Controlling the Structure and Antimicrobial Function of N-halamine

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Controlling the Structure and Antimicrobial Function of N-halaminebased Polyurethane Semi-Interpenetrating Polymer Networks Kemao Xiu, Jianchuan Wen, Jianhong Liu, Chuanxin He, and Yuyu Sun Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b03302 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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Industrial & Engineering Chemistry Research

Controlling the Structure and Antimicrobial Function of N-halamine-based Polyurethane Semi-Interpenetrating Polymer Networks

Kemao Xiu,2 Jianchuan Wen2, Jianhong Liu1, Chuanxin He1*, Yuyu Sun2*

1

College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen,

Guangdong, 518060, People’s Republic of China, 2Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854, USA

* Correspondence to Dr. Chuanxin He ([email protected]), and Dr. Yuyu Sun ([email protected]).

1

ACS Paragon Plus Environment


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For Table of Contents Only

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ABSTRACT A polymerizable N-halamine precursor, 3-(4’-vinylbenzyl)-5,5-dimethylhydantoin (VBDMH), was diffused into polyurethane (PU) in the presence of a cross-linker and an initiator. Post polymerization of VBDMH led to the formation of PU/PVBDMH semiinterpenetrating polymer networks (IPNs). Upon chlorination, the amide groups in PVBDMH were transformed into stable N-halamines. The presence of N-halamines in the semi-IPNs was confirmed by ATR-IR and energy dispersive X-ray spectroscopy (EDS) analysis. The N-halamine contents in the semi-IPNs could be readily controlled by changing reaction conditions. EDS analysis showed that the N-halamines mainly stayed on the semi-IPN surface, rather than in the bulk, which could be caused by the incompatibility of VBDMH with the amorphous regions in the original PU. Contact mode antimicrobial tests and SEM observations

demonstrated

that

the

semi-IPNs

had

potent

antimicrobial

and

biofilm-controlling effects against both Gram-positive and Gram-negative bacteria, pointing to great potentials of the new N-halamine-based semi-IPNs for a broad range of applications.

Keywords: Interpenetrating polymer network, N-halamine, Antimicrobial, Biofilm, Polyurethane

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Introduction In response to the serious problems caused by microbial biofilm formation on solid surfaces,1–6 antimicrobial polymers have attracted considerable research interests for a broad range of medical, environmental, industrial, and hygienic applicaitons.7–10 One strategy in the design of antimicrobial polymers is to incorporate N-halamines into the target materials to provide biocidal functions.10–25 N-halamines are compounds containing one or more nitrogen-halogen covalent bonds.10 Their antimicrobial potency is similar to that of chlorine bleach, one of the most widely use disinfectants, yet N-halamines are much more stable and less corrosive. Therefore, different approaches, including copolymerization, grafting, blending, and coating, etc.,11–22 have been used to introduce N-halamines into conventional polymers to achieve antimicrobial functions. In one of our previous studies, we developed a semi-interpenetrating polymer network (IPN) strategy to incorporate amine-based N-halamines into polyurethane (PU).15 PU is one of the most commonly used polymers for various applications because of its wide availability, low cost, and excellent physical and biological properties,26–29 but it is vulnerable for microbial colonization and biofilm formation. IPNs are alloys of crosslinked polymer networks (at least one of them is crosslinked).30–34 In our approach, an N-halamine monomer N-chloro-2, 2, 6, 6-tetramethyl-4-piperidyl methacrylate (TMPMCl) was diffused into PU with initiators and crosslinking agents. Thermal treatment led to the polymerization of TMPMCl

with

the

cross-linker,

forming

crosslinked

poly

(N-chloro-2,

2,

6,

6-tetramethyl-4-piperidyl methacrylate) (PTMPMCl) inside PU.15 This route significantly simplified the preparation process of antimicrobial PU, and the resulting semi-IPN showed 4


