Quaternized Q-PEIPAAm-Based Antimicrobial Reverse Thermal Gel: A

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Biological and Medical Applications of Materials and Interfaces

Quaternized PEI-PNIPAAm-based antimicrobial reverse thermal gel: a potential for surgical incision drapes Maria Bortot, Melissa Ronni Laughter, Madia Stein, Adam Rocker, Vikas Patel, and Daewon Park ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04020 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

Quaternized PEI-PNIPAAm-based antimicrobial reverse thermal gel: a potential for surgical incision drapes Maria Bortot1, Melissa Ronni Laughter1, Madia Stein1, Adam Rocker1, Vikas Patel2, Daewon Park*1 ––––––––– 1

Department of Bioengineering, University of Colorado Denver Anschutz Medical Campus, 12800 E. 19th Avenue, Aurora, CO 80045 USA 2

Department of Orthopedics, University of Colorado Denver Anschutz Medical Campus, 12800 E. 19th Avenue, Aurora, CO 80045 USA

* Corresponding Author (Email: [email protected], Address: Translational Biomaterials Research Laboratory, Department of Bioengineering, University of Colorado Denver Anschutz Medical Campus, 12800 E. 19th Avenue, P18-4403A, Aurora, CO 800045) ––––––––– Abstract A quaternized reverse thermal gel aimed at replacing current surgical incision drapes (SID) was designed and characterized. The antimicrobial efficacy of the quaternized reverse thermal gel was analyzed using both in vitro and in vivo models and was compared to standard SIDs. Polymer characterization was completed using both nuclear magnetic resonance (1H NMR) and lower critical solution temperature (LCST) analysis. Biocompatibility was assessed using a standard cell viability assay. The in vitro antimicrobial efficacy of the polymer was analyzed against four common bacteria species using a time-kill test. The in vivo antimicrobial efficacy of the polymer and standard SIDs were compared using a murine model aimed at mimicking surgical conditions. NMR confirmed the polymer structure and presence of quaternized groups and alkyl chains. The polymer displayed a LCST of 34°C and a rapid rate of gelation, allowing stable gel formation when applied to skin. Once quaternized, the polymer displayed an increase in kill-rate of bacteria compared to un-quaternized polymer. In experiments aimed at mimicking surgical conditions, the quaternized polymer showed statistically comparable bacteria-killing capacity to the standard SID and even surpassed the SID for killing capacity at various time points. A novel approach to replacing current SIDs was developed using an antimicrobial polymer system with reverse thermal gel properties. The reverse thermal gel properties of this polymer maintain a liquid state at low temperatures and a gel upon heating, allowing this polymer to form a tight coating when applied to skin. Furthermore, this polymer achieved excellent antimicrobial properties in both in vitro and in vivo models. With further optimization, this polymer system has the potential to replace and streamline pre-surgical patient preparations through its easy application and beneficial antimicrobial properties. Keywords: antimicrobial polymer, surgical drapes, surgical site infections, quaternized amine groups, reverse thermal-gel

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Introduction The human skin is host to several potentially harmful strains of bacteria. Under normal physiologic conditions, the intact surface of a healthy epidermis prevents these bacteria from penetrating. However, once the integrity of the epidermis is compromised, such as during surgery, this protection is lost. The result can be a surgical site infection (SSI), defined as an infection that develops in the body as a consequence of surgery, during or up to 30 days after a surgical procedure. Preventative measures are taken to reduce the risk of SSIs, and since the skin is one of the main sources of pathogens, antiseptic preparations prior to surgery are used along with surgical incision drapes (SIDs) to disinfect the skin and sequester bacteria that could have survived disinfection1,2. The use of SIDs has been common practice in surgeries for approximately 50 years; however, current SIDs have numerous drawbacks3. Among the most concerning is that some SIDs contain leachable antimicrobial agents and, as a result, lose their antimicrobial activity after a short period of time4. In addition, some patients show an allergic reaction to the leachable material contained within the SID or to the adhesive used to affix the drape to the skin5,6. Furthermore, the placement process of the drapes is time-consuming and, once placed, the drapes often do not remain properly attached to the skin surface, leading to increased infection risk7. Improper placement can easily lead to the formation of air pockets and wrinkles, creating space for microbes to proliferate8,9. Additionally, during the removal of the drape, abrasions may form on the skin close to the surgical site, further increasing the risk of infection. For these reasons, there has been growing interest in the development of an alternative to current SIDs. Polymers have become of particular interest in the field of antimicrobials because they can be fine-tuned and manipulated to possess antimicrobial properties as well as specific


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qualities necessary for certain medical applications. Specifically, polyethylenimine (PEI), a nontoxic, aliphatic, weakly basic, synthetic polymer, has garnered appeal due to its excellent intrinsic antimicrobial properties10. The antibacterial activity arises from the presence of positively-charged free primary amines in the PEI structure11,12. In addition, the antimicrobial action of the polymer can be enhanced by quaternization13. At the same time, due to the nature of this antimicrobial mechanism, studies have shown that bacteria failed to develop resistance to this lethal action over the course of many successive generations14. Additionally, PEI can be conjugated to other polymers to achieve various properties. Since the placement of SIDs can be troublesome, an alternative is to modify PEI to possess reversible thermal gelling (RTG) properties. These properties will allow the polymer to be sprayed as a liquid and undergo a physical liquid-to-gel transition when it comes into contact with the skin surface. One of the most prominent and well-studied reverse thermal gels is Poly(N-isopropylacrylamide) (PNIPAAm). When conjugated with other polymers, PNIPAAm may impart its thermosensitive behavior on otherwise non-stimuli-sensitive polymers (e.g., PEI)15,16. In this study, we synthesized PEI-PNIPAAm, a reverse-thermal gel (RTG) that exhibits a solution-to-gel transition close to body temperature. This polymer was subsequently quaternized to obtain Q-PEI-PNIPAAm, an RTG with enhanced antibacterial properties17. Ultimately, we developed an antibacterial polymer that may be sprayed onto the skin surface and will subsequently solidify into an antimicrobial gel layer. Once sprayed onto the skin, this gelled polymer layer can then act as a uniform antimicrobial surface during surgery. Moreover, it can be removed using cold water (or ethanol 70%) after surgery, thereby significantly reducing risk of skin irritation, blistering, and medical waste. Not only does this polymer have the potential to streamline the pre-surgical process by offering a “one size fits all” solution for surgical site



protection, regardless of patient size or type of procedure performed, but use of this polymer can ensure avoidance of any mistakes or difficulties during placement of SIDs (Figure 1).

