N-Halamine-Containing Electrospun Fibers Kill Bacteria via a Contact

Research Article www.acsami.org

N‑Halamine-Containing Electrospun Fibers Kill Bacteria via a Contact/Release Co-Determined Antibacterial Pathway Rong Bai,†,§ Qing Zhang,∥,§ Lanlan Li,⊥,§ Ping Li,# Yan-Jie Wang,Δ Oudjaniyobi Simalou,¶ Yanling Zhang,† Ge Gao,# and Alideertu Dong*,†,‡ †

College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot 010021, People’s Republic of China State Key Laboratory of Medicinal Chemical Biology, NanKai University, Tianjin 300071, People’s Republic of China ∥ Department of Chemistry, Tangshan Normal University, Tangshan 063000, People’s Republic of China ⊥ Affiliated Hospital of Inner Mongolia, University for the Nationalities, Tongliao 028000, People’s Republic of China # College of Chemistry, Jilin University, Changchun 130021, People’s Republic of China Δ Department of Chemical and Biological Engineering, University of British Columbia, 2360 East Mall, Vancouver, BC, Canada, V6T 1Z3 ¶ Département de Chimie, Faculté Des Sciences (FDS), Université de Lomé (UL), BP 1515 Lome, Togo ‡

S Supporting Information *

ABSTRACT: N-Halamine-based antibacterial materials play a significant role in controlling microbial contamination, but their practical applications are limited because of their complicated synthetic process and indistinct antibacterial actions. In this study, novel antibacterial N-halaminecontaining polymer fibers were synthesized via an one-step electrospinning of N-halamine-containing polymers without any additives. By adjusting the concentration of precursor and the molecular weight of polymers, the morphology and size of the as-spun N-halamine-containing fibers can be regulated. The as-spun fibers showed antibacterial activity against both Gram-positive and Gram-negative bacteria. After an antibacterial assessment using different biochemical techniques, a combined mechanism of contact/release co-determined killing action was evidenced for the as-spun N-halamine-containing fibers. With the aid of contact action and/or release action, this combined mechanism can allow N-halamines to attack bacteria, making the asspun fibers wide in the application of antibacterial fields, whatever it is in dry or wet environment. Also, a recycle antibacterial test demonstrated that the as-spun fibers can still offer antibacterial property after five recycle experiments. KEYWORDS: N-halamines, electrospun, antibacterial, mechanism, contact killing, release killing

■

healthcare products, and dyes and paints.13−17 To date, the antibacterial mechanism of N-halamines can be divided into two main pathways: (i) contact pathway, which is direct transfer of oxidative halogen (X+) from N-halamines to bacterial receptor,18,19 and (ii) release pathway, which is dissociation of halogen from N-halamines to solution with subsequent inactivation.20,21 In the study of antibacterial mechanism, some research groups preferred the contact pathway without freely released halogen, while others agreed with the release pathway. Because many research groups have devoted themselves to N-halamines fields, a variety of achievements have been reported up to now.22 Most research has been related to the use of chemical bonds to attach N-halamines onto inorganic or

INTRODUCTION As microbial contamination may pose a threat to public health worldwide,1 it is of great importance to develop innovative and creative strategies to control it. In response to the widespread occurrence of microbial contamination, various antibacterial agents with biocidal activity have been developed.2 To date, many sorts of antibacterial agents have emerged, such as free halogen, ozone, metal oxides, quaternary ammonium/phosphonium salts, peptides, guanidine, and N-halamines.3−10 Among these agents, N-halamines containing nitrogen− halogen covalent bonds are attracting growing interest due to their effective sterilization toward a wide range of microbes.11 In addition, N-halamines have many unique features, such as stability in wide ranges of temperature and humidity, durability over long-term usage, and regenerability upon rebleaching treatment.12 These merits make N-halamines attractive as promising disinfectants in various fields, including water treatment, air purification, textile products, medical and © XXXX American Chemical Society

Received: July 10, 2016 Accepted: November 3, 2016 Published: November 3, 2016 A

DOI: 10.1021/acsami.6b08431 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. (A) Chemical structure of 1,3-dichloro-5,5-dimethylhydantoin (DCDMH) and 1,3-dibromo-5,5-dimethylhydantoin (DBDMH). (B) Schematic illustration of the synthetic strategy to create as-spun N-halamine-containing polymer fibers using electrospinning. (C) Diagrammatic sketch of the as-synthesized DCDMH/DBDMH-containing poly(methyl methacrylate) (PMMA) fibers.