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antimicrobial efficacy against both Gram-positive and Gram-negative bacteria. However, the antimicrobial potency of the PTMPMCl-based semi-IPN was rather low (total kill of bacteria achieved after 4 hours of contact).15 This could be caused by (1) PTMPMCl is an amine-based N-halamine, which has weak antimicrobial potency because of the stability of the N-Cl bond;20 and (2) the N-halamine polymer PTMPMCl preferred to accumulate within the inside of the semi-IPNs, rather than on the surface, due to the good miscibility of TMPMCl with the soft segment of PU in the semi-IPN fabrication process.15 Since N-halamine/PU semi-INPs have many potential applications needing fast antimicrobial action, this study developed a new molecular design strategy in the preparation of N-halamine/PU semi-IPNs to achieve much more potent antimicrobial effects. We used 3-(4’-vinylbenzyl)-5,5-dimethylhydantoin (VBDMH) as a polymerizable N-halamine precursor to synthesize semi-IPNs. One rationale for this design was that the resulting VBDMH-containing N-halamine was an amide-based N-halamine, which would have more powerful antimicrobial effects than amine N-halamines.20 Another reason was that the VBDMH structure was very different from the ether-based soft segment of PU. The immiscibility would drive VBDMH, and consequently, the resulting polymeric N-halamine, to stay on the surface, rather than in the bulk of the semi-IPNs. This was expected to dramatically promote the contact between microbial cells and the N-halamines to further enhance antimicrobial action. To test this hypothesis, VBDMH-based amide N-halamine/PU semi-IPNs were fabricated and characterized, and the effectiveness of this design principle was confirmed by EDS studies and antimicrobial and biofilm-controlling tests.

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Experimental Section Materials. 4-vinylbenzyl chloride, 5,5-dimethylhydantoin (DMH), potassium iodide and trichloroisocyanuric acid (TCCA) were purchased from Sigma-Aldrich. Nutrient agar (BD 213000 and BD 236950) and nutrient broth (BD cat 234000 and BD cat 211825) were from Fisher Scientific. Di (ethylene glycol) dimethacrylate (DEGDMA) was obtained from Tokyo Chemical Industry (TCI). Polyurethane (PU) was provided by A-dec. Dicumylperoxide (DCP) was from Acros Organics. The microorganisms, Staphylococcus epidermidis (ATCC 35984) and Acinetobacter calcoaceticus (ATCC 31926) were provided by American Type Culture Collection. All other reagents were analytical grade.

Instruments. A Nicolet iS10 Mid-IR spectrometer was used to record ATR-IR spectra of the semi-IPN samples. All semi-IPN samples were discs with a diameter of 7.76 mm and a thickness of 1.0 mm. 1H NMR studies were performed on a 500-MHz spectrometer (Bruker, Switzerland). Scanning electron microscope (SEM) images and energy dispersive spectrometry (EDS) results were acquired from a JEOL JSM 7401 FE-SEM equipped with EDAX genesis XM2 imaging system. The sample discs were frozen and broken so that both the surface and the cross-section EDS spectra could be taken.

Synthesis of 3-(4’-vinyl benzyl)-5, 5-dimethylhydantoin (VBDMH). The synthesis of VBDMH followed the procedure we reported previously.14 Briefly, 6.4 g (0.05 mol) DMH and 2.8 g (0.05 mol) KOH were dissolved in 25 mL distilled water. A solution of 7.7 g (0.05 mol) 4-vinylbenzyl chloride in 10 mL methanol was added in a dropwise manner. The mixture was maintained at 65 °C under constant stirring for 3 h. After removing the solvent under reduced pressure at 45 °C, the solid was collected and recrystallized from 6


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methanol/water (5:1 v/v). VBDMH was obtained as white powders with a yield of 69.90%.

Preparation of PU/PVBDMH semi-IPN. A sequential strategy was used to synthesize the PU/PVBDMH (PUV) semi-IPN.15 VBDMH was dissolved in tetrahydrofuran (THF)/toluene (3:2 v/v) with a concentration of 0.1, 0.15, 0.2, 0.25 and 0.3 g/mL, respectively. DCP (initiator) and DEGDMA (cross-linker) were added with a molar ratio of 100:8:5 (VBDMH:DCP:DEGDMA). PU discs (diameter of 7.76 mm and thickness of 1.0 mm) were immersed in the monomer solutions at ambient temperature for 4 h to allow diffusion of the monomers. After evaporating the solvent, the discs were heated at 95 °C for 4 h to polymerize VBDMH and DEGDMA to form the crosslinked network. The discs were repeatedly washed with acetone to remove the unreacted monomers and initiator. After evaporation of acetone, the resulting semi-IPNs were weighed and the weight increment was calculated according to the following equation:

WI% =

(݉2 − ݉1) × 100% ݉1

where m2 and m1 were the weights of the PUV semi-IPN and the original PU, respectively.