Figure 1. Schematic showing placement and removal of the antimicrobial polymer (B) compared to the surgical incision drape (A). The polymer may be sprayed onto the surface of the skin where it will form a stable gel and can later be removed with water —compared to a surgical incision drape that requires manipulation and placement followed by disruption of skin cells upon removal. The first aim of this work was to synthesize and characterize the Q-PEI-PNIPAAm formulation chosen for this study. Nuclear magnetic resonance (NMR) was used to confirm the polymer structure and the presence of quaternary groups on the polymer backbone. Next, LCST was used to investigate the solution-to-gel transition rate and temperature. The second aim of this work was to evaluate the antimicrobial killing capacity of Q-PEI-PNIPAAm against five relevant bacterial strains: Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), Corynebacterium amycolatum (C.


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amycolatum), and Escherichia Coli (E. coli). Evaluation included the development of time-kill curves for Q-PEI-PNIPAAm, and an in vitro surface antibacterial assay. Next, we used a murine animal model to evaluate the performance of Q-PEI-PNIPAAm as a surgical drape compared to a positive control, IOBANTM drape, in killing and trapping the different species of bacteria. Materials and Methods Materials Polyethylenimine

(PEI

branched

10000

g/mol),

N-(3-dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), agar powder, lysogeny broth (LB) (Miller’s modification), and chloroform-d were obtained from Alfa Aesar (Ward Hill, MA). N-isopropylacrylamide (NIPAAm) was obtained from Acros Organics (Geel, Belgium). 4,4′-azobis (4-cyanovaleric acid) (ACA), methanol, sodium hydroxide (NaOH), 2-propanol, hexane, and diethyl ether were obtained from ThermoFisher Scientific (Pittsburgh, PA, USA). Staphylococcus aureus (ATCC 6538), methicillin-resistant Staphylococcus aureus (ATCC 700698), and Escherichia coli (ATCC 15597) were obtained from American Type Culture Collection (VA, USA). Staphylococcus epidermidis and Corynebacterium amycolatum were gifted by The Ronald G. Gill Laboratory (University of Colorado Denver, CO, USA). The Vybrant® MTT Cell Proliferation Assay Kit was obtained from Invitrogen (ThermoFisher Scientific, Pittsburgh, PA, USA). Fibroblasts were provided by (Cardiovascular Pulmonary Research Cell Repository – Nana Burns and Sandy Walchak, University of Colorado Denver Anschutz Medical Campus, CO, USA). Dimethylformamide (DMF) was obtained from BDH Chemicals (Poole, UK). IOBANTM surgical incision drape was obtained from 3M (St. Paul, MN, USA). Ketoprofen, saline, and isoflurane were obtained from MWI Veterinary Supply (Boise,



ID, USA). Coated Vicryl 4-0 sutures were obtained from Ethicon (Somerville, NJ, USA). Adult C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Equipment 1

H NMR was conducted with a Varian Inova 500 mHz NMR Spectrometer. LCST

measurements were determined using a Cary 100 UV-visible spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a temperature-controlled 6-cell stage. Finally, a Synergy Mx Microplate Reader (BioTek U.S., VT, USA) was used to measure absorbance in the MTT assay. Water contact angles were measured using a contact angle goniometer (KUDOS A-60, Future Digital Scientific Corporation). Polymer Synthesis PEI-PNIPAAm synthesis PNIPAAm was synthesized as previously described using radical polymerization with an azobis initiator17. Briefly, NIPAAm (44.19 mmol) and ACA (0.22 mmol) were dissolved in 25 mL of dry methanol. The reaction was purged with nitrogen for 30 mins and left to react for 3 hrs at 68°C. The product mixture was then precipitated once and washed twice in hot water (60°C). The final product was purified further via dialysis (molecular weight cutoff (MWCO) of 3500 Da) and lyophilized for storage. PNIPAAm-COOH to PEI conjugations were conducted using an EDC-NHS coupling reaction. PNIPAAm was first dissolved in 10 mL of DMF, and then EDCNHS (1.2 M excess to amine groups) were added. At the same time, in a separate flask, PEI was dissolved in 5 mL of DMF, and both reactions were left overnight. Then, the PEI solution was added drop-wise into the PNIPAAm-EDC-NHS and left to react for a period of 24 hrs under nitrogen. Subsequently, a rotary evaporator was used to remove the DMF, and precipitations in diethyl ether were used to remove unreacted polymer. Then, the polymer was dissolved in dH2O


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and added to a 12-14 kDa MWCO dialysis tube that was placed in a beaker with dH2O water for 48 hrs. Once the polymer was purified, it was lyophilized for a period of 48 hrs. Quaternization of amine groups After conjugation of PNIPAAm, the rest of amine groups in PEI-PNIPAAm were converted to quaternary ammoniums using alkylation agents. In short, the process involved converting amines to quaternary ammoniums by alkylation with 1-bromohexane followed by an iodomethane to complete the quaternization process. The alkylation by two steps (the first step with longer 1-bromohexane and the second step with shorter iodomethane) ensured higher density of quaternary ammonium groups in an unit area and higher bactericidal efficiency compared to the one step alkylation using single alkylation agent10. To achieve quaternization, PEI-PNIPAAm was first dissolved in 10 mL of DMF, and excess sodium bicarbonate was added. The reaction was set to a temperature of 95°C with reflux; while undergoing vigorous stirring, 1Bromohexane (20 M excess to remaining amine groups) was added and left for 48 hrs. The temperature was then lowered to 68°C and excess iodomethane was added for another 12-hour methylation. Three precipitations in diethyl ether were used to remove solvent and unreacted alkylating agents. Then, the polymer was dissolved in 10 mL of dH2O and dialyzed using 12-14 kDa MWCO dialysis tube in dH2O for 48 hrs. Finally, it was lyophilized for 48 hrs and stored appropriately. Polymer characterization Nuclear magnetic resonance 1

H NMR was conducted to identify the characteristic peaks of PEI, PNIPAAm, PEI-

PNIPAAm, and Q-PEI-PNIPAAm. Samples (3-5 mg) were dissolved in 600 µL of chloroform-d. The resulting spectra were processed and analyzed using iNMR reader software. Online



