Amide-Based Cationic Polymeric N-Halamines: Synthesis

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Amide-based Cationic Polymeric N-halamines: Synthesis, Characterization, and Antimicrobial and Biofilm-binding Properties Ze Jing, Kemao Xiu, and Yuyu Sun Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Industrial & Engineering Chemistry Research

Amide-based Cationic Polymeric N-halamines: Synthesis, Characterization, and Antimicrobial and Biofilm-binding Properties

Ze Jing, Kemao Xiu, Yuyu Sun∗

Department of Chemistry University of Massachusetts, Lowell, MA 01854, USA

* Correspondence to: Yuyu Sun ([email protected]).

ACS Paragon Plus Environment

Industrial & Engineering Chemistry Research 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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ABSTRACT A series of amide-based N-halamine precursors, poly 3-(4’-vinylbenzyl) - 5, 5dimethylhydantoin-co-trimethyl-2-methacryloxyethylammonium

chloride

(PVPT),

were

synthesized by copolymerizing 3-(4’-vinylbenzyl) - 5, 5-dimethylhydantoin (VBDMH) and trimethyl-2-methacryloxyethylammonium chloride (TMAC) with different compositions. Chlorine bleach treatment was used to transform the VBDMH moieties in the copolymers into amide N-halamines (Cl-PVPT). 1H-NMR, FT-IR, and dynamic light scattering (DLS) analysis were used to characterize the copolymers. With lower than 50% of VBDMH, the Cl-PVPT copolymers could be dissolved in water, and provided powerful antimicrobial function in killing Gram-positive as well as Gram-negative bacteria. Furthermore, Cl-PVPTs rapidly bound onto preexisting bacterial biofilms and eradicated adherent bacteria. The biofilm-binding kinetics were studied, and the parameters of the kinetics were provided.

Keywords: N-halamines, Cationic, Biofilm-binding, Antimicrobial


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Introduction Biofilms are structured communities containing microorganisms and polymeric extra cellular matrix (EPS) adhering to solid surfaces1-3. Protected by EPS, biofilm bacteria are much more resistant against disinfectants than non-biofilm bacteria, causing serious problems in medical, environmental, industrial, institutional and hygienic applications4,5. Common biocides, such as alcohols, iodine, hydrogen peroxides, quaternary ammonium compounds, etc, can be used to disinfect high-touch, high-risk surfaces containing biofilms 6, 7. However, these agents tend to have a short resident time on the biofilms, leading to suboptimal disinfecting efficacy against microbial cells embedded in biofilms. In our previous studies, we designed and synthesized a new class of cationic amine-based polymeric N-halamines as biofilm-binding disinfectants, which provided long-term and powerful antimicrobial activities to eradicate microbes in biofilms8. N-halamines are compounds containing oxidative halogens that are covalently bonded to nitrogen9. Upon contact, a halogen exchange reaction between the N-halamines and microbial cells could occur, leading to expiration of the microorganisms. While amine-based N-halamines are more stable, amide-based N-halamines provide more potent antimicrobial effects because of electron-withdrawing effect of the carbonyl groups9. To further broaden our selective options of biofilm-binding disinfectants, an amide-based N-halamine precursor, 3-(4’-vinylbenzyl) - 5, 5-dimethylhydantoin (VBDMH) was used to copolymerize with a cationic monomer, trimethyl-2-methacryloxyethylammonium chloride (TMAC), followed by bleach treatment to produce amide N-halamines (Cl-PVPT). In this design, the amide N-halamines were used to provide antimicrobial effects, and the quaternary ammonium monomer, TMAC, was employed to increase the water solubility of Cl-PVPT and act as “binders” toward negatively charged biofilms via electrostatic interactions to increase local N-halamine