Chlorination of the PUV semi-IPNs. The hydrophobicity of the PUV semi-IPNs made it difficult to chlorinate the samples with hypochlorite aqueous solutions. Thus, we used an organic chlorinating agent, trichloroisocyanuric acid (TCCA), in acetone with a concentration of 0.01 g/mL at ambient temperature for 30 min to transform the amide groups of PVBDMH into amide N-halamines. Afterwards, the discs were taken out and washed with acetone until the eluent contained no detectable TCCA (detected by potassium iodide-starch solution). The content of oxidative chlorine in the chlorinated semi-IPN was titrated with sodium thiosulfate aqueous solution. The chlorinated semi-IPN was weighed, cut into small pieces 7



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and immersed in ethanol in the presence of 0.5 g potassium iodide (KI) with constant shaking at ambient temperature for 5 h under nitrogen atmosphere. The iodine produced was titrated with 0.001 mol/L sodium thiosulfate aqueous solution. The same procedures were applied to the unchlorinated semi-IPNs to serve as controls. The chlorine content of the PUV semi-IPN was calculated according to the following equation:35

ሾClሿ ൬

݉‫݈݋‬ (‫ݒ‬1 − ‫ݒ‬0) × 10ି଺ ÷ 2 ൰= ݃ ݉

where v1 and v0 were the volume (mL) of Na2S2O3 solutions consumed in the titration of the chlorinated and unchlorinated semi-IPNs, and m was the weight of the chlorinated semi-IPN (g).

Contact mode antimicrobial function of the PUV N-halamine semi-IPNs. The antimicrobial efficiency of the chlorinated PUV semi-IPNs was evaluated with a contact mode test according to our previous work against S. epidermidis (Gram-positive bacteria) and

A. calcoaceticus (Gram-negative bacteria).15,21 The bacteria were cultured for 24 h following the supplier’s guidance, harvested by centrifugation, and resuspended in sterile PBS to reach a concentration of 108 to 109 colony forming units per milliliter (CFU/mL). In each test, 2.5 µL of the bacteria PBS suspension containing 0.05% TX-100 (to serve as a wetting agent) were pipetted onto the surface of a PUV N-halamine disc, which was then “sandwiched” using another identical disc.15 After different periods of contact time (15 min, 30 min, and 1 h), the “sandwich” was placed in 5 ml sterile PBS, vortexed to separate the “sandwich” and then sonicated for 10 min to transfer the adherent bacteria from the disc into the PBS. After serial dilution, 100 µL of each diluent were pipetted onto an agar plate and after 24-48 h of incubation, the CFUs on the plates were counted. The same procedure was applied to the 8


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original PU discs to serve as controls. Each test was repeated three times.

Biofilm-controlling function of the PUV N-halamine semi-IPNs. The bacteria (S. epidermidis and A. calcoaceticus, both of which have strong biofilm formation ability) were cultured, harvested, and resuspended in sterile PBS to reach a final concentration of 105–106 CFU/mL. The PUV N-halamine semi-IPN discs were immersed individually in 5 mL of the bacterial suspension for 30 min with constant shaking in a water bath (37°C for S.

epidermidis and 30°C for A. calcoaceticus) to allow bacterial initial adhesion.19 The discs were washed gently with sterile PBS three times to remove loosely attached bacteria, and then incubated in 5 mL nutrient broth for 24 h for bacterial colonization and biofilm formation. Afterwards, the discs were taken out, rinsed gently with sterile PBS to remove the planktonic bacteria, and then immersed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (SCB) at 4 °C for 24 h. After washing with SCB, the discs were put in sequential ethanol solutions following the concentrations of 30%, 50%, 75%, 90%, and 100% (30 min at each concentration) to dehydrate the bacteria. The resulting discs were mounted onto the sample holder, sputter coated with gold, and then observed under SEM. The original PU discs were tested with the same procedures to serve as controls.