Advanced Chemistry Development i-Lab (ACD/Structure Elucidator, version 12.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada) was used to predict and confirm the 1H NMR spectra peaks associated with each polymer at 5 MHz. Solution-to-gel phase transition The LCST was determined to characterize the gelling properties of the polymer. The polymer samples were dissolved in dH2O at 5 wt%. Then, transmittance values were measured at 500 nm, starting from a temperature of 25°C and increasing 0.5°C/min up to 45°C. Water contact angle Water contact angles were measured before (PEI-PNIPAAm) and after (Q-PEIPNIPAAm) the quaternization. For the measurement, glass slides (2.54 × 7.62 mm) were coated with each solution (5%, w/w) of PEI-PNIPAAm and Q-PEI-PNIPAAm. The water contact angle measurements were performed at 37oC by dropping water (~2 µL) on the coating surface and subsequent image analysis. The data were obtained from 50 measurements (5 samples per group × 10 different spots). Bacterial stock preparation The bacterial suspension was prepared using Miller’s LB broth (5 mL) that was inoculated with flash-frozen bacterial stock. The new stock was left for 16 hrs at 37°C in a shaker incubator (250 RPM). Subsequently, the bacterial suspension was centrifuged for 5 mins at 500 RPM, and the LB broth was poured off. The bacterial pellet was re-suspended in sterile phosphate buffered saline (PBS), and the concentration was obtained by measuring the absorbance of the solution at 600 nm. To conduct colony counting and determine the exact bacterial concentration, aliquots (20 µL) were taken from the bacterial suspension and diluted serially, 10-fold triplicates in a 96-well plate. The dilutions were used to obtain 40 µL aliquots


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from each well. The aliquots were then plated on LB agar plates and left at 37°C. Colonies were counted after 24 hrs. Antimicrobial tests using Q-PEI-PNIPAAm The antibacterial property of Q-PEI-PNIPAAm was evaluated against four clinicallyrelevant species of bacteria: S. aureus, MRSA, S. epidermidis, and E. coli. Using the methodology previously described, dilutions were performed using PBS to obtain a bacterial stock of 108 cells/ml. This stock was then used to fill four conical tubes, 3 mL each. Separately, PNIPAAm, PEI-PNIPAAm, and Q-PEI-PNIPAAm were each dissolved in DI water in conical tubes to form 1% w/w solutions to maintain the polymer in suspension and avoid gelling during the tests at 37°C. The fourth tube only contained the diluted bacterial stock to ensure bacterial survival. The tubes were placed at 37°C in a shaker incubator (250 RPM). At time 0, 30, 60 and 120 mins, 3 aliquots (20 µL) were taken from each tube and left to grown on agar plates at 37°C for 24 hrs. The colonies were then counted to quantify the killing capacity of each polymer. The experiment was repeated with each of the four bacterial strains listed above. Q-PEI-PNIPAAm activity as a surgical incision drape To test the killing capacity of the polymers when used as an SID, a surface experiment on agar plates was developed based on the well-known agar disk-diffusion method18. However, the approach we used does not rely on diffusion but rather on evaluating the antibacterial properties of the polymer surface. Using a grid pattern, four different agar plates were inoculated with a standardized inoculum of S. aureus, MRSA, S. epidermidis, E. coli, and C. Amycolatum. Each row of the grid pattern was dedicated to one bacterial strain with seven columns at varying concentrations from 103 to 109 colony-forming units (CFU)/mL. One of these grid plates served as a control plate and was incubated at 37°C for 48 hrs before being assessed for bacterial



growth. The other three plates were used to test the surface antimicrobial activity of the polymer against each bacterial strain. To begin, the plates were placed on a 37°C hot plate and, after polymer optimization,1 mL of 5 wt% PNIPAAm, 5 wt% PEI-PNIPAAm, or 5 wt% Q-PEIPNIPAAm was sprayed on each plate respectively and allowed to gel. The plates were then incubated at 37°C for 48 hrs. Colony counting was conducted to evaluate bacterial growth between the polymer and the agar surface. In vitro cytotoxicity using the MTT Assay An MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide assay (MTT assay) was chosen to evaluate the biocompatibility of PNIPAAm, PEI-PNIPAAm, and Q-PEIPNIPAAm. This assay is commonly used to evaluate cell metabolic activity and has also been used extensively to assess cell toxicity19. The three polymers were dissolved in ethanol at 1, 3, and 5 wt%. The solutions were added to separate wells of a 96-well plate and allowed to gel at 37°C. Fibroblasts (7,000 cells/well) were then placed on top of each polymer film. Cells were left to grow for a period of 72 hrs. Several controls were used, including plain media, PEIPNIPAAm, PNIPAAm, Chlorhexidine 2% (a common skin-preparation solution), and media with 5% DMSO. All samples were tripled for statistical significance. The Vybrant® MTT Cell Proliferation Assay Kit assay was then performed per manufacturer’s instructions. In short, cell culture media was changed, and 10 µL of the 12 mM MTT stock solution was added to each well. As a negative control, 10 µL of the MTT stock solution was added to 100 µL of medium alone. The plate was then left at 37°C for 4 hrs. Subsequently, all but 25 µL of medium was removed from the wells. 50 µL of DMSO was added to each well and mixed thoroughly. Finally, absorbance was read at 540 nm on a microplate reader. All results were normalized and expressed as a percentage of the negative control.


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Animals All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Denver Anschutz Medical Campus. A total of 48 adult C57BL/6J male mice ranging in age from 12 to 24 weeks were allowed to acclimate for 1 week prior to any surgical procedures. Mice were maintained on a 14/10-hour light/dark cycle with access to water and food ad libitum. Mice were then separated into groups of twelve based on the treatment tested: Q-PEI-PNIPAAm/bacteria spray, IOBANTM drape/bacteria spray, no drape/bacteria spray, no drape/no bacteria spray. Each group of 12 mice was further separated into groups of three depending on the strain of bacteria being tested. For the in vivo biocompatibility component of the study a total of 18 C57BL/6J mice were used. In like manner to 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. Mouse skin incision surgery To begin, adult C57BL/6J males were anesthetized with 5% isoflurane in oxygen and maintained on 2% isoflurane in oxygen for the duration of the procedure. Before beginning the surgical procedures, a subcutaneous injection of ketoprofen (5 mg/kg) was administered to minimize any pain felt following the procedure. Next, an area of fur was removed with clippers starting below the base of the neck and moving 3 cm down the back of the mouse. The shaved area was then washed with warm water and disinfected with an alcohol. After prepping the skin, 0.5-1 mL of 108 CFU/mL bacterial solution of one of the following bacterial strains, C. amycolatum, E. coli, MRSA, S. aureus, or S. epidermidis, was sprayed onto the shaved area and allowed to dry. After inoculation of the bacteria, 5 wt% Q-PEI-PNIPAAm in 70% ethanol (0.5-1



