Efficient Nanofibrous Membranes for Antibacterial Wound Dressing

Oct 18, 2016 - functional wound dressing materials.3,4 First, the conformable and flexible electrospun nonwovens can provide an effective physical bar...
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Efficient Nanofibrous Membranes for Antibacterial Wound Dressing and UV Protection Shuai Jiang,†,‡,§,⊥ Beatriz Chiyin Ma,†,⊥ Jonas Reinholz,† Qifeng Li,‡ Junwei Wang,‡ Kai A. I. Zhang,† Katharina Landfester,† and Daniel Crespy*,†,∥ †

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001, China § University of Chinese Academy of Sciences, Beijing 100049, China ∥ Department of Materials Science and Engineering, School of Molecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 21210, Thailand ‡

S Supporting Information *

ABSTRACT: Materials with a hierarchical structure often demonstrate superior properties with combined and even synergistic effects of multiple functions. Herein, we report the design of a new class of material with a multicompartment nanofibrous structure as a promising candidate for antibacterial wound dressing and functional textile applications. The design consists in first synthesizing nanocapsules loaded with functional payloads and subsequently embedding the nanocapsules into polymer nanofibers by using the colloid-electrospinning technique. The nanocontainer-in-nanofiber structure allows for a selective and separate loading of different functional agents with different polarities, and it offers a flexible combination of the properties of nanocontainers and nanofibers. An example of the potential for these multicompartment materials is demonstrated here, in which the synergistic antibacterial effect against E. coli K-12 and B. Subtilis combined with anti-UV property is shown. KEYWORDS: antibacterial, anti-UV, electrospinning, nanofibers, wound dressing

1. INTRODUCTION

Previous works have reported the incorporation of antibacterial agents into nanofibrous scaffolds, such as Ag nanoparticles, chitosan, metal oxide nanoparticles, and antibiotics.2,12−14 However, due to the toxicity of conventional antibacterial agents15,16 and antibiotic multiresistance among bacteria, much attention has been given to the benefits of combining several antibacterial agents.17−19 The advantage of the combination strategy is based on the fact that compounds with different mechanisms of action may act synergistically by targeting multiple sites in the cell,20 hence broadening the spectrum of antibacterial properties, reducing drug dosage, lowering side effects, shortening treatment duration, and minimizing or delaying the onset of drug resistance.19,21−23 Essential oils have been proved to be effective against a large variety of organisms including bacteria, virus, fungi, protozoa, and parasites.23,24 Combinations of essential oils with conventional synthetic antimicrobial agents have shown enhanced antibacterial effect in vitro.23−25 For example, combining chlorhexidine digluconate (CHG) with essential oils, in particular with cinnamon oil, the oil of the plant Leptospermum

Skin injuries especially chronic wounds are a global healthcare issue,1 and the healing process of a wound is highly influenced by the wound dressing material.2−6 Electrospun polymeric nanofibrous nonwovens, possessing a high porosity with excellent pore interconnectivity, exhibit unique advantages for functional wound dressing materials.3,4 First, the conformable and flexible electrospun nonwovens can provide an effective physical barrier to protect the open wound from further physical damages and contaminations from exogenous microorganisms. Second, the fibrous scaffolds can serve as a template for the skin cells in the self-repairing process, which is beneficial to minimize the formed scar.7 Third, electrospun nanofibrous mats are permeable to moisture and air, thereby allowing the extraction of extra body fluid from the wound area to avoid infection and maintain a moisturized environment.8 The moisture could reduce the time for wound healing and formation of scars since in a wet environment skin is regenerated without the formation of a scab.9 In addition, antibacterial and therapeutic agents can be loaded in the mats to fabricate active wound dressing material, which could then release the agents to the wound area and further promote the healing process.3,10,11 © 2016 American Chemical Society

Received: July 25, 2016 Accepted: October 18, 2016 Published: October 18, 2016 29915

DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922

Research Article

ACS Applied Materials & Interfaces

fibers (see Table 2). The electrospinning process was carried out using a setup fabricated by IME Technologies with an applied voltage of 15 kV, a working distance between the spinneret and the collector of 10 cm, and a feeding rate of 0.2 mL·h−1. Cross-Linking of PVA Nanofibers. The electrospun nanofibrous mats were first carefully detached from the aluminum foil collector. The detached nonwovens were then exposed to GA/HCl vapor in a vacuum desiccator at 6 × 10−2 MPa for 1 h. An amount of 1 mL of 50 wt % GA aqueous solution was used as the source of GA vapor, and 20 μL of 37 wt % HCl aqueous solution was used as the source of HCl vapor which catalyzes the cross-linking reaction between the −OH of PVA and the −CHO of GA to form acetal bridges.28 Afterward, the cross-linked nanofibrous mats were exposed to air flow in the fume hood for 24 h in order to evaporate the unreacted GA and HCl. Antibacterial Test. All glass apparatuses used in the experiments were autoclaved at 121 °C for 20 min to ensure sterility. The bacterial cells were cultured in nutrient broth (Sigma-Aldrich) and agitated at 300 rpm for 16 h at 37 and 30 °C for E. coli K-12 and B. subtilis, respectively. The cells were then washed with sterilized saline solution (0.9 wt % NaCl), and the final cell density was adjusted to about 3.0 × 106 cfu (colony forming units) mL−1. Then, 10 μL of the nanocapsule dispersion or 10 mg of nanofibers were added to the cell suspension with a final volume of 2 mL and kept at room temperature. At selected time intervals, aliquots of the sample were collected and serially diluted with sterilized saline solution. An amount of 0.1 mL of the diluted sample was then immediately spread on nutrient agar (Sigma-Aldrich) plates and incubated at 37 °C for 16 h to determine the number of viable cells (in colony forming units, cfu). For comparison, control experiments with only E. coli K-12 and B. subtilis bacterial cells were carried out in the absence of nanocapsules or nanofibers. The percentage of the survival fraction was determined by dividing the number of cfu of the samples incubated with nanocapsules or nanofibers by the number of the cfu of the control group. All treated and control experiments were performed in independent triplicates. Biofilm Formation Assay. The B. subtilis cells were cultured in nutrient broth (Sigma-Aldrich) and agitated at 300 rpm for 16 h at 30 °C. The final cell density was adjusted to about 3.0 × 106 cfu (colony forming units) mL−1. Then, 10 μL of the nanocapsules dispersion or 10 mg of nanofibers was added to the cell suspension with a final volume of 1 mL in each well of a 24-well polystyrene microtiter plate (Greiner, USA) and incubated at 30 °C for 24 h. After that, the solutions of bacteria, nanocapsules, or nanofibers were carefully removed. The remaining formed biofilm and wells were rinsed with sterile distilled water. Positive control samples with bacterial cells in the absence of nanocapsules or nanofibers and negative control samples without bacteria were also prepared. The samples were then stained with 0.4% (w/v) crystal violet for 30 min, washed with sterile distilled water to remove the excess of unbound dye, and eluted with 30% (v/v) acetic acid, and the absorbance at 590 nm was measured. Biofilm formation was quantified by measuring the difference between the absorbance of untreated and treated bacteria samples. All treated and control experiments were performed in independent triplicates. Culture of NIH/3T3. NIH/3T3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Scientific Fisher No. 11880036) supplemented with 10% fetal bovine serum, 1% L-glutamin, 100 U/mL penicillin, and 100 μg/mL of streptomycin. Cells were incubated at 37 °C and 5% CO2 in a humidified atmosphere. The medium was refreshed every other day, and cells were split regularly before reaching 100% confluence using trypsin (Gibco, USA) and a split ratio of 1:3. Release of Antibacterial Agents from Multicompartment Nanofibers. To study the release profiles of antibacterial agents from the cross-linked nanofibers, 20 mg of the fiber mats and 1.0 mL of demineralized water were placed in a dialysis bag (MWCO 14000) with both ends sealed. The dialysis bag was then immersed in 9 mL of water in a 20 mL glass vial under stirring. At given time intervals, 0.4 mL of the release medium was withdrawn for UV−vis measurement to determine the concentration of released antibacterial agents. An equal amount of demineralized water was added to keep the volume constant.