concentration and extend resident time. The kinetic parameters of Cl-PVPT binding were also investigated. Experimental Section Materials. Trimethyl-2-methacryloxyethlammonium chloride (TMAC), 4-vinylbenzyl chloride, 5, 5-dimethylhydantoin (DMH), azobisisobutyronitrile (AIBN), poly (ethylene glycol) from Aldrich or Acros. Tubes for dialysis (1000D) were provided by Spectrum labs. Staphylococcus epidermidis (S. epidermidis; ATCC 35984) and Escherichia coli (E. coli, ATCC 15597) were obtained from the American Type Culture Collection (ATCC). The nutrient broth and agar were purchased from Fisher Science Education. Instruments. A Nicolet™ iS™10 spectrometer was used to obtain ATR-IR spectra. Proton NMR studies were carried out using a Bruker & Spectrospin Avance spectrometer. A Delsa™Nano HC DLS particle analyzer (Beckman Coulter) was used to determine molecular weights and the surface zeta potentials. Ultraviolet (UV) studies were performed on a Beckman Coulter DU 520 spectrophotometer. Preparation of 3-(4’-vinylbenzyl) - 5, 5-dimethylhydantoin (VBDMH). 3-(4’-vinylbenzyl) - 5, 5-dimethylhydantoin (VBDMH) was prepared following a previously reported procedure by us as shown in Scheme 110. Briefly, 0.05 mol DMH and 0.05 mol KOH was placed in 25mL deionized water, and 0.05 mol 4-vinylbenzyl chloride was mixed with 10 mL MeOH. The two solutions were combined and the reaction continued at 65 °C for 3 hours. The solvent was evaporated at 40 °C, and, the resulting solid was recrystallized from MeOH/H2O. DMH powders were obtained after drying under vacuum at 40 °C overnight. The yield was 71.70%. Synthesis

of

poly

3-(4’-vinylbenzyl)

-

5,

5-dimethylhydantoin-co-trimethyl-2-

methacryloxyethylammonium chloride (PVPT). A series of polymeric amide-based N-


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halamine precursors (PVPT) were synthesized by adjusting the molar ratios of VBDMH and trimethyl-2-methacryloxyethlammonium chloride (TMAC), as shown in Scheme 1. The copolymerization of VBDMH and TMAC were carried out at 93 °C for 5 h under N2 in ethanol, using azobisisobutyronitrile (AIBN, 1 wt%) as initiator. The contents of VBDMH were varied between 10% and 90% (mole %), and the resulting copolymers were designated as PVPT1090, PVPT 3070, PVPT 5050, PVPT 7030, and PVPT 9010. When the VBDMH content was less than 50%, the product could be dissolved in water. In order to obtain biofilm-binding biocides, PVPT 7030 and PVPT 9010, which were insoluble in water, were not included in the following studies. After removal of ethanol, impurities in PVPT1090, PVPT3070 and PVPT5050 were removed using the dialysis tube in water. After rotary evaporation in the presence of ethanol and toluene, the PVPTs were dried under vacuum at 40 °C for 24 h. The yields of PVPT1090, PVPT 3070 and PVPT 5050 were 86.12%, 87.38% and 66.72%, respectively. The structures of the PVPTs were confirmed in ATR and NMR analyses. 1H NMR was used to calculate the monomer compositions11, and DLS was used to determine molecular weights12-14. Synthesis of Cl-PVPT. The PVPTs were treated in a 1:30 dilution of regular chlorine bleach at pH=4, 60°C for 120 min. The solution was dialyzed with water to remove impurities. The solvent was evaporated with the presence of ethanol and toluene at 40 °C in a rotary evaporator. The resulting Cl-PVPTs were denoted as Cl-PVPT1090, Cl-PVPT3070 and Cl-PVPT5050, respectively. The presence of nitrogen-halogen structure was detected by UV study

10, 15, 16.

Titration was used to determine chlorine contents in Cl-PVPTs by following procedures reported in previous research15, 17. Minimum inhibitory concentration of the Cl-PVPTs. Minimum inhibitory concentrations (MIC) of the Cl-PVPTs were determined against S. epidermidis (ATCC#35984) and E. coli



(ATCC#15597). Briefly, 10 mL soy broth containing a colony of freeze-dried bacteria was placed in a microbial cabinet at 37 °C for 28 hours. The Cl-PVPTs were serially diluted into 12 concentrations using soy broth. One (1) mL of each Cl-PVPT was added into a 24-well sterile cell culture plate, 1 mL bacteria suspension with a density of 106 CFU (colony forming unit)/mL was added, and the bacteria grew at 37 °C for one day. MIC value was determined as the lowest ClPVPT concentration leading to the absence of viable bacteria (clear broth) after incubation. The same procedure was followed to determine the MICs of the TMAC homopolymer (PolyTMAC). Each experiment had three repetitions18. Biofilm-binding properties. Microscope glass slides were cut into 1x1 cm2 squares and washed with ethanol and air-dried. The squares were put individually into 3 mL 106 CFU/mL of the bacteria in nutrient broth. The temperature was maintained at 37 °C. The vials were gently shaken for 80 hr to form bacterial biofilms. The squares were washed 3 times with non-flowing PBS, and placed individually into a Cl-PVPT solution in PBS at 25°C. The concentration of the Cl-PVPT in the PBS solution was tested periodically for 24 hr with a UV spectrometer to determine the binding amount of Cl-PVPT onto the biofilms16. Zeta potential analysis. Zeta potentials of the original glass square, glass with preformed biofilm, and biofilms treated with the Cl-PVPT were determined on the DLS zeta potential apparatus. Each test was repeated 3 times16. Biocidal effects of the Cl-PVPT on bacterial cells in the biofilms. Biofilm were put into 2 mL Cl-PVPT3070 solutions (0-5 mg/mL) at 37°C16. Periodically, the biofilm samples were collected and cleaned three times with sterile PBS. In order to remove the adherent bacteria from glass, the glass slides were treated by sonication for 5 min followed by vortex for 1 min. Dilutions of the