(Figure 1B). As expected, we obtained a fibrous morphology, and then examined the morphology and size evolution. Subsequently, the antibacterial action of the as-spun fibers and their biocidal mechanism was investigated to assess their potential as antibacterial agents in factual applications. We believe that our proposed strategy should have a significant implication in disinfection and other relevant fields.

polymeric substrates, rendering them antibacterial properties. The general route involved in N-halamine attachment includes (1) preparation of N-halamine precursor, (2) attachment of precursor onto substrate, and (3) transformation of precursor into N-halamine. In most cases, the route looks somewhat complicated and the productivity usually seems unsatisfactory. Thus, it is urgently required to simplify these fabrication steps. Instead of the chemical bonding approach, the physical methods could be recommended as an effective choice. It has been well-known that electrospinning is a successful and efficient physical method to synthesize various composite fibers in antibacterial fields.23−26 In N-halamines fields, the electrospinning techniques used mainly fall into two scopes:27−31 (i) synthesis of N-halamine electrospun via copolymerization of N-halamine-containing monomers with acrylic, substituted-acrylic, or vinyl monomers, followed by electrospinning treatment,29 or (ii) modification of electrospun fibers with presynthesized N-halamines.30,31 Both pathways are performed with the aid of complicated chemical reactions. To avoid complicated chemical procedures, our group used a simple method to synthesize N-halamines using electrospinning. However, it is not easy to form fibrous N-halamines directly using electrospinning because most commercially available N-halamines can be hardly electrospun in a direct way. Consequently, poly(methyl methacrylate) (PMMA) was selected in our study as a polymeric matrix which was blended first with N-halamines and then electrospun to obtain Nhalamine-containing PMMA fibers. Undoubtedly, PMMA is available facilely and its existence can favor the formation of stable and size-controllable N-halamine-containing fibers. With the help of PMMA as a matrix in this electrospinning system, antibacterial N-halamines could be brought into the fibrous form. In this study, the N-halamine-containing polymeric fibers were fabricated for antibacterial applications using electrospinning method. As illustrated in Figure 1, 1,3-dichloro-5,5dimethylhydantoin (DCDMH) and 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) were selected as two model N-halamines (Figure 1A), and the PMMA fibers bearing N-halamines (Figure 1C) were synthesized by electrospinning technique

■

EXPERIMENTAL SECTION

Materials. 1,3-Dichloro-5,5-dimethylhydantoin (DCDMH) and 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) were purchased from Aladdin Industrial Inc. Methyl methacrylate (MMA), N,N-dimethylformamide (DMF), and potassium persulfate (KPS) were obtained from Tianjin Chemical Reagent Plant. All the chemicals were used without purification. Electrospinning of N-Halamine-Containing Polymers. MMA was added into 150 mL of ultrapure water containing 0.1 g of KPS, and the polymerization reaction was run at 75 °C under the condition of N2 gas inlet. After reaction, the as-prepared PMMA was added into DCDMH and/or DBDMH containing DMF, and stirred overnight to prepare transparent and achromatous electrospinning solution. A buret with an inserted copper rod to connect with high voltage of 12 kV was filled with the electrospinning solution. An aluminum foil, as the counter electrode, was fixed with a distance of about 20 cm from the buret tip. The electrospinning was carried out at room temperature to obtain DCDMH- and/or DBDMH-containing PMMA fibers. For comparison, the PMMA fibers without N-halamines were also synthesized in the same way. Determination of Oxidative Halogen Content. Oxidative halogen content in the as-spun fibers was determined by the modified iodometric/thiosulfate titration procedure.32 The percentage of oxidative halogen (X+ %) was calculated according to the following equation. X +% =