mL), an IOBANTM drape or no drape was applied on top of the shaved area depending on the assigned group of each mouse. As a positive control, a group of mice were prepped in a similar way as described above; however, no bacteria were inoculated on the skin and no drape was applied to the shaved area. Next, a 1 cm skin incision was performed in the center of the prepared area on the back. The incision was then irrigated with 0.5 mL of sterile saline to simulate surgical conditions. The incision site was swabbed with a sterile cotton swab and used to inoculate LB agar plates to access the bacterial load present at the incision site. The incision was closed with 1-2 continuous sutures, and the mice were monitored closely and allowed to recover. The incision site was swabbed with a sterile cotton swab for bacterial presence at time points 0, 10, 20, 30, 40, 50, 60, 75, 90, 105, and 120 mins post-closure. For the IOBANTM drape, swabs were taken around the incision site as well as the drape itself. Each swab was used to inoculate an LB agar plate, which was then placed in a 37°C warm room for 24 or 48 hrs in the case of C. amycolatum to allow bacteria to grow. After the 120 min time point, mice were euthanized by CO2 inhalation followed by cervical dislocation. Analysis of incision site bacterial presence Agar plates that had been inoculated using a swab of the incision site were placed in a 37°C warm room for either 24 or 48 hrs depending on the bacteria sample collected. Following bacterial growth, all LB agar plates were photographed for analysis of bacterial colony presence. Bacteria agar cultures were quantified by counting visible CFUs on each plate. This was completed using Image J software to process photos and detect particles. Biocompatibility of Q-PEI-PNIPAAm in vivo


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Aside from the in vitro MTT assay, a secondary in vivo test was conducted to determine if the polymer would elicit an immune response if it were to enter the body. Subcutaneous injections of Q-PEI-PNIPAAm were used to assess the biocompatibility of the polymer after 24 hrs and, 7 and 28 days. Mice were split into two equal size groups. The 9 mice in the experimental group were given 60 µL of 5 wt% Q-PEI-PNIPAAm in saline using subcutaneous injections on the right dorsal flank. The 9 mice in the control group were given subcutaneous injections of 60 µL of sterile saline. At each time point (24 hrs, 7 and 28 days) 3 mice from each group were euthanized by CO2 inhalation followed by cervical dislocation. After euthanasia, tissue surrounding the injection site was harvested. Tissue was fixed overnight in 10% formalin. Subsequently, the tissue samples were washed 3 times with 1X PBS (3 mins per wash) and placed in 30% sucrose for 48 hrs to cryoprotect the sample. The samples were then cut crosswise and embedded in OCT compound with the cut edge facing downward in the mold. The embedded tissue was then frozen at -80°C and cryosectioned into 5 µm sections, which were then placed on glass slides. Macrophage presence was chosen as a marker for immune response, hence, the presence of these cells in the injection site was quantified using immunohistochemistry. The tissue sections were then fixed in 10 % formalin for 10 mins and washed 3 times for 5 mins each in a washing buffer (1X PBS with 0.1% m/v Tween). Next, tissue sections were permeabilized for 10 mins 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 mins each followed by blocking with a blocking buffer (0.25% Triton X-100, 2% BSA, and 4% γ globulins in 1X PBS) for 60 mins to prevent non-specific binding. Next, tissue sections were stained with anti-CD68 antibody (1:500 in blocking buffer) overnight at 4°C, washed 3 times with the washing buffer for 5 mins each, and



stained with Alexa Fluor 594 (1:500 in 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. To quantify macrophage presence confocal images of stained slides were taken using a Zeiss LSM 780. Five representative sample images were 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. Analysis of variance (ANOVA) followed by two-tailed t-tests when applicable were used to determine significant differences between groups. Differences between groups were considered significant when p < 0.05. Results and Discussion Polymer characterization 1

H NMR was used to confirm the structure of the polymer and the presence of

quaternized groups. Since the quaternization by two alkylation agents randomly occurs at all amine groups (including primary, secondary and tertiary amine groups) in PEI-PNIPAAm, many studies used simplified structures that have been widely accepted20,10,21. Based on these wellknown studies, we presented the simplified Q-PEI-PNIPAAm structure with representative quaternary ammonium. As shown in Figure 2, the 1H NMR spectrum of Q-PEI-PNIPAAm confirmed quaternization. The presence of protons corresponding to alkyl chains was verified with peaks at 0.9 and 1.3 ppm, which is in well agreement with other studies22,23. Peak assignments were supported by NMR predictions from the Advanced Chemistry Development.


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Figure 2. 1H NMR spectrum of Q-PEI-PNIPAAm. Spectral analysis confirmed the conjugation of alkyl chains and the presence of peaks at 0.9 and 1.3 ppm. R: negative counter ions (R = I -

when the quaternization terminated by iodomethane; R = Br when the quaternization terminated during the first step using hexyl bromide. In this case, the methyl group in quaternary ammonium would be replaced with a hexyl group). The LCST was analyzed to ensure that Q-PEI-PNIPAAm exists as a liquid at room temperature but transitions to a gelled form when in contact with body temperature. The LCST represents the temperature at which hydrophobic interactions cause contraction and subsequent physical gelation of the polymer scaffold24,25,26,27. Sub-physiologic transition of Q-PEIPNIPAAm is critical for the application of this polymer as an SID as it must transition to a gel upon exposure to the skin’s surface. Although the LCST of PNIPAAm has been extensively studied, the incorporation of both PEI and quaternary groups could impact the gelation properties of the final polymer product28. Figure 3A displays the results from the LCST study for PEIPNIPAAm and Q-PEI-PNIPAAm. Q-PEI-PNIPAAm and PEI-PNIPAAm both show similar