morrisonii, and manuka oil at a lower level of inhibition, resulted in a substantial reduction in the amount of CHG required to achieve the same level of growth inhibition compared to CHG used alone.24 Herein, we report the development of a new class of antibacterial material with a multicompartment nanofibrous structure for wound dressing. The design principle of the material consists of the synthesis of silica nanocapsules (SiNCs) loaded with functional payloads and subsequent embedding of the SiNCs in polymer nanofibers via colloid electrospinning. This designed nanostructure offers a multiple nanoscale platform for selective and separate delivery of functional agents with different polarities, either in the hydrophobic core of nanocapsules, as the amphiphilic surfactant on capsules surface, or dispersed in the hydrophilic polymer matrix of nanofibers. In this work, hydrophobic peppermint oil (PO), amphiphilic octenidine·2HCl (OCT), and hydrophilic CHG were used as a model combination for the antibacterial system, and enhanced disinfection effect on Escherichia coli K-12 and Bacillus subtilis was demonstrated. The presented strategy allows for a flexible combination of functional agents, thereby providing a platform for the synthesis of multifunctional materials. As an example, hydrophobic UV-absorber octyl methoxycinnamate (OMC) was introduced in the antibacterial fibers via loading it in the SiNCs, and anti-UV property of the nanofibrous mats was investigated.

2. EXPERIMENTAL SECTION Materials. Tetraethoxysilane (Alfa Aesar, 98%), hexadecane (Sigma-Aldrich, 99%), olive oil (Sigma-Aldrich), octyl methoxycinnamate (Sigma-Aldrich, 98%), peppermint oil (Sigma-Aldrich, natural, from Mentha piperita L.), cetyltrimethylammonium chloride (CTMACl, Acros Organics, 99%), octenidine·2HCl (TCI, >98%), chlorhexidine digluconate (Sigma-Aldrich, 20 wt % aqueous solution), crystal violet (Sigma-Aldrich, 90%), and glutaraldehyde (GA, Merck KGaA, 50 wt % aqueous solution) were used as received. Poly(vinyl alcohol) (Polysciences Inc., 88 mol % hydrolyzed) with a molecular weight Mw of 125 000 g·mol−1 was used as received. Escherichia coli K-12 (E. coli K-12) and Bacillus subtilis (B. subtilis) were purchased from DSMZ Leibniz-Institute (Braunschweig, Germany). Demineralized water was used through all the experiments if not specifically mentioned. All chemicals and solvents were used as received unless otherwise specified. Synthesis of Silica Nanocapsules. Silica nanocapsules were synthesized in miniemulsion. The surface of miniemulsion droplets was used as a soft template for the hydrolysis and condensation of alkoxysilanes.26,27 An amount of 2.0 g of tetraethoxysilane was first mixed with 125 mg of hexadecane and 1 g of OO, PO, and OMC as control sample, antibacterial agent, and UV absorber, respectively. The hydrophobic mixture was then added to 30 mL of 0.77 mg·mL−1 aqueous solution of CTMA-Cl or OCT under stirring. After a preemulsification by stirring at 1000 rpm for 1 h, the emulsion was sonicated for 180 s with cooling ice by using a Branson 450 W sonifier with a 1/2″ tip at 70% amplitude in a pulse regime (30 s of sonication, 10 s of pause). The resulting miniemulsions were stirred at 1000 rpm at room temperature for 12 h to obtain silica nanocapsules. The capsules containing functional agents PO, OCT, PO + OCT, and OMC + OCT are denoted as SiNC-PO, SiNC-OCT, SiNC-PO/OCT, and SiNC-OMC/OCT, respectively. Fabrication of Multicompartment Nanofibers. Multicompartment nanofibers were prepared by electrospinning an aqueous solution of PVA containing SiNCs. The weight ratio of PVA and SiNCs in the electrospun nanofibers is 3.2:1. In the case of nanofibers containing hydrophilic antibacterial agents, CHG was dissolved in the electrospinning solution. The fibers were denoted NF-0, NF-PO, NF-OCT, NF-CHG, NF-PO/OCT, NF-PO/CHG, NF-OCT/CHG, NF-PO/ OCT/CHG, or NF-OMC/OCT according to the composition of the 29916

DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922

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Figure 1. (a) Schematic illustration of the synthesis of SiNCs by using miniemulsion polymerization; (b1−e1) SEM and (b2−e2) TEM micrographs of the SiNCs: (b1, b2) SiNC-0, (c1, c2) SiNC-PO, (d1, d2) SiNC-OCT, (e1, e2) SiNC-PO/OCT. dimensions of 13 mm × 2 mm × 80 μm (gage length × width × thickness). Water absorption ratio of the prepared nanofibrous mats was defined as the swelling degree in this study. Swelling degree of the fibrous mats is measured by first weighing the dry fibrous samples and then immersing them in demineralized water. The equilibrium of water absorption was considered to be reached when the weight of wet samples does not change. The weight of swollen samples was measured after the excessive water on the surface was removed with filter paper. The swelling degree of the fibrous mats is defined as follows m − mo Swelling degree = s × 100% mo (1)

Anti-UV Test. The anti-UV property of the OMC-loaded nanofibrous mats was evaluated based on the UV absorbance of the materials. The electrospun nanofibrous mats were placed between the ring shape frames, which were then positioned on the sample holder of a PerkinElmer Lambda 25 UV−vis spectrophotometer. The UV absorption spectra were collected at wavelengths in the range of 200− 600 nm. Analytical Methods. The morphologies of nanocapsules and nanofibers were examined with a Gemini 1530 (Carl Zeiss AG, Oberkochem, Germany) scanning electron microscope (SEM) operating at 0.35 kV and a Jeol 1400 (Jeol Ltd., Tokyo, Japan) transmission electron microscope (TEM) operating at an accelerating voltage of 120 kV. SEM and TEM samples of silica nanocapsules were prepared by casting the diluted nanocapsule dispersion on silicon wafers and carbon layer-coated copper grids, respectively. The samples of nanofibers for SEM and TEM were prepared by depositing electrospun nanofibers directly on silicon wafers and carbon layercoated copper grids, respectively. The hydrodynamic diameter of the capsules was evaluated by dynamic light scattering (DLS) using a Nicomp particle sizer (model 380, PSS, Santa Barbara, CA) at a fixed scattering angle of 90°. The shell thickness of the capsules was estimated by counting 100 capsules from TEM micrographs. The diameter of electrospun nanofibers was estimated by counting 100 nanofibers from SEM micrographs. The concentration of released antibacterial agents was tracked by recording their UV−vis absorption using PerkinElmer Lambda 25 UV−vis spectroscopy. The calibration curves of the antibacterial agents are given in Figure S1. The encapsulation of peppermint oil and octyl methoxycinnamate in silica nanocapsules was investigated by Fourier transform infrared (FTIR) spectroscopy. The dispersions of silica nanocapsules were freeze-dried, and the powders were pressed with KBr to form a pellet. Transmittance between 4000 and 500 cm−1 was recorded in a Spectrum BX spectrometer from PerkinElmer. The contents of peppermint oil and octyl methoxycinnamate in the freeze-dried samples were measured by thermogravimetry. The thermogravimetric analysis (TGA) measurements were performed with a NetzschGerätebau TG 209 under nitrogen atmosphere. The temperature range is between 25 and 900 °C with a heating rate of 10 K·min−1. Mechanical properties of the nanofibrous mats were tested at ambient temperature by using a ProLine table-top testing machine Z005 from the company Zwick/Roell. The test was conducted at a crosshead speed of 10 mm/min by using dumbbell specimens with the

