a pliable layer that can be peeled off more easily under mechanical strain.

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54 Figure 24 Q PEI PNIPAAm accumulation following 120 minute surface test with shaking at 250 rpm. A: Slide coated with 1.25% QPP B: Slide coated with 7.5% QPP. For this experiment, polymer solutions were sprayed onto the glass slides to ensure even coating of the po lymer. Due to the viscosity of 7.5% Q PEI PNIPAAm, several experiments were delayed due to difficulty spraying the polymer. In those instances, the problem was frequently solved by changing spray bottles or priming the spray nozzle with a lower concentrati on polymer followed by the more viscous 7.5% Q PEI PNIPAAm. However, it was determined that due to difficulty with the application, 7.5% Q PEI PNIPAAm was not ideal for application at a surgical drape. Based on the results of this study, it was decided th at a 5% Q PEI PNIPAAm formulation was ideal for use as a surgical drape. This solution had very good surface antimicrobial activity, the polymer surface created at this concentration showed good durability, and this concentration was easily sprayable, Ther efore, is was determined that approximately 100 L of Q PEI PNIPAAm should be applied for every 4 cm 2

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55 In vitro Q PEI PNIPAAm Activity as a Surgical Incision Drape The previous experiment demonstrated that the surface of Q PEI PNIPAAm has antimicrobial activity. However, bacteria was readily able to move around in the bacterial solution, which maximized bacterial contact with the antimicrobial surface. It was therefore necessary to demonstrate the Q PEI PNIPAAm surfaces have the ability to kill stat ionar y layers of bacteria such as those on the skin. Thus, an in vitro study was developed to simulate Q PEI PNIPAAm activity when used as a surgical drape. In this study, LB agar was used to simulate the epidermis. The same five relevant bacterial strains wer e used in this simulation: C. amycolatum E. coli MRSA S. aureus and S. epidermidis For the purpose of this test, a grid pattern was developed in which all five bacterial strains could be tested at once with seven initial stock bacterial concentrations for each (Figure 25). As one of the controls, bacterial with starting concentrations varying from 10 3 to 10 9 CFU/mL grown on agar with no polymer treatment. Figur e 2 5 Growth of bacteria in grid pattern after 48 hour incubation on agar with no polymer.

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56 The goal of t his surface test was to determine if Q PEI PNIPAA m demonstrated antimicrobial activity against bacteria trap p ed between the drape and the ski n and against bacteria that fall on top of the drape. This was com pleted as described in the methods, and as expected Q PEI PNIPAAm killed bacteria trapped between itself and agar and bacteria on its surface (Figure 26). Figur e 26 In vitro simulation of Q PEI PNIPAAm antimicrobial activity when used as a SID. Growth after 48 hours is shown. Bacteria were plated in the grid pattern below and above the polymers on separate plates. In this simulation agar represents the skin and the polymer represents the drape. Bacteria grew on both PNIPAAm p lates, and PEI PNIPAAm had some bacterial growth. Q PEI PNIPAAm inhibited all bacterial growth except for a small untreated area.

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57 Bacteria plated on agar then covered with PNIPAAm, PEI PNIPAAm and Q PEI PNIPAAm was used to simulate the presence of bacteri a under a surgical incision drape. To simulate bacterial contamination on top of a SID, the polymers were gelled on top of agar and bacteria was plated on top. PNIPAAm was used as a control, and as expected bacterial growth was observed on both plates. In the image in Figure 26, it is difficult to see bacterial growth between PNIPAAm and agar, but upon closer examination MRSA, S. aureus and S. epidermidis colonies are present. A s previously mentioned, PEI PNIPAAm has some antimicrobial activity [91] In this in vitro simulation PEI PNIPAAm had some bacterial growth under the polymer where the polymer was poo rly applied around the plate edges and E. coli growth was observed on top of the polymer. In the case of Q PEI PNIPAAm all bacterial growth was inhibited except for a small untreated area where E. coli growth was observed Based on this in vitro simulation, Q PEI PNIPAAm shows promising potential for use as a surgical drape. Murine Skin Incision Mo del Skin incisions were completed with one of four different skin treatments (Figure 27) As a positive control, an incision was performed after skin cleansing with no drape. Inoculation of the incision site with b acteria and no surgical incision drape was used as a negative control. Q PEI PNIPAAm applied to the incision site after inoculation with bacteria was used as the experimental SID, and bacterial inoculatio n followed by placement of IOBAN TM 2 was used as a compa rison S ID All groups except for the positive control were completed five times each for C. amycolatum E. coli MRSA S. aureus and S. epidermidis bacterial strains.