LCST trends with a transition temperature of approximately 34°C. The slight decrease in the gelation temperature of Q-PEI-PNIPAAm could be due to the incorporation of hydrophobic alkyl groups. A study reported that the alkylation using hydrophobic alkylation agents increased the hydrophilicity of PNIPAAm-based RTG, leading to decrease in the transition point29. The increase in the hydrophobicity of Q-PEI-PNIPAAm was also confirmed by a surface contact angle measurement (Table 1). The contact angle of PEI-PNIPAAm was 32 ± 6, which increased to 51 ± 5 after the quaternization (Q-PEI-PNIPAAm). Thus, the increase in the hydrophobicity might cause the slight decrease in the gelation temperature of Q-PEI-PNIPAAm. Despite this slight difference in gelation temperature, both Q-PEI-PNIPAAm and PEI-PNIPAAm are fullyformed gels at and above 35°C (Figure 3B). Normal body temperature ranges between 36.1°C and 37.2°C, meaning that this formulation of Q-PEI-PNIPAAm will be a gel at temperatures slightly lower than body temperature and is therefore ideal for use as a surgical drape.

Figure 3. LCST graphs of the polymers (A) LCST of Q-PEI-PNIPAAm and PEI-PNIPAAm polymer samples (B) Image of the solid gel stability at 37°C .

Table 1. Water contact angles of PEI-PNIPAAm and Q-PEI-PNIPAAm Sample PEI-PNIPAAm

Contact angle (θ) 32 ± 6


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Q-PEI-PNIPAAm

49 ± 5

Antimicrobial suspension test using Q-PEI-PNIPAAm Gram-positive bacteria are the major inhabitant of the skin, so three common strains, S. epidermidis, S. aureus, and MRSA, were chosen to test the antibacterial properties of the polymer30. Also, although not as prevalent on the skin surface, a gram-negative bacterium, E. coli was also included in the study. Gram-negative bacteria have a similar cell wall but also have an additional outer membrane that can make them more resistant to antimicrobials that function through cell wall disruption31. As can be observed in Figure 4, Q-PEI-PNIPAAm was capable of achieving 8-Log10 bacterial reduction in MRSA, S. epidermidis, E. coli, and S. aureus. The results also showed that PEI-PNIPAAm itself possesses certain antibacterial activity. This is consistent with the previous literature as PEI itself has been shown to kill S. aureus32,14. The antibacterial properties of PEI result from the presence of protonated ammonium groups and non-protonated amine groups. The ethylene backbone in the polymer acts as a hydrophobic group, which creates repeating cationic, amphiphilic structures along the polymer backbone at a neutral pH without any further chemical modification. However, this result was not consistent in all bacterial strains. PEI-PNIPAAm did not exhibit antibacterial properties against S. aureus and MRSA. The reason behind this could be resistance conferred by differences in the bacterial cell wall33. Specific to the MRSA strain tested, Hanaki et. al. studied the activated cell-wall synthesis associated with vancomycin resistance in methicillin-resistant S. aureus clinical strains Mu3 and Mu50. They demonstrated that cell-wall synthesis and turnover are up-regulated in vancomycin resistance; S. aureus isolates, leading to thicker and more disorganized cell walls34. Thus, to kill these strains, the intrinsically protonated ammonium group alone in PEI may not be enough. The



difference of antimicrobial efficiency between Q-PEI-PNIPAAm and PEI-PNIPAAm may be explained by the well-accepted antimicrobial action mechanisms, which 1) the cationic domains in a quaternized polymer are adsorbed onto anionic site the cell wall by an electrostatic interaction and 2) and a lipophilic component (long alkyl chains) acts as a surfactant, promoting diffusion (penetration) through the cell wall, disrupting the cytoplasmic membrane and resulting in the death of the cells35,36. Thus, the synergistic actions of cation domains and hexyl chains in Q-PEI-PNIPAAm might lead to better antimicrobial efficiency than PEI-PNIPAAm.

Figure 4. Antibacterial time-kill curves. PEI-PNIPAAm and Q-PEI-PNIPAAm were added to stationary-phase bacteria, and samples (n=3) were taken at 0, 30, 60, and 120 mins to determine bacterial concentrations. Kill-curves were constructed for the following bacteria: MRSA (A), S. epidermidis (B), E. coli (C), and S. aureus (D). Antimicrobial surface test using Q-PEI-PNIPAAm Q-PEI-PNIPAAm proved to have antimicrobial activity when in suspension, however, this involves maximum bacterial contact with the antimicrobial polymer. The polymer was developed to be used as a surgical drape and, due to this, it was necessary to ensure that the


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material had surface antibacterial properties. Therefore, a second in vitro antibacterial assay was developed to test Q-PEI-PNIPAAm antibacterial surface capacity and to ensure the polymer could kill stationary layers of bacteria, such as those on the skin. The agar disk-diffusion method was used as guidance in the development of the new surface assay18. Although the polymer did not diffuse into the agar surface, the general methodology of the agar disk-diffusion assay was followed to simulate bacterial contamination on top of an SID. Figure 5A shows the agar plate that served as a positive control for the bacterial grid pattern described in the Methods section.



Figure 5. In vitro surface analysis of Q-PEI-PNIPAAm antimicrobial activity. (A) This plate served as a control plate and was incubated at 37°C for 48 hrs before being assessed for bacterial growth (B) Column one shows the polymer sprayed on top of the bacteria plated in grid pattern. Column two shows bacterial growth after 48 hrs, and as shown, Q-PEI-PNIPAAm inhibited all bacterial growth except for one small untreated area.


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To test the polymer surface killing capacity, as shown in Figure 5B, the polymer was sprayed and allowed to gel on top of the previously inoculated bacteria. The bacteria growth surrounding the agar plate might be from the coating defect that was not covered by the polymer during the spraying.The agar surface was used to simulate skin and to enable evaluation of the antibacterial surface capacity of the polymer. As shown in Figure 5B, Q-PEI-PNIPAAm was the only polymer able to inhibit the growth of C. amycolatum, E. coli, MRSA, S. aureus, and S. epidermidis. However, there was one small area of agar that was not covered with polymer, and thus E. coli growth was observed. In vitro cytotoxicity using the MTT Assay Determination of cell growth rates is a common approach for testing the effects of cytotoxic agents. The MTT Assay developed by Mossman is still one of the most common and versatile assays37. This assay involves the conversion of the water-soluble MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble formazan. The formazan is then solubilized, and the concentration is determined by optical density at 540 nm. As shown in Figure 6, increasing polymer concentrations reduced cell viability. However, even at the highest polymer concentration (5%), there was no statistically significant difference between QPEI-PNIPAAm and chlorhexidine 2% (FDA-approved positive control), which is the current standard surgical skin preparation. These results show that the polymer had no statistical difference in terms of biocompatibility when compared to the standard skin preparation solution, indicating its potential as a viable replacement.



