Bilayered Antimicrobial Nanofiber Membranes for Wound Dressings

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Bioengineering

Bilayered antimicrobial nanofiber membranes for wound dressings via in-situ cross-linking polymerization and electrospinning Yanping Huang, Nianhua Dan, Weihua Dan, Weifeng Zhao, Zhongxiang Bai, Yining Chen, and Changkai Yang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b03122 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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Industrial & Engineering Chemistry Research

Bilayered antimicrobial nanofiber membranes for wound dressings via in-situ cross-linking polymerization and electrospinning Yanping Huang,a Nianhua Dan,a Weihua Dana,b* Weifeng Zhao,c Zhongxiang Bai,a Yining Chen,a and Changkai Yanga a

College of Light Industry & Textile & Food Engineering, Key Laboratory for

Leather Chemistry and Engineering of the Education Ministry, Sichuan University, Chengdu 610065, China. bResearch

Center of Biomedical Engineering, Sichuan University, Chengdu 610065,

China. cCollege

of Polymer Science and Engineering, State Key Laboratory of Polymer

Materials Engineering, Sichuan University, Chengdu 610065, China.

* Corresponding author, E-mail: [email protected]; Tel.: +86 28 85408988: Fax: +86 28 85408988;

Abstract: The aim of this study was to fabricate a novel bilayered wound dressing with excellent antibacterial performance through electrospinning and in-situ cross-linking polymerization.

Quaternary

ammonium

salts

([2-(methacryloyloxy)ethyl]

trimethylammonium, MTA) were first polymerized and cross-linked in the presence of polycaprolactone (PCL) to form PCL/PMTA composites. Then, PCL was 1

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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

electrospun as the cytocompatible inner layer that directly contact the wound, and the PCL/PMTA layer was placed as the outer layer as a defense against bacteria. The PCL/PMTA layer exhibited a hydrophilic surface and showed a strong antibacterial effect on E. coli. and S. aureus. All bilayered antimicrobial nanofiber membranes did not release any toxic agents to cells, and the PCL layer exhibited better effects on cell growth. The electrospun bilayered nanofiber membranes are promising candidates for antimicrobial wound dressings. Key words: Electrospinning, In-situ cross-linking polymerization, Wound dressing, PCL, Antibacterial activity

Introduction Skin is a considerably important part of the human body due to its capacity for temperature regulation, water retention and protection of underlying organs1, 2. However, traumatic injury or illnesses, for example, diabetes, often lead to a lack of skin structure and functions3. During wound healing, bacterial infections often result in healing delays and increases in morbidity4, 5. Therefore, it is imperative to fabricate bactericidal wound dressings that are able to avoid microorganisms growth or prevent the permeation of bacteria into the wound. Many researchers worldwide have made efforts6,

7

to address this health issue. Historically, doctors used animal fats, honey

pastes, gauze and even plant fibers as treatment materials to help patients with wound issues8. However, they have only little effect on defenses against bacteria. Furthermore, the conditions that are suitable for wound healing are in favor of the 2


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invasion and replication of bacteria9. Currently, films, microspheres and other dressing materials, carrying diverse kinds of releasable bacteria-killing agents, such as antimicrobial drugs5,

10-12

and silver nanoparticles13-16, have been produced to resist

infections. Topical delivery of antibiotics agents can be effective. However, the addition of these agents might lead to a decrease in mechanical properties17, limitation of the duration of antimicrobial activity17, and potential toxicity (which results from uncontrolled release)18, and can even increase the risk of antimicrobial resistance19. The disadvantages mentioned above have hindered the application of release agents. It is well established that polyquaternary ammonium salts can be very effective at inhibiting the proliferation of yeast, fungi, gram-negative and gram-positive bacteria, which is attributed to their potent antimicrobial activities9,

20.

Polyquaternary

ammonium salts, which contain alkyl chain, exhibit superior bactericidal performance and overcome the shortcomings of weak chemical stability and potential antimicrobial resistance

shown

trimethylammonium

by

other

chloride

antibiotics solution

agents21.