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resulting suspensions were incubated on agar plates at 37 ° for 24-48 h for CFU determination of recoverable bacteria. Results and Discussion Synthesis and characterization of the Cl-PVPT copolymers. The monomer, VBDMH, was synthesized by reacting 4-vinylbenzyl chloride with DMH as we reported in a published study10. The 1H NMR and FT-IR spectra of VBDMH were shown Figures S1 and S2 (Supporting Materials), confirming synthesis of VBDMH. Copolymerization of VBDMH with TMAC produced PVPT. The samples were characterized in ATR-IR and 1H NMR studies. In the ATR-IR spectra (Fig. 1), both VBDMH and TMAC showed peaks at 1630 cm-1, which were attributable to the C=C double bonds. In the spectra of the copolymers, these bands disappeared because of the polymerization reactions. In 1H NMR analysis using PVPT3070 as an example (Fig 2), the 1H attached to the carbon-carbon double bonds in VBDMH had peaks at 5.24, 5.8 and 6.7 ppm, and the protons of the C=C in TMAC had peaks at 5.78 and 6.17 ppm. After copolymerization, these signals disappeared in the spectrum of the PVPT3070 copolymer. The 1H NMR spectra of other PVPTs were shown in Figures S3 and S4 with similar results. The molecular weights and compositions of the PVPTs were provided in Table S1 in Supporting Materials. Chlorination transferred the amide groups in PVPT into N-halamines. Using the UV spectrum of PVPT3070 as an example (Fig 3), before chlorination, PVPT 3070 showed a peak at 275 nm, and this was caused by the benzene ring and the hydantoin ring structures of the VBDMH moiety. After chlorination, the UV absorption peak shifted to 290 nm in the spectrum of ClPVPT3070, confirming the formation of N-Cl bonds after chlorination10. Other PVPTs and ClPVPTs demonstrated similar changes in UV studies (curves are provided in Figure S6).



The contents of Cl in the Cl-PVPTs were presented in Table S2 (Supporting Materials). It could be seen that the experimental chlorine contents was much lower than the theoretical values (if the N-H groups in PVPT were completely transferred to N-Cl). This observation could be explained by the hydrophobic nature of the VBDMH moieties in the copolymers. Since the TMAC moieties were hydrophilic, these moieties will wind around the hydrophobic VBDMH structures in the random-coils of the polymer in aqueous solutions. Thus, portions of the VBDMH moieties could “hide” inside the random-coils and not all of them had the opportunities to be chlorinated. Besides, after chlorination, the VBDMH moieties in the copolymers tended to be more hydrophobic, making it difficult to expose the oxidative chlorine during the titration process. Minimum inhibitory concentrations (MIC). The MIC values of polyTMAC and the Cl-PVPT copolymers against S. epidermidis and E.coli were summarized in Table 1. As a cationic polymer, the TMAC homopolymer showed certain antimicrobial ability, with a MIC of 100 mg/ml for E. coli and 20.83 mg/ml for S. epidermidis. The antimicrobial activities of quaternary ammonium based cationic polymers were caused by the adsorption of the cationic polymer onto the anionic bacteria surface, which could compromise bacteria cell membrane, increase permeability, and cause leakage of intercellular substances10. The water soluble Cl-PVPT copolymers showed much lower MIC values than polyTMAC, suggesting that the N-halamine structures could be the major contributor to the antimicrobial activity. The MIC values of polyTMAC and the Cl-PVPT copolymers against E.coli was much higher than those against S .epidermidis. E.coli is Gram negative and S. epidermidis is Gram positive. Unlike Gram-positive bacteria, Gram-negative bacterial cells are protected by a lipid bilayer. This could prevent/reduce the penetration of disinfectants through the cell walls22-30, rendering them more resistant against disinfection.