Figure 6. Cell viability assay comparing toxicity of the polymers with the standard surgical preparation (2% Chlorhexidine). No statistical difference between cells exposed to 2% Chlorhexidine and Q-PEI-PNIPAAm. Mouse skin incision model For the murine skin incision model, various strains of bacteria were introduced to the cleaned and prepped skin. Next, depending on the group assignment of each mouse, either QPEI-PNIPAAm (Figure 7A) or the IOBANTM iodine-impregnated drape (Figure 7B) was applied over the bacteria. As a negative control, the skin was prepped and cleaned followed by bacteria inoculation onto the skin with no subsequent surgical incision drape placement as shown in Figure 7C. Finally, as a positive control, the skin was prepped and cleaned with no bacteria inoculation and no surgical incision drape placement (Figure 7D). No complications arose for any of the mice during the skin incision surgery. Samples taken from the incision site were cultured on LB agar and quantified using the method described above.


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Figure 7. Murine model treatment groups. (A) Q-PEI-PNIPAAm applied as a surgical drape over bacteria. (B) IOBANTM 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.

Five agar plates that were inoculated from each time point and corresponding groups were analyzed at each time point. Figure 8 reports the number of CFUs detected by bacterial swabs following the skin incision surgery for each of the five bacteria species: C. amycolatum, E. coli, MRSA, S. aureus, and S. epidermidis. For all five species, the largest number of bacterial colonies were cultured from the negative controls that received the bacteria spray with no drape placement. This trend was consistent throughout each time point. For experiments using C. amycolatum, the use of either Q-PEI-PNIPAAm or IOBANTM drape resulted in reduced detection of CFUs. Furthermore, there was no statistical difference observed between the use of Q-PEI-PNIPAAm compared to IOBANTM at most time points. However, at 75 mins post-suturing, mice treated with Q-PEI-PNIPAAm had significantly fewer detected colonies compared to mice treated with IOBANTM (Figure 8A). Analysis of MRSA



cultures showed similar results when comparing the Q-PEI-PNIPAAm and IOBANTM, with a significant difference between the number of CFUs present at 0, 10, 20, and 75 mins. However, at the remaining time points, there was no significant difference between the Q-PEI-PNIPAAm and IOBANTM groups. Interestingly, there was an increase in bacteria CFUs seen with samples taken from the IOBANTM (incision swab) for all of the time points. It is important to note that there is an unrepresentative low number of CFUs reported at time 0 for the negative control due to the formation of fused colonies representative of large amounts of bacteria (Figure 8B). Figure 8C shows the number of E. coli CFUs following inoculation and appropriate treatment. Based on the number of detected CFUs, E. coli displayed poor survival on the mouse epidermidis, showing a rapid decline in bacterial survival over the 120 min course. Despite this observation, Q-PEI-PNIPAAm showed a significant difference in the killing capacity of E. coli when compared to both IOBANTM swabs (drape and incision) up until the first time point. At the remaining time points, no statistical difference was observed between Q-PEI-PNIPAAm compared to IOBANTM. Similar results were observed for experiments using S. aureus and S. epidermidis (Figures 8D and 8E). Significantly fewer CFUs were detected for the Q-PEIPNIPAAm mice at time 0 compared with the IOBANTM drape; however, there was no statistical difference at the remaining time points.


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Figure 8. The number of CFUs for C. amycolatum (A), MRSA (B), E. coli (C), S. aureus (D), and S. epidermidis (E) observed following skin incision surgery. Error bars represent the standard error of the mean. Results from this animal model consistently show lower numbers of detected CFUs with the use of Q-PEI-PNIPAAm as a surgical drape compared to IOBANTM. However, in most of these cases, the difference in CFUs detected with the use of these two drapes is not statistically different. With that being said, data from all five bacterial strains indicated that the performance of Q-PEI-PNIPAAm as a surgical drape is comparable to that of IOBANTM. Furthermore, it should be noted that, while Q-PEI-PNIPAAm remained on the mouse skin following surgery, the IOBANTM drapes were removed, and thus Q-PEI-PNIPAAm had more time to kill the bacteria.



Notably, however, Q-PEI-PNIPAAm is intended to remain on the skin following surgery, so this study ultimately tested this polymer as it is intended to be used. Biocompatibility of Q-PEI-PNIPAAm in vivo The polymer was designed to be used for topical use as a surgical incision drape. However, due to the nature of surgical procedures traces of Q-PEI-PNIPAAm could enter the body cavity. For this reason, an in vivo biocompatibility test was conducted. As described in the methods, subcutaneous injections containing either Q-PEI-PNIPAAm or saline were administered to 18 mice. The tissue surrounding the injection area was collected and treated to determine the number of macrophages in the area. As shown on Figure 9A, the presence of macrophages was confirmed with the use of a double stain that was conducted with an antiCD68 antibody conjugated to an Alexa Fluor 594 (red stain) and DAPI to stain cell nuclei (blue stain). Although Figure 9A shows that Q-PEI-PNIPAAm had a higher number of macrophages present compared to saline, the quantification of CD68+ cells shown in Figure 9B demonstrates that there was no significant difference after 28 days. Similarly, it is important to mention that prior studies conducted to assess biocompatibility of chlorhexidine (current standard antiseptic) in rats have shown significant adverse reactions38,39.


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Figure 9. Biocompatibility of Q-PEI-PNIPAAm in vivo. (a)Representative confocal images of the harvested tissue. 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 µm. (b) The graph shows the comparison of CD68+ cells per unit area following 1, 7, and 28 days after subcutaneous injections of saline and Q-PEI-PNIPAAm (QPP). Error bars represent the standard error of the mean.