([MTA][Cl])

[2-(Methacryloyloxy)ethyl] is

capable

of

radical

polymerization because this quaternary ammonium salt monomer has a reactive methacryloyl group. [MTA][Cl] has been applied to modify reduced graphene oxide22 and stainless steel23, and shows outstanding germicidal activity. However, there exists a potential risk of cytotoxicity from quaternary ammonium salts, which may owe to the destruction of the phospholipid bilayer of the cell membrane caused by the quaternary ammonium group. To achieve the goal of excellent germicidal activity and to avoid underlying cell toxicity, a bilayered wound 3



dressing is designed here. Among the methods of preparing bilayered membrane, electrospinning has attracted much attention from academia24. A high voltage power supply system, a collecting system and a spinneret system are the main parts of a typical electrospinning apparatus. Changing of the apparatus varies the structures and morphologies of fibers. Using high-voltage electric fields, electrospinning is the most efficient and straightforward method for producing fiber membranes in micron even nano scale, which could mimic the structure of extracellular matrix (50 - 500 nm) well 25-33.

Previous researches have shown that nanofibrous architecture is beneficial for

retainning moisture, facilitating gas permeation, and stopping bleeding, which are important to wound dressings34-42. Moreover, electrospun nanofiber mats hold promise for antibacterial applications because of their high surface-to-volume ratio, conductivity and chemical reactivity43. The pore size shown by electrospun fiber mats is quite small, so that bacteria cann’t penetrate into the wound. In addition, serving as a free-standing material or a conformal surface coating, nanofiber mats offer a controlled interaction with microorganisms, and could impart their antibacterial properties to the underlying substrate44. Several studies have focused on bilayer electrospun wound dressings. Figueira et al.45 developed polycaprolactone-hyaluronic acid/chitosan-zein electrospun bilayer nanofibrous membranes to cover wounds and promote the wound healing process. Results showed that the produced electrospun membrane displayed good physicochemical properties and exhibited no toxic effects on normal human dermal fibroblasts (NHDF) cells, implying the potential for wound dressing applications. Pal et al.46 had reported a bilayer skin graft comprising a porous 4


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cotton-wool-like electrospun 3D layer with a membranous structure of PCL-chitosan nanofibers. The data demonstrated that the scaffold promoted efficient fibroblast cell attachment, infiltration, and proliferation, and assisted in extracellular matrix protein synthesis and keratinocyte stratification in vitro. Compared to the standard Tegaderm dressing™, the bilayer exhibited a faster healing speed in third-degree burn wound margins created in rat models. Yao47 fabricated a bilayer membrane as a novel wound dressing, with a commercial polyurethane wound dressing as an outer layer and an electrospun gelatin/keratin nanofibrous mat as an inner layer. Animal studies showed that the bilayer membrane could accelerate the formation of blood vessels and reduce in the wound area. Improved wound repair at 14 days with a thicker epidermis and a larger number of newly formed hair follicles were also found in the bilayer membrane case. These investigations proved the good prospects for the application of bilayer electrospun wound dressing. PCL, a synthetic biocompatible polymer with excellent mechanical stability, that has been approved by FDA for biomedical applications and is commonly utilized to heal wounds, deliver drugs and repair bone25, is involved in this research. Previously, antibacterial electrospun PCL wound dressings were developed to carry antibiotic drugs, such as silver sulfadiazine48, ciprofloxacin/laponite49, and curcumin50. Despite the cell compatibility and bactericidal activity demonstrated by drug-loaded PCL membranes, there are still some shortcomings related to the drug release period and drug resistance51. To our knowledge, there have been few reports about electrospinning of PCL modified with polyquaternary ammonium salts. 5



In-situ cross-linking polymerization is a method using functional monomers to fabricate functional polymer materials52. During the reaction, polymerization and cross-linking of the functional monomers, acting as the functional units, take place to form network macromolecules. The in-situ cross-linking polymerization method displays merits by saving time, averting tedious purification and, lowering the dosage of raw materials53-55, and it is easier to perform than blending, coating, grafting and other membrane modification methods56-60. In addition, it is demonstrated by many works that the modified materials manufactured by the one-pot and simple method show robust and distinguished performances53,

55, 61-64.