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58 Figure 27 Murine model treatment g roups. A: Q PEI PNIPAAm applied as a surgical drape over bacteria B: IOBAN TM 2 iodine impregnated drape applied over bacteria C: Positive control group with no drape or bacteria. D: Negative control group with applied bacteria and no drape. The skin incision surgery was completed successfully for 48 mice with 3 mice in each group. For each mouse, s wabs of bacterial growth were successfully taken and plated on LB agar after incision and at 11 additional time points up to 2 hours after incision closure. No complications arose for any of the mice during the skin incision surgery Growth on LB agar was quantified as described in the methods section and data was compiled into plots of bacterial colony forming units (CFUs) as a function of time following the skin surgery. Figure 28 reports the number of C. amycolatum CFUs detected by bacterial swabs following skin incision surgery. Trends show that the largest amount of bacterial colonies

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59 were detected for the negative control with no drape Use of Q PEI PNIP AAm and IOBAN TM 2 resulted in reduced detection if CFUs. However, due to large standard deviations there is no statistical difference between most groups. In the case of Q PEI PNIPAAm compared with I OBAN TM 2 no statistical difference was observed at most time points. However, at 75 minutes mice treated with Q PEI PNIPAAm had significantly fewer detected colonies compa red with mice treated with IOBAN TM 2 Figur e 28 The number of C. amycolatum colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. The number of E coli CFUs detected by bacterial swabs following skin incision surgery is shown in Figure 29 Based on detected CFUs, E coli survived poorly on the mouse epidermidis. At 0 minutes there were statistically fewer CFUs positive control mice compared with negative control mice In the case of Q PEI PNIPAAm compared with IOBAN TM 2 no statistical difference was ob serve d

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60 Figure 29 The number of E coli colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. Figure 30 shows the number of MRSA CFUs detected following skin incision surgeries. It should be noted that the low number of CFUs reported at time 0 for the negative control was due to the presence of large fused MRSA colonies These large fused colonies indicated the presence of large amounts of bacteria, and therefore CFUs at this time point should be higher. Trends once again indicated that the negative control group had t he highest CFUs. The polymer mice had consistently fewer MRSA colonies when compared with IOBAN TM 2 mice. At times 0, 10, 20, and 75 minutes Q PEI PNIPAAm drape mice had statistically significant lower numbers of CFUs compared with the IOBAN TM 2 drape group and at all other times there was no statistical difference between the two groups This data suggests that the use of Q PEI PNIPAAm as a SID is comparable to IOBAN TM 2 use, and that Q PEI PNIPAAm may even be a more effective SID than IOBAN TM 2

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61 Figure 30 The number of MRSA colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the stan dard error of the mean. indicates statistical difference with p < 0.05. The number of S aureus CFUs following murine skin incision surgeries is shown in Figure 31. Significantly fewer CFUs were detected for the polymer mice at time 0 com pared with the IOBAN TM 2 drape, but there was no statistical difference at other time points Figure 31 The number S a ureus colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the stan dard error of the mean. indicates statistical difference with p < 0.05.