Results and Discussion Preparation and characterization of the PUV N-halamine semi-IPNs. VBDMH was synthesized following a procedure we reported previously.14 The 1H NMR spectrum of VBDMH in deuterated DMSO was shown in Figure 1, confirming the chemical structure and purity of the monomer used in this study. VBDMH was swollen into PU together with the thermal initiator (DCP) and the crosslinking agent (DEGDMA). The polymerization of 9



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VBDMH and DEGDMA led to the formation of PUV semi-IPNs consisting of linear PU and crosslinked PVBDMH. As a result, although THF could readily dissolve the original PU, it could only swell, but could not dissolve, the PUV semi-IPNs. The effects of VBDMH content in the solution on the weight increment of the PUV semi-IPNs were shown in Figure 2. With the increase of VBDMH concentration in the solution, the weights of the semi-IPNs increased rapidly initially; at higher than 0.2 g/mL of VBDMH, however, this trend became unobvious, suggesting that a saturated value was reached, which could be caused by the limited solubility of VBDMH in the amorphous region of the original PU. In the following studies, the semi-IPNs with a weight increment of 31.97 %, 44.35 %, and 56.09 % were selected as representative samples, which were denoted as PUV1, PUV2 and PUV3, respectively. The amide groups of PVBDMH in the semi-IPNs were transformed into N-halamines through chlorination. Trichloroisocyanuric acid (TCCA) was used as the chlorinating agent instead of hypochlorite bleach. This was because the PUV semi-IPNs were very hydrophobic, and our screening tests showed that aqueous bleach solutions led to low active chlorine content in the resulting PUVs. After chlorination with TCCA, the content of oxidative chlorine in the N-halamine semi-IPNs (PUV1, PUV2 and PUV3) was iodometrically titrated, and the results were summarized in Figure 3. The oxidative chlorine content was 2.90, 2.97 to 4.23 (10-5 mole per gram of semi-IPN) for the N-halamines of PUV1, PUV2 and PUV3, respectively, which could provide potent antimicrobial activity, as shown below. Figure 4 showed the ATR-IR spectra of the samples. Little difference could be observed between the spectrum of the original PU and PUV3. However, after subtracting the 10


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spectrum of PU from the spectrum of PUV3, two strong peaks at 1773.35 cm-1 and 1712.60 cm-1 appeared, which were attributed to the imide and amide carbonyl groups of the newly introduced PVBDMH in the semi-IPNs. Similar results were obtained in the study of PUB1 and PUV2, further confirming the synthesis of the PUV semi-IPNs. The EDS spectra of PUV N-halamine semi-IPNs were recorded for C (0.277 keV), N (0.392 keV), O (0.525 keV), and Cl (2.621 keV). Figure 5 displayed the spectrum of PUV3 N-halamine as an example. Based on the data, the oxidative chlorine weight ratios in the surface and the bulk could be calculated (The table in Figure 5). All the PUV N-halamine semi-IPNs showed a much higher surface chlorine weight contents (from 4.48 % to 5.53 %) than in the bulk (from 0.29 % to 0.69 %). As the original PU did not contain any chlorine atoms, the distribution of chlorine represented the distribution of the N-halamines. Thus, these results strongly suggested that the PVBDMH-based N-halamines were dominantly on the surface of the semi-IPN rather than in the bulk, causing more chlorine on the surface and less on the cross section. This phenomenon could be explained by the molecular structure of VBDMH and PU. The original PU contained amorphous soft segments composed of polyether, and crystalline hard segments consisted of aromatic rings. The soft and hard segments were immiscible, leading to micro-phase separations. VBDMH had benzene ring in its structure, which was very different from the amorphous soft segment in PU. The hard segment was the crystalline region that VDBDH could not diffuse into. Therefore, it would be difficult for VBDMH to stay in the bulk of PU, resulting in the surface accumulation of the PVBDMH crosslinked polymer as well as the subsequent amide-based N-halamine structures. Our results thus 11



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confirmed that in the design of functional semi-IPNs, we can control the distribution of the second polymer (in the bulk or on the surface) by matching or mismatching the miscibility between the diffusing monomer (the monomer to form the second polymer) and the amorphous region of the first, pre-existing polymer.

Antimicrobial and biofilm-controlling function of the PUV N-halamine semi-IPNs. The antimicrobial efficiency of the PUV N-halamine semi-IPNs was evaluated with two representative disease-causing, biofilm-forming bacteria, A. calcoaceticus (Gram-negative) and S. epidermidis (Gram-positive). As shown in Figure 6, all the PUV N-halamine samples provided a total kill of the testing bacteria after 30 min of contact, which was much more potent than the amine-based N-halamine semi-IPNs (a total kill cold be only be achieved after 4 h of contact) we reported previously.15 This could be caused by two factors: (1) the amide N-halamines provided by PVBDMH in the current study were much more potent than amine-based N-halamines, because the electron-withdrawing effect of the amide carbonyl groups could stabilize the Nwhen Cl+ left the N-halamine structure;20 and (2) the amide N-halamines in the PUV semi-IPNs stayed on the surface rather than in the bulk, and this could significantly promote the contact between the testing bacteria with the N-halamines, leading to fast antimicrobial action. Thanks to the powerful antimicrobial effect, the PUV N-halamine semi-IPNs demonstrated potent biofilm-controlling ability. As shown in Figure 7, in the biofilm-controlling tests, both A. calcoaceticus and S. epidermidis could form layered biofilm structures on the original PU surface. However, the surface of the PUV N-halamine 12