Hence, via the method of

in-situ cross-linking polymerization, [MTA][Cl] could be introduced into PCL films to obtain antibacterial activity. Herein, the aim of this study is to fabricate a novel bilayered wound dressing with excellent contact antibacterial activities through in-situ cross-linking polymerization and electrospinning. PCL was introduced as the cytocompatible inner layer that directly contact the wound, and the polyquaternary ammonium salts modified PCL (PCL/PMTA) layer was placed as the outer layer as a defense against bacteria. Subsequently, bilayered dressing was further evaluated for surface hydrophilicity, morphology, antibacterial activity and cytocompatibility.

Experimental section Materials Polycaprolactone (PCL) with a molecular weight of 80,000 Da was bought from Sigma-Aldrich. [2-(Methacryloyloxy)ethyl] trimethylammonium chloride solution 6


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([MTA][Cl],

75

wt%

H2O

solution

with

the

monomer),

2,2'-azobis

(2-methylproronitrile) (AIBN, 90%) and N,N'-methylene bisacylamide (MBA; 99%) were supplied from Aladdin Chemical Company. 2,2,2-Trifluoroethanol (TFE, 99.5%) was obtained from Chengdu Best Reagent Co., Ltd. The LIVE/DEAD Baclight Bacterial Viability Kit L-7012 was used to visually investigate the bactericidal

capability.

3-(4,5-Dimethylthiazol-2-yl)

-2,5-diphenyltetrazolium

bromide (MTT) and phosphate buffer solution (pH 7.4) were coming from Thermo Fisher Scientific Inc. If not specifically mentioned, other reagents and chemicals were purchased from Sigma-Aldrich. Preparation of the bilayered antibacterial nanofibrous membranes First, through in-situ cross-linking polymerization, the PMTA chains were physically intertwined with chains of PCL. Subsequently, electrospinning was performed. In brief, PCL (7% w/w) was dissolved in TFE and placed into a 25 mL reagent bottle. Then specified amounts of AIBN, MBA and [MTA][Cl] were added to the above PCL solution. Homogeneous and transparent solutions were acquired after magnetic stirring. Afterward, under a stirring speed of 120 rpm, the bottle was kept at 65 oC in an oil bath for 8 h. The solution removed from the oil bath and placed at room temperature to stop the polymerization. The summarized details of the chemical dosages and sample names are displayed in Table 1. Continuous electrospinning technology was adopted to prepare the bilayered nanofiber membranes. The as-prepared samples were placed into 5 mL plastic syringes, which were equipped with 21 G stainless steel blunt needles and ejected at 7



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feed rates of 1 mL/h. The vertical distance between the collector and needle was fixed at 30 cm. The electrospinning voltage was set at 30 kV. During the electrospinning process, the relative humidity and temperature were held at 45 ± 5% and 25 ± 2°C, respectively. Collection of the nanofibers was conducted on aluminum foil with rotation speeds of 140 rpm. Table 1. The chemical dosages and sample names for the prepared solutions. Samples

PCL (g)

[MTA][Cl](g)

AIBN (mg)

MBA (mg)

TFE (g)

PCL

1.4

-

-

-

18.6

1.4

0.35

35.0

17.5

18.6

1.4

0.70

70.0

35.0

18.6

1.4

1.40

140

70.0

18.6

PCL/PMTA1 PCL/PMTA2 PCL/PMTA3

The electrospinning procedure was performed in two stages: (1) after collecting PCL fibers, the PCL/PMTA solution was spun sequentially based on the aforementioned parameters; (2) Finally, the electrospun films were soaked in water to remove any unreacted monomer, initiator and crosslinker for 3 days, and then dried at 40°C in a vacuum oven for at least 2 days. The synthesized bilayered membrane was kept at 4°C until further use. Scheme 1 depicts the diagrammatic sketch of the preparation of the electrospinning solution; as well as the schematic illustration for the 8


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electrospinning process.

Scheme 1 Diagrammatic sketch of the setup for the modification of PCL with MTA (a) and electrospinning of bilayered structured membranes (b).