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62 Figure 32 shows the number of Staph epidermidis CFUs detected by bacterial swabs following skin incision surgery. Trends show that the highest amount of bacterial colonies was obser ved for the negative control with no drape. There was no statistical difference between the use of Q PEI PNIPAAm or IOBAN TM 2 drapes. Figure 32 The number Staph epidermidis colony forming units (CFUs) picked up by bacterial swabs following the skin incision surgery. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. Results from this animal model consistently show lower numbers of detected CFUs with t he use of Q PEI PNIPAAm as a su rgical drape compared with IOBAN TM 2 However, in most of these cases the difference in CFUs detected with the use of these two drapes is not statis tically different Data from all five bacterial strains indicated that the pe rformance of Q PEI PNIPAAm as a surgical drape is comparable with the use of IOBAN TM 2 It should be noted that while Q PEI PNIPAAm remained on the mouse s kin following surgery, the IOBAN TM 2 drapes were removed, thus Q PEI PNIPAAm had more time to kill ba cteria. However, Q PEI PNIPAAm is intended to remain on the skin following surgery, so this study tests this polymer as it is intended to be used.

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63 A power a nalysis to estimate the sample size required for the detection of the mean within 25% with a power o f 0.80 was completed for each bacterial strain. A sample size (n) required to meet the above parameters was calculated at each time point based on the negative control data as this data frequently exhibited the highest variance. A minimum, maximum, and mea n n value were calculated and those values are reported for each bacterial strain in Table 4. Based on these calculations if it is desired to determine the mean within 25% of its value with a power of 0.80 at every time point, then the study would need to be completed for the maximum n value. However, the maximum n value is quite high for some of these bacterial strains and it would be costly to complete such a study. Table 4 Sample size (n) values calculated by the power analysis for each bacterial strain Subcutaneous Injections Although Q PEI PNIPAAm was designed for topical use as a surgical drape, trace amounts of the polymer are likely to enter the body cavity during surgery. To assess the immune response instigated by the polymer subcutaneous injections were successfully conducted for 12 mice. Samples of s terile saline and Q PEI PNIPAAm were injected into mice for time points of 1 and 7 days with 3 mice in each experimental group. Tissue harvested from the area surrounding the inje ction site was then stained for macrophage presenc e with an anti CD68 antibody. Stained tissue samples were imaged with confocal microscopy (Figure 33).

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64 Figure 33 Representative confocal i mages o f immunostaining for macropha ges after 1 and 7 days. Macrophages were stained with anti CD68 antibody and Alexa Fluor 594 which appears in red DAPI was used to stain cell nuclei, which appear in blue Cells containing both colors were identified as macrophages. Scale bars represent 100 m. Macrophages were observed in all subcutaneous injection group s. Immune response was quantified by determining the number of macrophages per unit are a. Figure 34 shows a graph of macrophages per mm 2 for the polymer and saline at both time points. Q PEI PNIPAAm injections at both time points had higher macrophage counts per area than saline. There was no statistical difference between Q PEI PNIPAAm and saline macrophages per area for 1 day subcutaneous injections. For 7 day subcutaneous injections, Q PEI PNIPAAm

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65 had statistically higher counts of macrophages per area. While macrophages per area increased for Q PEI PNIPAAm and saline injections between 1 and 7 days, the increases were not statistically significant. Fig ure 34 Macrophages per unit area following 1 and 7 day subcutaneous injections of saline and Q PEI PNIPAAm. Error bars represent the standard error of the mean. indicates statistical difference with p < 0.05. Results from subcutaneous injections indicate that both saline and Q PEI PNIPAAm injections prompt an immune response. In the case of biocompa tible materials, any initial immune response should dissipate over time. Saline is commonly used as a sham injection in research, and it has been shown that an y immune response to saline disappears with time [139] In this study, the immune response to saline injections increased from 1 to 7 days. Therefore, there was no control group in which immune response returned to baseline. This means that althou gh it can be conclude d the Q PEI PNIPAAm prompts on immune response, no conclusions can be made about the biocompatibility or toxicity of the polymer.