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As expected, in the testing of E. coli, higher VBDMH contents led to more potent antimicrobial effects, which could be due to more available N-halamine structures. Also, E. coli surface is more hydrophobic due to the presence of the lipid bilayer, making it easier for hydrophobic N-halamines (e.g., Cl-PVPT5050) to bind. On the other hand, in the testing of S. epidermidis, Cl-PVPT3070 showed much more potent antibacterial effects than both ClPVPT1090 and Cl-PVPT5050. Compared with Cl-PVPT1090, Cl-PVPT3070 contained more Nhalamine structures to provide antimicrobial function. Compared with Cl-PVPT5050, ClPVPT3070 is more hydrophilic and has a much lower molecular weight (see Table S1 in Supporting Information). Thus, it was easier for Cl-PVPT3070 to diffuse into the bacteria and make contact with intercellular proteins and enzymes to provide antimicrobial effects 19-21. Biofilm-binding behaviors. Cl-PVPT3070 was used as an example for biofilm-binding studies, which had limited binding amount on the original glass slides (Fig 4a). With the presence of bacterial biofilms on the glass, the binding amounts of Cl-PVPT3070 were much higher, confirming that Cl-PVPT3070 mainly bound onto the bacterial biofilms. The binding of Cl-PVPT3070 onto the biofilms was investigated by zeta-potential analysis. As shown in Fig. 4b, glass slides with biofilms had a negative charge of -19.07 mV (E. coli biofilm) and -11.98 mV (S. epidermidis biofilm) respectively, which were much lower than the zeta potential of the original glass slides (-4.24 mV). After Cl-PVPT3070 treatment, the biofilmcontaining glass slides showed positive charges, which were +19.55 mV (E. coli biofilm) and +15.39 mV (S. epidermidis biofilm), respectively. This phenomenon further confirmed that the cationic Cl-PVPT3070 bound onto the negatively charged biofilms. Kinetics of Cl-PVPT3070 binding. As described in our prior study8, we used Equation 1 to determine the amount of Cl-3070PMPQs bound to the biofilms16:



𝑉

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(1)

Г = (𝐶0 ― 𝐶𝑡) ∗ 𝐴

Г is the amount of Cl-PVPT3070 adsorption (mg/cm2) at time t (h), C0 and Ct represent the contents of Cl-PVPT3070 (mg/ml) at time t0 and tt (h). V is the solution volume (ml), and A is the surface area of glass slides used in the studies. Fig. 5 showed the adsorption curves of Cl-PVPT3070 onto the original glass slides and the biofilm-containing glass. The adsorption of polyelectrolytes can be simulated with a three-step model31, 32. In the case of Cl-PVPT3070, Step 1 occurred within the first 0.5 h. This step was mass transportation, controlled by convection and/or diffusion31, 33. The second step was between 0.5 h to 3 h. The adsorbed cationic Cl-PVPT3070 repelled the adsorption of other cationic Cl-PVPT3070 polymers, which significantly reduced the rate in adsorption. Step 3 occurred after 3h of adsorption, which contained the reorganization of the adsorbed Cl-PVPT3070 polymer chains to form adsorbed layers and achieve adsorption equilibrium31, 32, 34-36. The steps described above might overlay during the adsorption of charged polymers. Thus, we also used a bimodal model shown in Equation 2 to characterize this process31, 37.

(

Г = A1 ∗ 1 ― 𝑒

―𝑡 𝜏1

) + A2 ∗ (1 ― 𝑒

―𝑡 𝜏2

)

(2)

Here, A1 and A2 represent the amplitudes, and τ1 and τ2 represent the time in the fast and slow steps in the adsorption processes, respectively8. Based on the equation (1)16 and the bimodal model equation (2)38, 39, Fig, 6 showed the simulation of the adsorption process in the binding of Cl-PVPT3070 onto glass slides containing adherent S. epidermidis. Based on the R-square values, the bimodal model fitted the adsorption curve nicely. We obtained similar results in investigating the binding of Cl-PVPT3070 onto the original glass and glass containing E. coli biofilms. The data were presented in Figures S6 and S7.