Characterization of the bilayered nanofibers Scanning electron microscopy (SEM, JSM-7500F, Japan) was utilized in this study to observe the morphologies of samples, and thermogravimetric analysis (TGA, Netzsch Co., Germany) and attenuated total reflection flourier transformed infrared spectroscopy (ATR-FTIR, Nicolet 560, America) were used to investigate the chemical and physical compositions. Meanwhile, the surface wettability was determined by measuring water contact angles (WCA) using video capture (Attension Theta, Biolin Scientific, Sweden). The morphology of surface and section of prepared nanofiber mats were observed by SEM. The samples were all examined at an accelerating voltage of 5 kV after being coated with gold. Nano Measure software was used to measure the average fiber diameters.

9



The chemical characteristics of the bilayered nanofiber mats were evaluated by ATR-FTIR. The spectra were acquired with a resolution of 4 cm -1 in the range of 625–4000 cm-1. Samples of approximately 3 mg were evaluated by a thermal analyzer. The analysis test was carried out under a N2 atmosphere. The parameters of analysis were set as follows: started from ambient temperature and, raised to 800 oC at a heating rate of 15 oC/min.

To measure the WCA, each nanofiber mat was cut to a 1.5 mm2 square and attached to a glass slide. Using video capturing, WCAs of samples were recorded and calculated. With 10 μL of DI water being dropped on the surface of the nanofiber mats, the static WCA results were measured after 5 s. Antibacterial activity tests The bactericidal properties of the nanofibrous mats against Staphylococcus aureus (S. aureus, gram-positive) and Escherichia coli (E. coli, gram-negative) bacteria were investigated in the current study. The shake flask test, SEM analysis and inhibition zones were performed to investigate the antimicrobial activity according a previous report 9. For the evaluation of bacterial attachment on the nanofiber mats, the different PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 samples were cut into 1 cm × 1 cm pieces and stored in 24 well plates. After treating with UV overnight, each well was fed 2 mL of bacterial suspensions at a concentration of 106 cfu/mL. The cultivation occurred for 12 h at 37 °C. A live/dead staining assay was performed to evaluate the 10


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antibacterial performance of the mat surfaces. The staining solution was composed of normal saline, SYTO9 and propidium iodide (1000:1.5:1.5 in volume). Membranes were soaked in the staining solution and incubated for 15 min in the dark. The membranes were then examined by fluorescence microscopy (Olympus IX53, Japan) after being rinsed with normal saline. For the shake flask test, the membranes (area of 2 cm2) were immersed into a bacterial suspension of 105 cfu/mL and shaken in an incubator shaker at 130 rpm for 3 h. A CFU counting method was used to estimate the number of viable E. coli and S. aureus cells remaining in suspension. From the suspension, 100 μL was serially diluted 10-fold, and 50 μL was pipetted onto freshly prepared agar plates. The agar plates were incubated at 37 oC for 20 h, and developed bacterial colonies were counted. After cultivation, the membranes were washed with normal saline and immobilized by 2.5% glutaraldehyde. Then, 0, 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol solutions were used to dehydrate the membranes. SEM was applied to observe the morphology of attached bacteria. Cytotoxicity of electrospun bilayered nanofiber mats The biocompatibility of the investigated samples was assessed by a cell proliferation assay and SEM analysis. Briefly, electrospun bilayered mats were cut into 25 cm2 squares and immersed in 8.3 mL of culture medium without serum. These samples were incubated at 37 oC for 24 h before use. Meanwhile, L929 fibroblast cells were cultivated in cell culture medium with both calf serum and antibiotic solution and seeded in 96 well plastic tissue culture plates. Approximately 1 × 104 cells were 11



seeded in every well. The incubator was set at 37 oC with 5% CO2. After cultivation for 24 h, 200 μL of the extract sample solution was added to each well. Then, the plates were cultivated for another 1, 3, and 5 days in the incubator. Next, 20 μL of MTT was added to each well of the culture plate at each time point. At 4 h of cultivation at 37 oC, formazan crystals had already formed. Subsequently, 200 μL of dimethylsulfoxide was introduced and the samples were incubated for 15 min in the dark. Finally, a microplate reader was applied to measure the optical density (OD) at 492 nm to evaluate the grade of cytotoxicity. For the SEM analysis, the sterilized samples were cut into 1 cm2 pieces and placed in a 24 well culture plate. L929 fibroblast cells were seeded onto the nanofiber film at approximately 0.5 × 104 cells/cm2 per well. The cells were incubated in the incubator for 3 days. On the second day, the culture medium was changed. After slight washing 3 times with phosphate buffer solution, the samples were immobilized by 4% paraformaldehyde at 4 oC. Subsequently, a series of decreasing grades of ethanol was used to dehydrate the samples seeded with L929 fibroblasts. After drying, samples were observed by SEM. Statistical analysis SPSS was employed here to perform the statistical analysis. The significant differences between the groups were determined by ANOVA test. If p < 0.05, the results were considered significant.