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semi-INPs were much cleaner. Only scattered adherent bacteria could be observed, and no biofilms were formed. The N-halamines in the PUV semi-IPNs showed excellent durability. Under normal lab conditions (25 oC, 30-90 % RH), the samples have been stored for more than 6 months without any significant changes in the active chlorine contents as well as the antibacterial efficacies against the Gram-positive and Gram-negative bacteria, pointing to long antimicrobial durations for a wide range of applications.

Conclusions An amide-based polymerizable N-halamine precursor, VBDMH, was used to prepare PU/PVBDMH semi-IPNs. Upon chlorination, the amide groups in PVBDMH were transformed into N-halamines. Because of the poor miscibility of VBDMH with the amorphous soft segments of the original PU, the resulting amide N-halamines mainly stayed on the surface, rather in the bulk of the semi-IPNs. This structural characteristic led to potent antimicrobial and biofilm-controlling efficacy of the semi-IPNs against both Gram-negative and Gram-positive bacteria. The molecular design principal used in the current study provided a new strategy in controlling the structure and function of semi-IPN-based antimicrobial and other classes of functional polymers. That was, by matching or mismatching the miscibility of the diffusing monomer with the amorphous region of the first polymer, one can readily control the distribution of the second polymer in the resulting semi-IPNs (surface or the bulk), and thus, the functions of the final products.

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Acknowledgements: Dr. Sun thanks the support from UMass Lowell (startup support and internal funding). Dr. He thanks and acknowledges the support from National Natural Science Foundation of China (21374064 and 21574084). Dr. Xiu thanks the grant from Independent University Alumni Association at Lowell.

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Sun, X.; Zhang, L.; Cao, Z.; Deng, Y.; Liu, L.; Fong, H.; Sun, Y. Electrospun Composite Nanofiber Fabrics Containing Uniformly Dispersed Antimicrobial Agents as an Innovative Type of Polymeric Materials with Superior Antimicrobial Efficacy. ACS Appl. Mater. Interfaces 2010, 2, 952. Lin, J.; Jiang, F.; Wen, J.; Lv, W.; Porteous, N.; Deng, Y.; Sun, Y. Fluorinated and Un-Fluorinated N-Halamines as Antimicrobial and Biofilm-Controlling Additives for Polymers. Polymer (Guildf). 2015, 68, 92. Sun, X.; Cao, Z.; Porteous, N.; Sun, Y. Amine, Melamine, and Amide N -Halamines as Antimicrobial Additives for Polymers. Ind. Eng. Chem. Res. 2010, 49, 11206. Cao, Z.; Sun, Y. Polymeric N-Halamine Latex Emulsions for Use in Antimicrobial Paints. ACS Appl. Mater. Interfaces 2009, 1, 494. Cerkez, I.; Kocer, H. B.; Worley, S. D.; Broughton, R. M.; Huang, T. S. N-Halamine Biocidal Coatings via a Layer-by-Layer Assembly Technique. Langmuir 2011, 27, 4091. Chen, Z.; Luo, J.; Sun, Y. Biocidal Efficacy, Biofilm-Controlling Function, and Controlled Release Effect of Chloromelamine-Based Bioresponsive Fibrous Materials. Biomaterials 2007, 28, 1597. Chen, Z.; Sun, Y. N-Halamine-Based Antimicrobial Additives for Polymers: Preparation, Characterization, and Antimicrobial Activity. Ind. Eng. Chem. Res. 2006, 45, 2634. Cao, Z.; Sun, Y. N-Halamine-Based Chitosan: Preparation, Characterization, and Antimicrobial Function. J. Biomed. Mater. Res. - Part A 2008, 85, 99. Szycher, M. Biostability of Polyurethane Elastomers: A Critical Review. In Journal of Biomaterials Applications; 1988, 3, 297. Lamba, N. M.; Woodhouse, K. A.; Cooper, S. L. Polyurethanes in Biomedical Applications; CRC press, 1997. Griesser, H. J. Degradation of Polyurethanes in Biomedical Applications-A Review. Polym. Degrad. Stab. 1991, 33, 329. Planck, H.; Egbers, G.; Syré, I. Polyurethane in Biomedical Engineering. II. Prog. Biomed. Eng. 1987, 3, 213. Zhao, N.; Liu, S. Thermoplastic Semi-IPN of Polypropylene (PP) and Polymeric N-Halamine for Efficient and Durable Antibacterial Activity. Eur. Polym. J. 2011, 47, 1654. Liu, S.; Zhao, N.; Rudenja, S. Surface Interpenetrating Networks of Poly(ethylene Terephthalate) and Polyamides for Effective Biocidal Properties. Macromol. Chem. Phys. 2010, 211, 286. Dragan, E. S. Design and Applications of Interpenetrating Polymer Network Hydrogels. A Review. Chem. Eng. J. 2014, 243, 572. Karabanova, L. V.; Lloyd, A. W.; Mikhalovsky, S. V.; Helias, M.; Phillips, G. J.; Rose, S. F.; Mikhalovska, L.; Boiteux, G.; Sergeeva, L. M.; Lutsyk, E. D.; et al. Polyurethane/poly(hydroxyethyl Methacrylate) Semi-Interpenetrating Polymer Networks for Biomedical Applications. J. Mater. Sci. Mater. Med. 2006, 17, 1283. Gitsov, I.; Zhu, C. Novel Functionally Grafted Pseudo-Semi-Interpenetrating Networks Constructed by Reactive Linear-Dendritic Copolymers. J. Am. Chem. Soc. 16