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Table 2 presented the calculated kinetic parameter values of Cl-PVPT3070 binding to different glass slides (with or without bacterial biofilms) using the bimodal model. The adsorption rate was calculated based on the slope of the tangent line to the adsorption curve 8: A1

―𝑡

A2

―𝑡

Г′ = 𝜏1 ∗ 𝑒 𝜏1 + 𝜏2 ∗ 𝑒 𝜏2

(3)

Based on the parameter data (Table 2), the adsorption rate (Г′) of Cl-PVPT3070 onto the glass slide samples could be calculated, as shown in Table S3. According to this model, the adsorption of Cl-PVPT3070 onto each substrates contained a fast step and a slow step. The fast step occurred in the first 3h which contained the two initial adsorption steps described in the threestep model, suggesting that diffusion and adsorption occurred concurrently in the fast adsorption step. The bound cationic Cl-PVPT30/70 repelled the approaching of new cationic Cl-PVPTs, leading to non-diffusive adsorption. After 3h, the binding process became the slow-step. The adsorption and repelling forces achieved balance, and the Cl-PVPT chains reorganized to reach equilibrium31. Effects of Cl-PVPT3070 adsorption on adherent bacterial cells. The binding of Cl-PVPT3070 led to powerful biocidal effects against adherent biofilm bacteria. Without Cl-PVPT3070 treatment, the original E. coli and S. epidermidis biofilms contained 1.4 x 105 CFU/cm2 and 8.8 x 103 CFU/cm2 of recoverable bacteria, respectively. Upon treatment with Cl-PVPT30/70 for 24h, only 0.5 mg/mL of Cl-PVPT3070 was needed to totally kill all the adherent bacteria in either of the bacterial biofilms. Biocidal effects were affected by treatment time. With 1.5 mg/mL of Cl-PVPT3070, the Nhalamine killed all the adherent S. epidermidis in 0.5 h and all the adherent E. coli in 1 h. This concentration was much lower than the MIC value of Cl-PVPT3070 against free-floating E. coli



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(see Table 1). It was believed that the cationic TMAC moieties in Cl-PVPT3070 interacted with the negative charges on biofilms. This effect could increase local N-halamine concentrations and extend resident time of Cl-PVPT3070 on the biofilms, resulting in potent antimicrobial action at low Cl-PVPT concentration. As expected, the amide-based Cl-PVPT N-halamine showed much faster and much more powerful antimicrobial effects against biofilm bacteria than the previously reported amine-based N-halamines8. This could be mainly because that the N-Cl bonds in Cl-PVPT are connected to electron-withdrawing carbonyl groups, which could loosen the nitrogen-halogen covalent bond and stabilize the nitrogen anion as chlorine cation leaves the structure, increasing the oxidative reaction rate and antimicrobial potency of the amide-based N-halamines. Thus, the Cl-PVPT based N-halamines could be good candidates for applications that need fast antimicrobial actions. Conclusion Amide-based cationic N-halamine precursors, poly 3-(4’-vinylbenzyl) - 5, 5dimethylhydantoin-co-trimethyl-2-methacryloxyethylammonium

chloride

(PVPTs),

were

synthesized through the copolymerization of 3-(4’-vinylbenzyl) - 5, 5-dimethylhydantoin (VBDMH) with trimethyl-2-methacryloxyethylammonium chloride (TMAC). Chlorine treatment transferred the N-H structure in the VBDMH moieties into amide-based N-halamines (Cl-PVPT). At less than 50 mole% of VBDMH, the resulting Cl-PVPTs were soluble in water. Cl-PVPT solutions showed powerful antibacterial activity against free floating S. epidermidis or E. coli, and were less effective in killing the Gram-negative bacteria than the Gram-positive ones. Cl-PVPT rapidly bound onto preexisting bacterial biofilms. The adsorption kinetic parameters fitted to the bimodal model well. The adsorbed Cl-PVPT demonstrated rapid and powerful biocidal effects


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against biofilm bacteria, suggesting that the new amide-based cationic polymeric N-halamines for are attractive candidates for rapidly disinfecting microorganisms embedded in biofilms.



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interferometry resolves mechanistic aspects of polyelectrolyte adsorption. Langmuir. 2008, 24, 10633. (36) Rojas, O. J.; Neuman, R. D.; Claesson, P. M. Desorption of low-charge-density polyelectrolyte adlayers in aqueous sodium n-dodecyl sulfate solution. J. Colloid Interface Sci. 2001, 237, 104. (37) Guzmán, E.; Ortega, F.; Baghdadli, N.; Luengo, G. S.;Rubio, R. G. Colloids. Effect of the molecular structure on the adsorption of conditioning polyelectrolytes on solid substrates. Colloids Surf. A. 2011, 375, 209. (38) Chiang, C.-Y.; Starov, V.M.; Lloyd, D.R. Crystallization kinetics of a polymer-solvent system.1. Derivation of model-equations. Colloid J. 1995, 57,715. (39) Chiang, C.-Y.; Starov, V.M.; Hall, M.S.;Lloyd, D.R. Crystallization kinetics of polymersolvent systems. 2. Experimental verification of the model. Colloid J. 1997, 59,236.