Results and discussion In the in-situ cross-linking polymerization reaction, the MTA[Cl] monomer polymerized in the solution. The PCL polymer did not react during the polymerization 12


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of MTA[Cl]. To prevent the elution of the hydrophilic polymer PMAT (the polymer of MTA[Cl]), the cross-linking method was hence applied. In-situ cross-linking was employed in this study for saving time, averting tedious purification, and lowering the dosage of raw materials53. In addition, the reaction temperature needed for the in-situ cross-linking polymerization of MTA[Cl] was 65 oC, which exceeded the melting point of PCL. To maintain the nanofibrous structure, the reaction was performed before electrospinning. After the reaction, the PMTA chains were physically entwined with the chains of PCL and the PMTA-modified PCL (PCL/PMTA) solution was prepared.

SEM analysis of the electrospun films The morphology of the electropsun mats examined by SEM is presented in Fig. 1. Smooth surface and nonbeaded structures could be found in all mats. However, a more aligned array was shown by PCL/PMTA mats, compared with the PCL fiber. Quaternary ammonium groups were positively charged. It was speculated that, in the course of in-situ cross-linking polymerization, positive charge formed on the PCL chains, which heightened the charge density of the prepared solution. Due to the electrical attraction, the positive charge gathered on the point of a needle. The accumulated charge was beneficial for overcoming the restriction of the surface tension. When the charged solution passed through the electric field, it stretched into fibers if the electric field was strong enough. With increasing concentrations of MTA, the distribution of diameters widened, and the diameters of the nanofibers increased, as shown in Fig. 1. The average diameters 13



of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 were 360 ± 140, 380 ± 110, 540 ± 210 and 660 ± 240 nm, respectively. During the reaction, polymerization and cross-linking of the functional monomers took place on the PCL matrix to form network macromolecules, which lead to the growth of molecular weight and viscosity. This behavior might be the reason for the variation in fiber diameter and distribution. From the perspective of Fig. 1(e) and (f), the layers could be easily distinguished from each other. SEM examination of the bilayered membrane revealed that the two layers integrated well (Fig.1 (g)). Franco et. al65 had reported a novel bilayer scaffold composed of electrospun polycaprolactone and poly(lacto-co-glycolic acid) (PCL/PLGA) membrane and glutaraldehyde (3.5% v/v) cross-linked chitosan/gelatin hydrogel. More than 60% of the prepared fibers had diameters below 1000 nm. Yao et. al66 prepared a bilayer membrane with a commercial polyurethane wound dressing as an outer layer and electrospun gelatin/keratin nanofibrous mat as an inner layer, the average diameter of the fibers was approximately 160.4 ± 45.8 nm. In our study, the average fiber diameters of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 were 360 ± 140, 380 ± 110, 540 ± 210 and 660 ± 240 nm, respectively. It is worthy noted that the diameters of the produced fibers are within the range of collagen fibers found in natural extracellular matrix67, which suggest that the produced bilayered nanofibrous structure may mimic some structural features of the extracellular matrix, providing better microenvironment for cell adhesion, proliferation, differentiation and ultimately improved skin tissue regeneration at wound site68 .

14


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Fig. 1. SEM images of the electrospun PCL(a), PCL/PMTA-1(b), PCL/PMTA-2(c), and PCL/PMTA-3(d), and cross-section of PCL(e), PCL/PMTA(f) and bilayered(g) nanofiber membranes.