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66 CHAPTER VII CONCLUSION The first specific aim of this study was to characterize the formulation of Q PEI PNIPAAm intended for use as a surgical drape. Successful synthesis of the desired structure of Q PEI PNIPAAm was confirmed with 1 H NMR. Analysis of th verified that Q PEI PNIPAAm forms a stable gel at a temperature slightly be low body temperature. The antimicr obial activity of the two dimensional surface of Q PEI PNIPAAm was investigated with two studi es. The first study demonstra ted that the Q PEI PNIPAAm surface has antimicrobial activity against all five bacterial strains used in this research. From this study, it was also concluded that a 5 wt% solution of Q PEI PNIPAAm in 70% ethanol was ideal for use as a spray a ble gelling su rgical drape. The second surface test was an in vitro simulation of Q PEI PNIPAAm antimicrobial activity as a surgical drape. This study demonstrated Q PEI PNIPAAm has the potential to kill bacterial trapped between the polymer and the skin during surger y as well as any bacterial contamination that may fall onto the drape surface. The effectiveness of Q PEI PNIPAAm used as a surgical drape was compared to the use of a commercially available widely used iodine impregnated SID in a murine skin incision mode l In this model, the incision site was cleansed, inoculated with a particular strain of bacteria, and an IOBAN TM 2 drape or Q PEI PNIPAAm was applied to the area. Drape performance was evaluated based on the relative differences in detectable CFUs between the tested groups. Although at most time points there was no statistical difference between the number of CFUs detected in m ice where Q PEI PNIPAAm or IOBAN TM 2 we re

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67 applied as a drape overall data trends and a small number of statistical points indicated that Q PEI PNIPAAm may be mo re effective as a SID than IOBAN TM 2 Data from this murine model supports that hypothesis that Q PEI PNIPAAm performance as a surgica l drape is comparable to a commercially available drape. Subc utaneous injections of sterile saline and Q PEI PNIPAAm were administered for 1 and 7 days to determine if Q PEI PNIPAAm instigates an in vivo immune response. It was found the between 1 and 7 days, both saline and Q PEI PNIPAAm injections result in an increase in macrophages per area. This indicates that both prompt and immune response. However, no conclusions can be made about the biocompatibility of the polymer without a full cytotoxici ty assay where the control group displays a dissipation of the initial immune response.

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68 CHAPTER VIII FUTURE WORK Additional Polymer Optimization Although during the animal model it was observed that Q PEI PNIPAAm gelled on the mouse skin surface, the gelli ng temperature of the polymer should be further tailored. The Q PEI PNIPAAm formulation used in this study was repo rted to form a stable gel by 35 However, the skin surface temperature varies between approximately 33 [140], [141] Ther efore, to ensure that the polymer remains gelled on the skin during surgery, the polymer length of the alkyl chains conjugated the PEI PNIPAAm, which would increase the amount of hydrophobic groups on the pol ymer. Increasing the hydrophobic groups will result in gelation at a lower temperature. Increase Mouse Model Sample Size Although data trends of the current study suggest that Q PEI PNIPAAm has t he potential to o utperform IOBAN TM 2 high variability in reported CFUs rendered many of the trends statistically insignificant. In many cases, there was no statistical differences even between the negative and positive control. An increased sample size is thus necessary and should reduce some of the variability. It is expected that with an increased sample size, more statistical differences will be seen between the groups. A power analysis was completed to calculated the required sample size to detect the number of CFUs w ithin 25% of its true mean with an 0.8 power, and results of the analysis were reported in Table 4. The analysis indicated that increased sample size would improve the significance of the data.

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69 In Vivo Cyt oxicity Assay Q PEI PNIPAAm is not intended for injection into the body cavity. However, trace amounts of the polymer are likely to enter the body cavity through the incision site during surgery For this reason, it is important to conduct an in vivo cytotoxicity where it can be determined if Q PEI PNIP AAm is biocompatible. Biocompatibility can be assessed with the addition of longer duration subcutaneous injection groups. Skin Irritation Test Surgical drapes may remain on the skin surrounding the incision site for long periods of time during surgery. Fo r this reason, it would be useful to assess whether Q PEI PNIPAAm causes skin irritation after prolonged contact with the epidermis. As mentioned in the background previous skin irritation tests of a quaternized polymer showed no skin irritation for a 7 da y duration. However, a similar test should be completed with the polymer formulation used in this study.

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