Page 17 of 25 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


(40) Qian, L.; Sun, G. Durable and regenerable antimicrobial textiles: Synthesis and applications of 3-methylol-2, 2, 5, 5-tetramethyl-imidazolidin-4-one (MTMIO). J. Appl. Polym. Sci. 2003, 89, 2418-2425. O

Methanol and Deionized water +

65 0C, 3h KOH

NH HN O

N

O

O NH Cl

(a) AIBN + O

N

O

n

m

93 0C, 5h Ethanol N2 Protection

O

O

O

CH2

CH2

CH2

CH2

O N

N

NH

Cl

N

O

Cl

O NH

(b) bleach n

m O

N

O

O

n

m

60°C 2h

O

O

O

CH2

CH2

CH2

CH2 N

N

Cl

O

O

NH

N Cl

(c)


N

Cl


Scheme 1. Preparation of Cl-PVPT: (a) Synthesis of VBDMH10 ; (b) Synthesis of PVPT11; and (c) chlorination of PVPT

Fig. 1. ATR-IR spectra of TMAC, VBDMH, and the PVPT copolymers


Page 18 of 25

Page 19 of 25 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


Fig 2. 1H NMR spectra of VBDMH, TMAC, and PVPT3070



3 2.5

Absorbance

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 25

2 1.5 1 0.5 0 190

210

230

250

270

290

310

330

350

(nm)

Fig. 3. UV spectra of PVPT3070 (solid line) and Cl-PVPT3070 (dotted line)


Page 21 of 25

binding amount(ug/cm2)

350.00

Binding amounts biofilm E.coli biofilm S.epidermidis control

300.00 250.00 200.00 150.00 100.00 50.00 0.00 0.5h

1h

3h 6h time/h

18h

24h

a.

Zeta protentials

25 20 15 Zeta potential(mV)

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


10 5 0 -5

A

B

C

D

E

-10 -15 -20 -25 b. Fig. 4a Binding amounts of Cl-PVPT3070 onto biofilms;

4b Zeta potential values of A. Pure glass slides; B. Glass slides with E.coli biofilms; C. Glass slides with S. epidermidis biofilms; D. Cl-PVPT3070 treated glass slides containing E.coli biofilms; and E. Cl-PVPT3070 treated glass slides containing S. epidermidis biofilms



500 450 400 350 Г(ug/cm2)

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 22 of 25

300 control

250

E Coli

200

S epidermidis

150 100 50 0 0

5

10

15 t(h)

20

25

30

Fig. 5. Adsorption amount (ug/cm2) of Cl-PVPT 3070 as a function of time t (h)


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


Amount of adsorbed Cl-PVPT3070 (ug/cm2)

Page 23 of 25

300

200

―𝑡

(

―𝑡

)

Г = 169.02 ∗ 1 ― 𝑒0.34 + 169.02 ∗ (1 ― 𝑒0.34 ) 2

R =0.90

100

0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h)

Fig. 6 Adsorption amount Cl-PVPT 3070 onto S. epidermidis biofilm vs time fitted by the bimodal model



Page 24 of 25

Table 1. Minimum inhibition concentrations (MIC) of the Cl-PVPT copolymers against E.coli and S. epidermidis (mg/mL) MICs MIC against E. coli* MIC

against

Cl-PVPT1090

Cl-PVPT3070

Cl-PVPT5050

29.22

12.72

5.63

1.83

0.1

5.63

S.

epidermidis**

* The minimum inhibitory concentration of the TMAC homopolymer against E.coli was 100 mg/mL ** The minimum inhibitory concentration of the TMAC homopolymer against S. epidermidis was 20.83mg/mL


Page 25 of 25 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


Table 2. Kinetic parameters of bimodal model for adsorption of Cl-PVPT3070

Substrate Control glass slides E.coli biofilm S. epidermidis biofilm

A1

A2

ᴛ1

ᴛ2

R2

28.33 98.24

28.33 78.84

0.21 56.89

0.21 0.003

0.87 0.99

169.02

169.02

0.34

0.34

0.90


Amide-Based Cationic Polymeric N-Halamines: Synthesis

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