Physical and chemical properties There were some differences between the thermal properties of pure PCL and PCL/PMTA, as displayed in Fig. 2. It was determined by the DTG curves (Fig. 2(a)) that the decomposition temperatures of PCL/PMTA and PCL were all approximately 410 oC, which was higher than the autoclave sterilization requirements for clinical treatments. TGA curves (Fig. 2(b)) revealed that when the temperature exceeded the decomposition temperature, the modified membranes overlapped. The results implied that the copolymers would be completely decomposed if the temperature exceeded the decomposition temperature. According to the literature52, the DTG peaks of PMTA appear at approximately 240 oC

and 420 °C. The presence of PMTA had no effect on the degradation temperature

of PCL. Compared with those of PCL, DTG curves of the modified membranes 15



showed one additional peak, at 234 oC. At this temperature, PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 lost 1.02%, 1.42%, 3.45% and 7.64% of their weight, respectively, which accounted for bound water. For PCL/PMTA-1, the amount of PCL/PMTA was not high enough to distinguish its own degradation step in the thermogram from that of PCL (Fig. 2 (b)). However, for PCL/PMTA-2 and PCL/PMTA-3, it is possible to observe two degradation steps: The first one is related to the bound water, and the second one is related to PCL/PMTA.

Fig. 2. DTG (a) and TG (b) curves of PCL and PCL/PMTA electrospun films.

The ATR-FTIR spectra of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 are displayed in Fig. 3. The peaks at 2942 and 2865 cm-1 were attributed to the symmetric and asymmetric stretching vibrations of the CH2 group. The intense sharp peak at 1723 cm-1 was assigned as the C=O stretching vibrations. The peaks representing CH2 bending vibrations appeared at approximately 1471, 1418, and 1365 cm-1, and COO vibrations at approximately 1239 cm-1 were due to the PCL structure. According to reports in the literature, the characteristic peaks of PMTA are at approximately 1718, 1472 and 954 cm-1 and, belong to the C=O stretching vibration69, C-N stretching of amide groups70 and the vibration absorption peak of the group of – 16


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N+(CH3)3 groups71, respectively. Unfortunately, the characteristic peaks of PMTA overlapped with the peaks of PCL, making it difficult to ascertain the presence of PMTA. However, the appearance of a wide peak from 3000 - 3750 cm-1 demonstrated the presence of bound water absorbed by PCL/PMTA.

Fig. 3. ATR-FTIR spectra of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3.

XRD analysis As displayed in Fig. 4, the PCL and PCL/PMTA nanofiber membranes polymerized with various MTA contents were evaluated by XRD. Two sharp characteristic peaks, at angles of 2θ=21.4

o

and 23.8 o, were found in all cases,

corresponding to the typical semicrystalline structure of the PCL biopolymer. These two peaks were related to the (110) and (200) crystallographic planes of PCL, respectively72. Two characteristic peaks of pure PCL were still retained in PCL/PMTA films, but there were no new peaks in PCL/PMTA films, which meant that in-situ cross-linking polymerization with MTA did not significantly affect the crystalline structure of PCL.

17



Fig. 4. XRD traces of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3.

WCA analysis WCA measurements were performed here to evaluate the surface wettability of the electrospun nanofiber membrane. The pure PCL membrane was hydrophobic, showing a WCA of 129o. However, after in-situ cross-linking polymerization with MTA, the WCA of the prepared PCL/PMTA films decreased. With increasing proportions of MTA, the WCA values of the films decreased to 108o, 81o and 39o for PCL/PMTA-1, PCL/PMTA-2, and PCL/PMTA-3, respectively (Fig. 5), due to the hydrophilicity of the quaternary ammonium groups on MTA. Herein, the introduction of MTA increased the hydrophilicity of the mats.