where ms and m0 are the weights of the swollen and dry samples, respectively. Confocal laser scanning microscopy was performed on the LSM SP5 STED Leica Laser Scanning Confocal Microscope (Leica, Germany). A small piece of the fibers (0.5 cm × 0.5 cm) was cut, washed with PBS, and placed in a 35 mm μ-Dish (Ibidi, Germany). Afterward, NIH/3T3 cells were seeded out on top of the fibers in a density of 2 × 105 cells/mL. The experimental setup was incubated for 24 h at 37 °C and 5% CO2 in a humidified atmosphere. After incubation, the fibers were carefully removed and placed upside down in a fresh 35 mm μ-Dish and fixed with a coverslip. The cell membrane of the cells was stained with 1 μg/mL of CellMask Orange Plasma membrane Stain (Thermo Fisher Scientific Inc., USA), and the nucleus was stained with 5 μM DRAQ5 (Cell Signaling Technology Inc., USA) for 5 min. The material was immediately analyzed via confocal laser scanning microscopy. For excitation, λ = 561 nm and λ = 633 nm lasers were used. Dyes were detected at 572−600 nm (plasma membrane; pseudocolored in red) and 644−700 nm (nucleus; pseudocolored in blue). The fibers were monitored via transmission. Finally, pictures were analyzed with the Leica software LAS AF Lite.

3. RESULTS AND DISCUSSION Synthesis of Silica Nanocapsules Loaded with Antibacterial Agents. Silica nanocapsules were synthesized in miniemulsion by using the surface of miniemulsion droplets as a template for the hydrolysis and condensation of tetraethoxysilane (Figure 1a). The antibacterial agent PO was 29917

DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922

Research Article

ACS Applied Materials & Interfaces first mixed with hexadecane and tetraethoxysilane to form the dispersed phase which was then converted to miniemulsion droplets after emulsification. PO was hence encapsulated in situ in the core of the nanocapsules (SiNC-PO and SiNC-PO/ OCT). The encapsulation of PO in the capsules was characterized by using FTIR spectroscopy. The presence of characteristic peaks (e.g., 2958, 2925, 2871, and 1709 cm−1) of PO in the spectra of freeze-dried SiNC-PO samples confirmed the encapsulation of PO in the capsules (see Figure S2a). The content of PO in the capsules was determined to be ∼28% by TGA measurement (see Figure S3a). The encapsulation of PO in the capsules can protect the lipophilic bioactive components of PO from degradation, oxidation, or interactions with the surrounding environment.29 Furthermore, the main constituents of the peppermint oil are menthol and menthone. The main functional groups of these molecules are hydroxyl and ketone groups, and therefore no strong interaction with the silica shell is expected. Olive oil (OO) was used as the core control material since it does not present any specific antibacterial properties. CTMA-Cl and octenidine·2HCl (OCT) were used as cationic surfactants for the stabilization of SiNC-0, SiNC-OCT, and SiNC-PO/OCT capsules. Welldefined core−shell morphology for the SiNCs was identified by SEM and TEM as shown in Figure 1b−e, indicating the successful condensation of alkoxysilanols at the interface of miniemulsion droplets. Detailed information including the composition, hydrodynamic diameter, and shell thickness of the synthesized SiNCs is given in Table 1. Table 1. Composition, Average Hydrodynamic Diameter, and Shell Thickness of SiNCs

a

entry

core

surfactant

diameter (nm)

SiNC-0 SiNC-PO SiNC-OCT SiNC-PO/OCT

OO PO OO PO

CTMA-Cl CTMA-Cl OCT OCT

209 230 187 242

± ± ± ±

a

82 61 51 68

Figure 2. Inactivation efficiency of E. coli K-12 (a) and B. subtilis (b) in the presence of different SiNCs (0.28 mg·mL−1). Data represents the results of three independent experiments (mean ± SE of the mean). The control sample contains neither capsules nor fibers.