(V + − V0) × 10−3 × 0.01 35.5 × X × 100 2 WX +

where VX+ and V0 are the volumes (mL) of sodium thiosulfate solutions consumed in the titration of the N-halamine-containing PMMA fibers and pure PMMA fibers, respectively, and WX+ is the weight of the N-halamine-containing PMMA fibers (g). Characterizations. Morphology and size of samples were examined by scanning electron microscopy (SEM, Shimadzu SSXB


Research Article


Figure 2. SEM (A and B), size distribution (insert of A), TEM (C), and EDX result (insert of C) of PMMA−DCDMH (1), PMMA−DBDMH (2), and PMMA−DCDMH/DBDMH (3). 550) and transmission electron microscopy (TEM, Hitachi H-8100). The energy-dispersive X-ray (EDX) was performed during the scanning electron microscope measurements. The high-angle annular dark-field scanning TEM (HAADF-STEM) characterization was performed on a FEI-Tecnai G2F20S-TWIN. FTIR spectra were recorded by using a Thermo Nicolet (Woburn, MA) Avatar 370 FTIR spectrometer. 1H NMR spectra were recorded on a Bruker AVANCE III-500 NMR spectrometer in DMSO solution. X-ray photoelectron spectroscopy (XPS) measurement was performed on a PHI5000CESCA system with Mg Kα radiation. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of polymers were measured by size-exclusion chromatography (SEC) with a chromatographic system (Waters Division Millipore) equipped with a Waters model 410 refractive-index detector. Plate Counting Method. The samples were challenged with Staphylococcus aureus (S. aureus, ATCC 25923, Gram-positive bacteria) and Escherichia coli (E. coli, ATCC 8099, Gram-negative bacteria) using the plate counting method. Typically, bacteria were grown overnight at 37 °C in Luria−Bertani medium (LB, 10 g of tryptone and 5 g of yeast extract/liter), and the bacterial cells were harvested by centrifugation, washed with phosphate-buffered saline, and diluted to concentrations of 1 × 106 CFU/mL. The as-prepared 50 μL of bacteria suspension was mixed with 0.45 mL of sample suspension (100 mg/ mL), and incubated under constant shaking (200 rpm). After a certain period of contact time, 4.5 mL of 0.03 wt % sodium thiosulfate aqueous solution, sterilized by passing through 0.22-μm membrane and exhibiting no effect on the growth of bacteria, was added into the reaction suspension to neutralize the active chlorine and stop the antibacterial action of sample. After that, the mixture was serially diluted, and 100 μL of each dilution was dispersed onto Luria−Bertani (LB) growth medium. Survival colonies on LB plates were counted after incubation for 24−36 h at 37 °C. The plate counting tests were carried out in triplicate. Inhibition Zone Study. The N-halamine-containing PMMA fibers were mixed with KBr powder, and added into a circular mold with a

diameter of 1.0 cm. Under pressure at room temperature using a tablet machine, the sample discs were obtained and then moved onto the surface of LB agar plate overlaid with 500 μL of 106 CFU/mL of E. coli. After incubation at 37 °C for 24 h, the inhibition zone around the sample disc was visible. For comparison, a pure KBr disc was prepared as the negative control in the similar way. The inhibition zone studies were performed in triplicate. Contact Killing Assay. The freeze-dried bacterial cells were challenged with the N-halamine-containing PMMA fibers in the absence of liquid medium. In detail, E. coli and S. aureus were collected by centrifugation, washed twice with saline solution, frozen to −80 °C overnight, and then freeze-dried under vacuum for 24 h to a dry powder. Samples were added into a circular mold and compressed into small disks under pressure at room temperature using a tablet machine. Two sample disks were used and the dried bacteria mentioned above were placed between the two disks. After contacting for 24 h, the bacterial powder was removed aseptically from the disks and suspended in 10 mL of fresh LB nutrient broth. Finally, the survival of bacteria was determined using the plate counting method. The contact killing tests were carried out in triplicate. Release Killing Assay. The release killing assay was carried out according to Ahmed’s work.33 The suspension of the as-spun PMMADCDMH fibers were placed into a dialysis bag (molecular weight cutoff 100 Da) and immersed into phosphate-buffered saline at 37 °C in a constant temperature shaker for 1 week. To examine the release action, the dialysate solution was determined using the iodometric/ thiosulfate titration. In addition, the antibacterial evaluations were also conducted using the plate counting method to assess the bacterial killing action of the dialysate solution. The release killing tests were performed in triplicate.