Fig. 5. WCA of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3. 18


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In vitro antibacterial assay E. coli and S. aureus, the most widely investigated bacteria for antibacterial studies, were used as model bacteria to carry out the microbiological experiments, because these two bacteria are the main sources of infections in wound formation73. Each sample surface was soaked in 2 mL of model bacteria suspension and cultivated for 12 h at 37 oC, and then the antibacterial properties of all samples were evaluated by ﬂuorescence microscopy. Many live bacteria (both E. coli and S. aureus) were observed on the surface of PCL nanofiber mats and, tended to gather, but barely dead bacteria were discovered, which implied that the pure PCL membrane showed no germicidal activity, as shown in Fig. 6. The quantity of dead bacteria attached on the surface of PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 gradually increased compared those on the surface of PCL. The above results demonstrated the presence of quaternary ammonium groups on PCL/PMTA fibers that had been introduced by MTA on PCL via in-situ cross-linking polymerization. PCL/PMTA mats demonstrated excellent germicidal performance toward E. coli. The number of bacteria surviving on the surface of PCL/PMTA-1 decreased dramatically from their number on PCL, and the bacteria on the surfaces of PCL/PMTA-2 and PCL/PMTA-3 were completely dead (Fig. 6 (a)), which was ascribed to the increasing content of N+(CH3)3. Although S. aureus could not be entirely killed on PCL/PMTA films (Fig. 6 (b)), most bacteria were unable to live on the surface of PCL/PMTA membranes, which is beneficial for wound healing.

19



Fig. 6. Fluorescence microscopy images for PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 after incubation with E.coli (a) and S.aureus (b). The green spots represent live bacteria and the red spots represent dead bacteria.

Photographs of the bacterial colonies after culturing for 3 h at 37 oC are shown in Fig. 7. Compared with PCL, the modified membranes showed strong antibacterial activity. As shown in Fig. 7 and Table 2, when PCL was cultured with bacteria, a slight reduction (5.94% and 1.26%) in E. coli and S. aureus, respectively, was observed; however, the PMTA modified electrospun membranes showed strong bactericidal performance. PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 reduced E. coli growth by 53.83%, 88.45% and 99.85%, respectively (Fig. 7(a) and Table 2). For S. aureus, the modified mats also exhibited good antimicrobial activity (from 58.02% 20


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to 99.74%) (Table 2). The results demonstrated that the PMTA modified membranes have a powerful antibacterial effect, increasing with the amount of PMTA, which was in accordance with the results of fluorescent staining.

Fig.7 Photographs of agar plates on which (a) E. coli and (b)S. aureus suspensions were treated with PCL and PMTA-modified membranes.

Table 2 Calculated bactericidal activity of prepared electrospun membranes against E. coli and S. aureus.

Bacteria

PCL

PCL/PMAT-1

PCL/PMTA-2

PCL/PMTA-3

E. coli

5.94% ± 1.26

53.83% ± 1.91

88.45% ± 0.16

99.85% ± 0.26

S. aureus

1.26% ± 2.63

58.02% ± 0.88

89.14% ± 1.25

99.74% ± 0.44

To further investigate the mechanism of bacterial killing, the morphology of the attached bacteria was observed by SEM. The bacteria attached on the PCL surfaces showed the same morphology as healthy bacteria (Fig. 8). However, the bacteria attached on the PMTA-modified electrospun PCL nanofiber (PCL/PMTA) surfaces 21



Page 22 of 38

were changed, with considerable debris, indicating damage to their cellular envelopes (Fig. 8), which is consistent with the well-studied membrane-disrupting mechanism of bacterial killing by quaternary ammonium groups74, 75. Figueira et. al45 had reported a bilayered

electrospun

skin

substitute,

which

comprise

of

a

hyaluronic

acid-polycaprolactone (HA-PCL) upper layer and chitosan-zein-salicylic acid (CS-ZN-SA) bottom layer. Results showed that inhibitory effect of ∼99% for the S.aureus. Woo et. al76 had designed bilayer composites composed of an upper layer of titanium dioxide (TiO2)- incorporated chitosan membrane and a sub-layer of human adipose-derived extracellular matrix as a wound dressing. The chitosan TiO2 layer of bilayer composites had the ability to inhibit penetration of gram-negative E. coli and gram-positive S. aureus. Chanda et. al77 fabricated a biocompatible bilayered polymeric scaffold consisting of chitosan/polycaprolactone and hyaluronic acid. Antimicrobial property evaluation revealed reduction in bacterial adhesion on bilayered scaffolds. The good antimicrobial performance of the above bilayered wound dressings was attributed to the interaction between positively charged chitosan and the negatively charged bacterial surface, which was similar to the bacterial-killing mechanism of quaternary ammonium groups in this study. However, the eluting of chitosan could lead to the decreasing of bactericidal performance, implying the un-eluting PCL/PMTA might be more suitable to defense bacteria for long-term.