shell thickness (nm) 11 6 13 7

± ± ± ±

5 2 7 3

treatment for both types of bacteria. In this case, a combination of dual antibacterial agents in the capsules showed a higher antibacterial efficiency in comparison with each of them alone, which indicates a synergistic effect of the combination of different antibacterial agents. At the same time, antibiofilm properties of SiNCs were also evaluated. As it can be seen from Figure S4 (Supporting Information), SiNC-PO/OCT acted as the most efficient one without presenting any biofilm formation after 24 h of treatment. The biocompatibility of the fibers was investigated by cytotoxicity test using a direct contact method. NIH/3T3 cells were seeded out on top of the fibrous mats of NF-PO/ OCT/CHG. After 24 h of incubation, cells were found attached on the fibrous mats characterized by using CLSM (see Figure S6). Fabrication of Multicompartment Nanofibers via Colloid Electrospinning. Silica nanocapsules were then electrospun with poly(vinyl alcohol) (PVA) to form multicompartment nanofibers with a nanocapsule-in-nanofiber structure (see Figure 3a). The SiNCs/PVA dispersion is fed by a pump at a constant rate. When the electric field is applied, the liquid at the tip of the spinneret is charged, and a “Taylor cone” can form due to charge repulsion. Once the electrostatic repulsion overcomes the surface tension of the droplet, a charged jet of the dispersion is ejected toward the collecting plate. The solvent evaporates on the way to the collector, and uniform nanofibers with diameter of 200−300 nm are deposited as a nonwoven (see Figure 3b).31 SiNCs were

Measured by DLS at 90°.

Antibacterial Property of Silica Nanocapsules. The antibacterial property of the SiNCs was evaluated by using E. coli K-12 and B. subtilis as Gram-negative and Gram-positive model systems, respectively. A suspension of bacterial cells was incubated with SiNCs or nanofibers for 1, 3, and 6 h. The viability of the bacterial cells was assessed using the standard plating method (see Experimental Section for details). As shown in Figure 2, the sample SiNC-0 showed no toxic effect on either E. coli K-12 or B. subtilis despite the presence of CTMA-Cl, which is due to the low concentration (3.9 μg· mL−1) of CTMA-Cl in the dispersion compared to its minimum inhibitory concentration in bacteria (32 μg· mL−1).30 For the bacterial cells treated with SiNC-PO, SiNCOCT, or SiNC-PO/OCT, a significant decrease of 62%, 43%, or ∼99% in the viability of E. coli K-12 and 98%, 92%, or ∼99% in the viability of B. subtilis was observed in the first hour of treatment, respectively. This contrast can be due to the constitution of the outer membrane of the Gram-negative bacteria. The outer membrane acts as a barrier against the diffusion of molecules, thereby leading to a higher resistance to the antibacterial treatment in comparison to the Gram-positive one. Among all samples, SiNC-PO/OCT acted as the most efficient one, achieving ∼99% of inactivation in the first hour of 29918

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Figure 3. (a) Schematic illustration of the fabrication of multicompartment nanofibers by using the colloid electrospinning technique. Representative (b) SEM and (c) TEM micrographs of the fibers NF-PO/OCT/CHG.