Conclusion A novel polymer (Q-PEI-PNIPAAm) with antibacterial and thermogelling properties was designed. The time-kill curves showed excellent in vitro antibacterial properties against five different strains of bacteria, including gram-positive and gram-negative strains. In addition, in vivo tests with the mice skin model showed comparable, and in some cases superior, antibacterial activity over current SIDs. Similarly, the in vivo biocompatibility test showed no statistical difference in macrophage presence at 28 days post-subcutaneous injection between the mice treated with Q-PEI-PNIPAAm and saline. The biocompatibility test has shown more promising results than previously published biocompatibility studies conducted with the current standard antibacterial, 2% chlorhexidine. This polymer system may ensure ease of application, simplifying the surgical preparation process while also reducing surgical prep time. Also, this polymer may have the potential to mitigate some of the inherent drawbacks to the current SIDs, including a reduced risk of air bubble formation and a reduced risk of epithelial cell detachment upon removal. Furthermore, this polymer has the potential to be used in additional applications that would require antimicrobial properties or benefit from an antimicrobial coating (e.g. prevent catheter based infections). Although we have not investigated these additional applications, we are encouraged by the results we have obtained and the future potential of this material.


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Appendix/Nomenclature/Abbreviations AUTHOR CONTRIBUTIONS MB and MS performed aspects of the study including polymer synthesis, polymer characterization, animal surgeries, data collection, and data analysis. MB and ML performed data analysis and compiled the data/paper for publication. VP provided guidance for experiment direction. DP conceived the study and provided the means and direction for the synthesis and characterization of this polymer conduit. FUNDING Research supported by Bioscience Discovery Evaluation Grant UCD53252. ACKNOWLEDGEMENTS We would like to thank Ronald G. Gill (University of Colorado Denver Anschutz Medical Campus) for providing the S. epidermidis and C. amycolatum bacterial strains. We would also like to thank Nana Burns and Sandy Walchak (Cardiovascular Pulmonary Research Cell Repository – University of Colorado Denver Anschutz Medical Campus) for providing the fibroblast cells.



References (1)

Reichman, D. E.; Greenberg, J. A. Reducing Surgical Site Infections: A Review. Rev. Obstet. Gynecol. 2009, 2 (4), 212–221.

(2)

Greene, L. R. Guide to the Elimination of Orthopedic Surgery Surgical Site Infections: An Executive Summary of the Association for Professionals in Infection Control and Epidemiology Elimination Guide. Am. J. Infect. Control 2012, 40 (4), 384–386.

(3)

Webster, J.; Alghamdi, A.; Webster, J.; Alghamdi, A. Use of Plastic Adhesive Drapes during Surgery for Preventing Surgical Site Infection ( Review ) Use of Plastic Adhesive Drapes during Surgery for Preventing Surgical Site Infection. 2015, No. 4.

(4)

Milandt, N.; Nymark, T.; Jørn Kolmos, H.; Emmeluth, C.; Overgaard, S. IodineImpregnated Incision Drape and Bacterial Recolonization in Simulated Total Knee Arthroplasty: A Controlled, Randomized Experimental Trial. Acta Orthop. 2016, 87 (4), 380–385.

(5)

Zokaie, S.; White, I. R.; McFadden, J. D. Allergic Contact Dermatitis Caused by Iodophor-Impregnated Surgical Incise Drape. Contact Dermatitis 2011, 65 (5), 309.

(6)

Grove, G. L.; Eyberg, C. I. Comparison of Two Preoperative Skin Antiseptic Preparations and Resultant Surgical Incise Drape Adhesion to Skin in Healthy Volunteers. JBJS 2012, 94 (13).

(7)

Alexander, J.; Aerni, S.; JP, P. Development of a Safe and Effective One-Minute Preoperative Skin Preparation. Arch. Surg. 1985, 120 (12), 1357–1361.

(8)

Chiu, K. Y.; Lau, S. K.; Fung, B.; Ng, K. H.; Chow, S. P. PLASTIC ADHESIVE DRAPES AND WOUND INFECTION AFTER HIP FRACTURE SURGERY. Aust. N. Z.


Page 30 of 35

Page 31 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J. Surg. 1993, 63 (10), 798–801. (9)

Harrop, J. S.; Styliaras, J. C.; Ooi, Y. C.; Radcliff, K. E.; Vaccaro, A. R.; Wu, C. Contributing Factors to Surgical Site Infections. J. Am. Acad. Orthop. Surg. 2012, 20 (2), 94–101.

(10)

Lin, J.; Qiu, S.; Lewis, K.; Klibanov, A. M. Bactericidal Properties of Flat Surfaces and Nanoparticles Derivatized with Alkylated Polyethylenimines. Biotechnol. Prog. 2002, 18 (5), 1082–1086.

(11)

Severina, I. I.; Muntyan, M. S.; Lewis, K.; Skulachev, V. P. Transfer of Cationic Antibacterial Agents Berberine, Palmatine, and Benzalkonium through Bimolecular Planar Phospholipid Film and Staphylococcus Aureus Membrane. IUBMB Life 2001, 52 (6), 321–324.

(12)

Lewis, K.; Klibanov, A. M. Surpassing Nature: Rational Design of Sterile-Surface Materials. Trends Biotechnol. 2005, 23 (7), 343–348.

(13)

Mancuso, R.; Amuso, R.; Armentano, B.; Grasso, G.; Rago, V.; Cappello, A. R.; Galiano, F.; Figoli, A.; De Luca, G.; Hoinkis, J.; Gabriele, B. Synthesis and Antibacterial Activity of Polymerizable Acryloyloxyalkyltriethyl Ammonium Salts. Chempluschem 2017, 82 (10), 1233–1234.

(14)

Milović, N. M.; Wang, J.; Lewis, K.; Klibanov, A. M. Immobilized N-Alkylated Polyethylenimine Avidly Kills Bacteria by Rupturing Cell Membranes with No Resistance Developed. Biotechnol. Bioeng. 2005, 90 (6), 715–722.

(15)

Garay, M. T.; Alava, C.; Rodriguez, M. Study of Polymer – Polymer Complexes and Blends of Poly ( N -Isopropylacrylamide ) with Poly ( Carboxylic Acid ). 2 . Poly ( Acrylic Acid ) and Poly ( Methacrylic Acid ) Partially Neutralized. Polymer (Guildf).



2000, 41, 5799–5807. (16)

Zhang, X.; Wang, F.; Chu, C. Thermoresponsive Hydrogel with Rapid Response Dynamics. 2003, 4, 451–455.