Fig. 8 Representative SEM images of bacteria on different surfaces

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In vitro cytotoxicity For medical applications, it was crucial to investigate the biocompatibility of prepared materials. A colorimetric assay was performed in this study to evaluate the preliminary cytotoxicity of PCL, PCL/PMTA-1 PCL/PMTA-2 and PCL/PMTA-3 extracts. SEM analysis was applied here to investigate the cell adhesion on the nanofiber membranes. The effect of the extract of samples on the proliferation of L929 fibroblasts is illustrated in Fig. 9. With increasing culture time, the OD of all groups increased, as displayed in Fig. 9. The results showed that, after the fibroblasts had been incubated for 24 h, the OD values of all cases were slightly higher than that of the blank sample. This result was consistent with the the optical densities on days 3 and 5. However, there were no significant differences (p>0.05) between the group of samples and the blank, as well as between the groups with different contents of PCL/PMTA materials, which indicated that there were no toxic agents extracted from PCL/PMTA and that they thus did not reduce on cell viability. It was reported that the extract of hydroxypropyl-trimethyl ammonium chloride chitosan showed no side effects on L929 cells, regardless of the substitution of quaternary ammonium78.

23



Fig. 9. Proliferation of L929 cells cultivated in extracts of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3.

Fig. 10 shows the morphology of L929 fibroblasts and the interaction between the cells and films. After 3 days of culture, the cells exhibited a typical spindle shape and spread well on the surface of the PCL nanofibers. In addition, the cells not only integrated but also interacted well with the surrounding fibers (Fig. 10(a)). Furthermore, migration and proliferation in certain patterns of cells on the PCL nanofibers were observed, leading to the formation of a continuous monolayer. However, when PCL/PMTA films were cocultured with L929 cells, the number of cells on PCL/PMTA-1 was significantly less than that on the PCL film and the morphology of the cells was different from that of normal cells. Furthermore, no cells were observed to adhere on the PCL/PMTA-2 and PCL/PMTA-3 films (Fig. 10(c) and (d)). This result implied that if the quaternary ammonium groups on the PCL/PMTA had directly contacted the cells, they were able to break the cytomembrane of bacteria, as well as the cytomembrane of L929 cells. These results confirmed our initial hypothesis, making the design of bilayered wound dressing essential and indispensable.

24


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Fig. 10. SEM photographs of L929 fibroblasts cultured for 3 days on PCL(a), PCL/PMTA-1(b), PCL/PMTA-2(c) and PCL/PMTA-3(d).

Conclusions In summary, a novel bilayered wound dressing with excellent broad-spectrum contact antibacterial properties was fabricated through electrospinning and in-situ cross-linking polymerization. PCL was introduced as the cytocompatible inner layer that directly contacted the wound, and the polyquaternary ammonium salts-modified PCL (PCL/PMTA layer) was placed as the outer layer to kill the bacteria. The average fiber diameters of PCL, PCL/PMTA-1, PCL/PMTA-2 and PCL/PMTA-3 were 360 ± 140, 380 ± 110, 540 ± 210 and 660 ± 240 nm, respectively. With an increasing proportion of MTA, the WCA values of the films decreased from 129o (PCL) to 108o, 81o and 39o for PCL/PMTA-1, PCL/PMTA-2, and PCL/PMTA-3, respectively, due to the hydrophilicity of PMTA. The PCL/PMTA layer demonstrated significant antibacterial effects against the bacteria E. coli and S. aureus. All the PCL/PMTA membranes exhibited low cytotoxicity. When the quaternary ammonium groups on the PCL/PMTA had directly contacted the cells, they broke the cytomembrane of bacteria, as well as the cytomembrane of L929 cells. Thus, the bilayered electrospun nanofiber membranes could be an optimal choice for a new generation of antibacterial wound dressings. ASSOCIATED CONTENT *Supporting Information The Supporting Information is available free of charge on the ACS Publications 25



website at DOI:10.1021/acs.iecr.×××××××. Notes The authors declare no competing financial interest. Acknowledgements This study is financially supported by the National Natural Science Foundation of China (no. 51473001), and Key Technology Research and Development Program of Jiangyin City (JYKJ3369).

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Bilayered Antimicrobial Nanofiber Membranes for Wound Dressings

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