■

RESULTS AND DISCUSSION The N-halamine-containing PMMA fibers were synthesized by electrospinning as shown in Figure 1. The morphology and size C


Research Article


Figure 3. XPS Survey spectrum, C 1s spectrum, O 1s spectrum, N 1s spectrum, Cl 2p spectrum, and Br 3d spectrum of PMMA, PMMA−DCDMH, and PMMA−DBDMH.

Table 1. Elemental Analysis Results of PMMA−DCDMH and PMMA−DBDMH Fibers M1

M2

M1:M2 (g:g)

PMMA− DCDMH

PMMA

DCDMH

10:1

PMMA− DBDMH

PMMA

DBDMH

6:1

sample

a

analysis

C (%)a

H (%)a

N (%)a

Cl (%)b

Br (%)b

calculated found calculated found

57.30 55.55 ± 0.08 54.41 52.28 ± 0.05

7.597 7.848 ± 0.012 7.203 7.432 ± 0.014

1.293 1.480 ± 0.06 1.400 1.665 ± 0.005

5.931 5.008 ± 0.05 0 NDc

0 NDc 18.04 16.26 ± 0.07

Obtained from elemental analysis. bObtained from iodometric/thiosulfate titration. cNot determined.

DBDMH, which suggests the successful formation of Nhalamine-containing PMMA fibers. The formation of the asspun fibers was further confirmed by recognizing the characteristic groups in FTIR spectra (Figure S2). In addition to the PMMA peaks, the characteristic peaks of N-halamines (DCDMH and DBDMH) are visible in the FTIR spectra of the as-spun fibers. Chemical compositions of the as-spun fibers obtained from XPS spectra are shown in Figure 3. For comparison, the XPS spectrum of pure PMMA is given as well. Two elemental signals, Si 2s and Si 2p, are observed for all three spectra, which are likely attributed to the glass support used for sample immobilization.37 The PMMA shows only main peaks of C 1s and O 1s signals.38 As for N-halamine-containing PMMA fibers, additional peaks of N 1s, Cl 2p, and Br 3d can be seen, which are effective elemental markers for DCDMH and DBDMH.39 For further identification, the deconvolutions of C 1s, O 1s, N 1s, Cl 2p, and Br 3d were also performed.40,41 For pure PMMA, the C 1s peak is separated into three divided peaks including C−C, C−O, and CO bonds, whereas the N-halaminecontaining PMMA fibers have an additional divided peak of C− N bonds. The O 1s peak is composed of C−O and CO peak for all spectra. Importantly, the appearance of −N < , −NCl−, −NBr−, N−Cl, and N−Br bonds is well proven for the formation of N-halamine-containing PMMA fibers, which are well agreed with the 1H NMR and FTIR results. These characterizations mentioned above provide qualitative information about the as-spun fibers. To quantify the as-spun fibers, elemental analysis was performed. Results are shown in Table 1 with the examined contents of element C, H, N, Cl, and Br in fibers. It is obvious that the experimental results are generally close to the calculated ones, which suggests that there is no significant loss in materials during the synthesis process.