(17)

Laughter, M. R.; Ammar, D. A.; Bardill, J. R.; Pena, B.; Kahook, M. Y.; Lee, D. J.; Park, D. A Self-Assembling Injectable Biomimetic Microenvironment Encourages Retinal Ganglion Cell Axon Extension in Vitro. ACS Appl. Mater. Interfaces 2016, 8 (32), 20540– 20548.

(18)

Balouiri, M.; Sadiki, M.; Ibnsouda, S. K. Methods for in Vitro Evaluating Antimicrobial Activity: A Review. J. Pharm. Anal. 2016, 6 (2), 71–79.

(19)

Sun, K. H.; Liu, Z.; Liu, C.; Yu, T.; Shang, T.; Huang, C.; Zhou, M.; Liu, C.; Ran, F.; Li, Y.; Shi, Y.; Pan, L. Evaluation of in Vitro and in Vivo Biocompatibility of a Myo-Inositol Hexakisphosphate Gelated Polyaniline Hydrogel in a Rat Model. Sci. Rep. 2016, 6 (April), 1–11.

(20)

Behlau, I.; Mukherjee, K.; Todani, A.; Tisdale, A. S.; Cade, F.; Wang, L.; Leonard, E. M.; Zakka, F. R.; Gilmore, M. S.; Jakobiec, F. A.; Dohlman, C. H.; Klibanov, A. M. Biocompatibility and Biofilm Inhibition of N,N-Hexyl,methyl-Polyethylenimine Bonded to Boston Keratoprosthesis Materials. Biomaterials 2011, 32 (34), 8783–8796.

(21)

Lin, J.; Qiu, S.; Lewis, K.; Klibanov, A. M. Mechanism of Bactericidal and Fungicidal Activities of Textiles Covalently Modified with Alkylated Polyethylenimine. Biotechnol. Bioeng. 2003, 83 (2), 168–172.

(22)

Park, D.; Wang, J.; Klibanov, A. M. One-Step, Painting-like Coating Procedures to Make Surfaces Highly and Permanently Bactericidal. Biotechnol. Prog. 2006, 22 (2), 584–589.

(23)

Park, D.; Larson, A. M.; Klibanov, A. M.; Wang, Y. Antiviral and Antibacterial


Page 32 of 35

Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Polyurethanes of Various Modalities. Appl. Biochem. Biotechnol. 2013, 169 (4), 1134– 1146. (24)

Li, J.; Mo, L.; Lu, C.; Fu, T.; Yang, H.; Tan, W.; Sciences, M.; Genomics, F. Functional Nucleic Acid-Based Hydrogels for Bioanalytical and Biomedical Applications. 2017, 45 (5), 1410–1431.

(25)

Chaterji, S.; Kwon, I. K.; Park, K. Smart Polymeric Gels: Redefining the Limits of Biomedical Devices. Prog. Polym. Sci. 2007, 32 (8-9), 1083–1122.

(26)

Skrabania, K.; Kristen, J.; Laschewsky, A.; Akdemir, Ö.; Hoth, A.; Lutz, J. F. Design, Synthesis, and Aqueous Aggregation Behavior of Nonionic Single and Multiple Thermoresponsive Polymers. Langmuir 2007, 23 (1), 84–93.

(27)

Geever, L. M.; Nugent, M. J. D.; Higginbotham, C. L. The Effect of Salts and pH Buffered Solutions on the Phase Transition Temperature and Swelling of Thermoresponsive Pseudogels Based on N-Isopropylacrylamide. J. Mater. Sci. 2007, 42 (23), 9845–9854.

(28)

Schmaljohann, D. Thermo- and pH-Responsive Polymers in Drug Delivery. Adv. Drug Deliv. Rev. 2006, 58 (15), 1655–1670.

(29)

Wintgens, V.; Amiel, C. Physical Gelation of Amphiphilic poly(N-Isopropylacrylamide): Influence of the Hydrophobic Groups. Macromol. Chem. Phys. 2008, 209 (15), 1553– 1563.

(30)

Oda, Y.; Kanaoka, S.; Sato, T.; Aoshima, S.; Kuroda, K. Block versus Random Amphiphilic Copolymers as Antibacterial Agents. Biomacromolecules 2011, 12 (10), 3581–3591.

(31)

Delcour, A. H. Outer Membrane Permeability and Antibiotic Resistance. Biochim Biophys



Acta. 2009, 1794 (5), 808–816. (32)

Laverty, G.; Gorman, S. P.; Gilmore, B. F. The Potential of Antimicrobial Peptides as Biocides. Int. J. Mol. Sci. 2011, 12 (10), 6566–6596.

(33)

Bshena, O.; Heunis, T. D. J.; Dicks, L. M. T.; Klumperman, B. Antimicrobial Fibers: Therapeutic Possibilities and Recent Advances. Future Med. Chem. 2011, 3 (14), 1821– 1847.

(34)

Timofeeva, L.; Kleshcheva, N. Antimicrobial Polymers: Mechanism of Action, Factors of Activity, and Applications. Appl. Microbiol. Biotechnol. 2011, 89 (3), 475–492.

(35)

Park, D.; Finlay, J. A.; Ward, R. J.; Weinman, C. J.; Krishnan, S.; Paik, M.; Sohn, K. E.; Callow, M. E.; Callow, J. A.; Handlin, D. L.; Willis, C. L.; Fischer, D. A.; Angert, E. R.; Kramer, E. J.; Ober, C. K. Antimicrobial Behavior of Semifluorinated-Quaternized Triblock Copolymers against Airborne and Marine Microorganisms. ACS Appl. Mater. Interfaces 2010, 2 (3), 703–711.

(36)

Demirci, F.; Yildirim, K.; B. Kocer, H. Antimicrobial Open-Cell Polyurethane Foams with Quaternary Ammonium Salts; 2017; Vol. 135.

(37)

Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65 (1-2), 55–63.

(38)

Monteiro, A. S. F.; Macedo, L. G. S.; Macedo, N. L.; Feitosa, F. A.; Toyoshima, T. Biocompatibility of a Chlorhexidine Local Delivery System in a Subcutaneous Mouse Model. Med. Oral Patol. Oral Cir. Bucal 2011, 16 (2).

(39)

GF, C. Chlorhexidine Hypersensitivity: A Critical and Updated Review. J. Allergy Ther. 2013, 04 (04), 1–7.


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TOC Graphic


Quaternized Q-PEIPAAm-Based Antimicrobial Reverse Thermal Gel: A

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