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2003, 125, 11228. Dong, Q.; Cai, Q.; Gao, Y.; Zhang, S.; Gao, G.; Harnoode, C.; Morigen, M.; Dong, A. Synthesis and Bactericidal Evaluation of Imide N-Halamine-Loaded PMMA Nanoparticles. New J. Chem. 2015, 39, 1783.

17



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

6 2

1 3

4

8, 9

H2 O

5

Page 18 of 24

DMSO 7

7 8 9

1, 2 3, 4

Figure 5.1 1H NMR of VBDMH in DMSO 6́

6̋

5

Figure 1. 1H NMR spectrum of VBDMH in deuterated DMSO

18


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70

60

Weight increment %

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


50

40

30

20 0.10

0.15

0.20

0.25

0.30

Monomer concentration (g/mL)

Figure 2. The effect of monomer concentration in the solution on the weight increment of the semi-IPNs

19



6

5

4

-5

Chlorine content (10 mol/g of semi-IPN)

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

Page 20 of 24

3

2

1

PUV1

PUV2

PUV3

semi-IPNs

Figure 3. Titratable oxidative chlorine contents in PUV N-halamine semi-IPNs

20


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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


1712.60 cm-1

1773.35 cm-1

VBDMH PUV3 subtraction spectrum 2000

1000

PU

4500

4000

3500

3000

2500

2000

1500

1000

500

Figure 4. ATR-IR spectra of PU, PUV3, VBDMH and the subtraction spectrum between PUV3 and PU

21



surface bulk 5

Chlorine content (wt%)

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

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4

3

2

1

0

PUV1

PUV2 Semi-IPNs

PUV3

Figure 5. Representative EDS spectrum of PUV3 N-halamine and the chlorine weight ratios on the surface and in the bulk of the N-halamine semi-IPNs calculated from EDS spectra

22


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104

15 min 30 min

A. calcoaceticus

100.005

15 min 30 min

S. epidermidis

102 100.000

Percentage reduction (%)

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


100 99.995

98

96 99.990

94 99.985

92

90

99.980

PUV1

PUV2 Semi-IPN samples

PUV3

PUV1

PUV2 Semi-IPN samples

PUV3

Figure 6. Antimicrobial efficiency of the PUV N-halamine semi-IPNs against A.

calcoaceticus and S. epidermidis

23



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

(A) PU

PUV2

(B) PU

PUV2

Page 24 of 24

PUV1

PUV3

PUV1

PUV3

Figure 7. Representative SEM images of adherent bacteria on the surface of the original PU and the PUV N-halamine semi-IPNs (A) A. calcoaceticus and (B) S. epidermidis 24


Controlling the Structure and Antimicrobial Function of N-halamine

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