features of the DCDMH- and/or DBDMH-loaded PMMA fibers are presented in Figure 2. The as-spun fibers exhibit randomly oriented, straight, and continuous features (Figure 2A). Their sizes show narrow distribution with an average size of 891 nm for PMMA−DCDMH, 747 nm for PMMA− DBDMH, and 616 nm for PMMA−DCDMH/DBDMH, respectively (insert of Figure 2A). To clarify surface state, the magnified SEM images are given as well (Figure 2B). Unlike the quite smooth appearance of pure PMMA fibers, the Nhalamine-containing PMMA fibers offer coarse surface and serious barbotages (green arrow in Figure 2B). We speculated that this might be attributed to the heterogeneous distribution of N-halamines among the PMMA polymeric chains. Furthermore, it can be observed from TEM images in Figure 2C that quite uniform distributions are visible, and no aggregations are seen inside the fibers. So it can be concluded that the rough surfaces of as-spun fibers are attributed to the mixture system rather than the disordered distribution of Nhalamine molecules. Also, EDX analysis provided significant evidence for the combination of N-halamines with PMMA (insert of Figure 2C). Besides the elemental signals of C and O, the appearances of elements N, Cl, and Br are vital proof for the existence of N-halamines in the PMMA matrixes. According to 1H NMR spectra of the products in Figure S1, DMSO had a fitting signal at 2.5 ppm when it was selected as solvent.34 Both DCDMH and DBDMH have the similar chemical shifts because of their structural features, whose signals are completely from −CH3 groups located at 1−2 ppm.35 For PMMA, the signals at around 3.6 ppm can be associated with O−CH3, while a broad region at 0.4−2.0 ppm was mainly assigned to −CH2− from polymer chains.36 After electrospinning, the characteristic −CH3, O−CH3, and −CH2− signals are observed for PMMA−DCDMH and PMMA− D


Research Article


Figure 4. SEM images of (A−E) PMMA−DCDMH and (F−J) PMMA−DBDMH prepared with different concentrations of electrospinning precursor: (A and F) 10 wt %, (B and G) 11 wt %, (C and H) 12 wt %, (D and I) 13 wt %, and (E and J) 14 wt %.

controlling the precursor concentration. However, this size controllability is available only at lower concentration, and quite high concentration might lose morphology steadiness. As can be seen from Figure 4E-2, the products obtained with a concentration of precursor solution as high as 14 wt % could lead to some assemblages. Although fiber-like morphology can be kept, more serious aggregation (yellow region) resulted in the much rougher surfaces (green arrow). The molecular weight of PMMA matrixes also plays a vital role in determining the size of the as-spun fibers. To invesigate the effect of molecular weight on size, the N-halaminecontaining PMMA fibers were synthesized using PMMA with different molecular weights. As shown in Figure 4D and 4I, at the use of PMMA with a higher molecular weight (Mn = 23 kDa, Mw = 51 kDa, Mw/Mn = 2.2), the average sizes are 453 and 353 nm for PMMA−DCDMH and PMMA−DBDMH, respectively. In Figure S3, it can be clearly seen that a lower molecular weight (Mn = 12 kDa, Mw = 48 kDa, Mw/Mn = 4.1) can result in fiber-like products with straight appearance and uniform size. Obviously, their average sizes are only 224 and 208 nm for PMMA−DCDMH and PMMA−DBDMH, respectively. In general, the size decreases significantly when the molecular weight of PMMA matrixes is decreased. Even

However, the difference of the analysis results with those calculated data may result from the residual of solvent DMF used during the electrospinning process. Moreover, the obvious decrease in the halogen content should be possibly attributed to the halogen loss induced by the high voltage used during the electrospinning process. The size controllability of the as-spun fibers was evaluated by controlling the concentration of electrospinning precursor solution. In the synthesis, the precursor concentrations in DMF were varied from 10 to 14 wt %, and other parameters were kept constant. Figure 4 provides the SEM images and the corresponding size distribution of PMMA−DCDMH (A−E) and PMMA−DBDMH (F−J) synthesized with different precursor concentrations. In general, no significant change was observed in their morphologies with different concentration for either DCDMH- or DBDMH-loaded fibers. However, their size evolved significantly. In detail, the average size for PMMA−DCDMH and PMMA−DBDMH increases from 236 ± 77 nm to 813 ± 406 nm and from 130 ± 45 nm to 577 ± 182 nm, respectively, when the concentration rises from 10 to 14 wt %. Moreover, their size distribution broadens with increasing concentration. So it can be deduced that the size and size distribution of as-spun fibers could be modulated just by E


Research Article


Figure 5. Photographs for the bacterial culture plates of E. coli upon a 120 min exposure of (A) the control, (B) PMMA−DCDMH, and (C) PMMA−DBDMH. The results of the antibacterial kinetic tests for PMMA−DCDMH and PMMA−DBDMH against (D) E. coli and (E) S. aureus.

Figure 6. (A) Schematic illustration of antibacterial mechanism for N-halamines. (B) Survival of E. coli and S. aureus after contact killing assay of the control, pure PMMA, PMMA−DCDMH, and PMMA−DBDMH. (C) Optical images of the inhibition zone against E. coli for the samples (1) PMMA−DCDMH, (2) PMMA−DBDMH, and (3) PMMA−DCDMH/DBDMH.

F


Research Article


nism was put forward in Ahmed group,46 in which the mode of antibacterial action of N-halamines could not be by contact alone or release alone, but by a combined way. So the action of N-halamine-containing PMMA could be attributed to contact killing, release killing, or their combination. To check the possibility of the contact killing mechanism, freeze-dried bacterial cells were treated with N-halaminecontaining PMMA fibers in the absence of liquid medium. The antibacterial results are displayed in Figure 6B. In the comparison, pure PMMA fibers were used as control. It is palpable that the survival of bacteria treated with pure PMMA fibers is higher than 0.99. Unlike pure PMMA, the N-halaminecontaining PMMA fibers demonstrated survival lower than 0.4, evidencing the antibacterial capability of N-halamines against both E. coli and S. aureus in the absence of liquid medium. This antibacterial action performed in the dry condition is an effective evidence for the contact mechanism of N-halamines. Consequently, it can be demonstrated that the N-halamines should kill bacteria by the contact pathway. To further confirm the killing mechanism, the inhibition zone assay was carried out using E. coli as representative bacteria. It was acceptable that the inhibition zone not only could reflect the susceptibility of bacteria toward the antibacterial agents but also mirror their antibacterial mechanism to some extent.47 As confirmed in the previous reports,48 those antibiotics following the contact mechanism could hardly show the inhibition zone, consequently the inhibition zone study could be an effective route to test the release killing of antibiotics. As shown in Figure 6C, the control plate offers a robust growth showing crowded colonies without any aseptic region, while the PMMA−DBDMH, PMMA− DBDMH, and PMMA−DCDMH/DBDMH samples show the inhibition ring around the sample disc. The results indicated that the N-halamine-containing PMMA fibers kill bacteria by at least some of the oxidative halogen diffused from polymeric fibers. So the release killing mechanism is also one possible way for the N-halamines to perform antibacterial function. Furthermore, a closer observation in the inhibition zone assay shows that the DBDMH-containing fibers have a much broader inhibition area than the DCDMH-based ones. This also acts as another evidence for the release mechanism: because the N−Br bonds have a greater dissociation capability than the N−Cl bonds, the PMMA−DBDMH gives a larger aseptic ring than the PMMA−DCDMH under the same conditions. To determine the release mechanism of the as-spun fibers, whether resulting from the dissociation of just halogen ions from N−X bonds or from the release of whole N-halamine molecules from the PMMA matrixes, a dialysis test (Figure 7A) was carried out using the dialysis bag (molecular weight cutoff of 100 Da). In this way, only those molecules with a low molecule weight (

N-Halamine-Containing Electrospun Fibers Kill Bacteria via a Contact

Recommend Documents