agents covering a wider range of polarities can be offered by designing the fibers with a nanocapsule-in-nanofiber structure. The antibacterial activity of the multicompartment nanofibers is summarized in Figure 4. The sample NF-0, using CTMA-Cl as surfactant, showed no toxic effect on both types of bacteria cells up to 6 h of treatment (see Figure 4a and b). The fibers NF-CHG showed the highest disinfection efficiency against E. coli K-12 among the fibers loaded with a single antibacterial agent, reaching ∼99% of inactivation after 6 h (see Figure 4a), whereas the fibers NF-OCT exhibited the highest inactivation efficiency against B. subtilis, with ∼98% of inactivation in the first hour of treatment (see Figure 4b). Under the same conditions, the fibers loaded with antibacterial agents showed lower disinfection efficiency in comparison to their nanocapsule counterpart (see Figure 2 and Figure 4). This contrast is possibly related to the fiber matrix, which acts as an additional barrier for the release of the antibacterial content. The growth rate of bacteria in a wound is highly dependent on the contact time with the antibacterial agent and correspondingly on the rate of drug release from the nanofibers in a wound dressing material.3 A slow release of antibacterial agents from the wound dressing materials has both advantages and disadvantages. Ideal wound dressings are expected to exhibit a burst of drug release at the initial stage and then a sustained drug release for a longer period. The initial drug release is beneficial for suppressing infections and relieving pain of a wound in an early stage of infection. Meanwhile, sustained drug release is favorable as a long-term protection of the wound, which may secure the injury against further infections while the healing process takes place. For this, it is crucial that a wound dressing material should not change its features during a longer period of treatment.32 The antibacterial agent CHG dispersing in the fiber matrix of NF-CHG released ∼60% (0.006 mg·mL−1) of its initial loading amount in the first 1 h (see Figure 5). Comparatively, the OCT serving as surfactant for the synthesis of nanocapsules released only 15% (0.001 mg·mL−1) of its initial loading amount from the fibers NF-OCT due to its possible adhesion on the capsules. The combinations of antibacterial agents in nanofibers and their antibacterial activity were further investigated (see Figure 4c,d). In general, the different combinations of antibacterial agents resulted in a dramatic increase in the inactivation efficiency in comparison with that of the fibers loaded with a single antibacterial agent (see Figure 4a,b). In particular, a synergistic antibacterial effect of combination therapy was

found to be well distributed in the nanofibers as indicated by TEM (see Figure 3c). The core−shell structure of SiNCs can still be identified after being embedded in the fibers. Electrospun PVA nanofibers were rendered water insoluble by cross-linking them in GA/HCl vapor. Since the cross-linking process was conducted in vapor with no organic solvent used, a premature release of payloads during the cross-linking process was avoided. The cross-linked nanofibrous mat, NF-PO/OCT/ CHG, exhibits a tensile strength of 6.3 ± 0.3 MPa, a Young’s modulus of 0.7 ± 0.1 MPa, and a maximum strain of 210 ± 40% (see Experimental Section for the details of the mechanical test). Swelling degree of the cross-linked PVA nanofibers was determined to be ∼300%. The composition of the nanofibers is summarized in Table 2. Table 2. Composition of the Nanofibersa entry

antimicrobial agents

NF-0 NF-PO NF-OCT NF-CHG NF-PO/OCT NF-PO/CHG NF-OCT/CHG NF-PO/OCT/CHG

CTMA-Cl PO OCT CHG PO + OCT PO + CHG OCT + CHG PO + OCT + CHG

content [wt %] 0.33 13.34 0.33 0.47 13.34 13.34 0.33 13.34

+ + + +

0.33 0.47 0.47 0.33 + 0.47

a

PO: peppermint oil. OCT: octenidine·2HCl. CHG: chlorhexidine digluconate.

Antibacterial Property of Multicompartment Nanofibers. The unique nanocapsule-in-nanofiber structure allows for a selective loading of multiple antibacterial agents with different polarities, e.g., hydrophobic PO in the core of the capsules, amphiphilic OCT as the surfactant on the capsule surface, and hydrophilic CHG dispersed in the fiber matrix. The combinations of antibacterial compounds with different action mechanisms may target multiple sites in the cell, thereby achieving a synergistic antibacterial effect.20 In the case of SiNCs, hydrophobic peppermint oil and amphiphilic octenidine·2HCl were loaded in the oil core and as the surfactant of the nanocapsules, respectively. SiNCs with the same combinations of antibacterial agents were incorporated in electrospun NFs. Because of the hydrophilicity of the PVA fiber matrix, an additional hydrophilic antibacterial agent can be loaded in the fiber matrix. Therefore, extra combinations of antibacterial 29919

DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922

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ACS Applied Materials & Interfaces

Figure 4. Inactivation efficiency of E. coli K-12 in the presence of nanofibers embedded with SiNCs containing single (a) and combined (c) antibacterial agents. Inactivation efficiency of B. subtilis in the presence of nanofibers embedded with SiNCs containing single (b) and combined (d) antibacterial agents. Data represent the results of three independent experiments (mean ± SE of the mean).

materials, the UV absorber OMC was encapsulated in the core of the nanocapsules with OCT as the surfactant, thereby obtaining nanocapsules (SiNC-OMC/OCT) and nanofibers (NF-OMC/OCT) with combined anti-UV and antibacterial properties. The morphology of SiNC-OMC/OCT and NFOMC/OCT was characterized by SEM and TEM as shown in Figure 6. OMC is a widely used sunscreen in cosmetic formulations, and it is classified as a UV−B filter with the absorption in the range of 290−320 nm wavelength of the solar UV radiation.33 The encapsulation of OMC is beneficial to prevent its photodegradation as well as to reduce its skin absorption responsible for serious systemic side effects.33 The encapsulation of OMC in silica nanocapsules was confirmed by using FTIR spectroscopy (see Figure S2b). The encapsulation content of OMC in the capsules was determined to be ∼65% by TGA (see Figure S3b), which is consistent with the theoretical content of 60%. The UV absorbance of the dispersions of SiNCs was significantly increased due to the encapsulation of OMC (see Figure 6c). As expected, the intensity of absorbance increased as the concentration of SiNCs in the dispersions increased. As shown in Figure 6f, the anti-UV property of OMC-loaded SiNCs was preserved after being embedded in the PVA nanofibers. The antibacterial property of SiNC-OMC/OCT and NFOMC/OCT was also evaluated. As it can be seen from Figure 7, both SiNC-OMC/OCT and NF-OMC/OCT showed the highest disinfection potential against B. subtilis with nearly 99% of inactivation efficiency within the first hour of treatment in contrast to E. coli. Since only a single antibacterial agent (OCT) was present in the system, these results were expected and are comparable to the ones of SiNC-OCT and NF-OCT.

Figure 5. Cumulative release of OCT and CHG from fibers NF-OCT and NF-CHG.

observed on the inactivation of E. coli K-12 in the first hour. The specific synergistic effects of nanofibers NF-PO/OCT, NFPO/CHG, NF-OCT/CHG, and NF-PO/OCT/CHG are 67%, 38%, 46%, and 42%, respectively, in comparison with the sum of their individual counterparts. A similar trend was also observed in the evaluation of antibiofilm properties of the nanofibers. As shown in Figure S5 (Supporting Information), all nanofibers with a combination of antibacterial agents resulted in strong inhibition of biofilm formation in contrast to the fibers with only one antibacterial agent. The large initial suppression of bacterial growth by the nanofibers possibly prevented their anchoring and subsequently hindered a biofilm formation. Combined Antibacterial and Anti-UV Properties in the Fibers. To further increase the functionality of the 29920

DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922

Research Article

ACS Applied Materials & Interfaces

Figure 6. (a) SEM and (b) TEM micrographs of SiNC-OMC/OCT; (c) UV absorbance of dispersions of SiNC-OCT and SiNC-OMC/OCT with different concentrations; (d) SEM and (e) TEM micrographs of NF-OMC/OCT; (f) UV absorbance of the nanofibers NF-OCT and NF-OMC/ OCT.

4. CONCLUSIONS We report a new class of multicompartment nanofibrous material with a nanocapsule-in-nanofiber structure. The design principle of the material consists in first synthesizing nanocontainers loaded with functional payloads and subsequently embedding the capsules in a polymer nanofiber by using the colloid-electrospinning technique. This designed nanostructure offers multiple nanoscale platforms for selective and separate delivery of functional agents with different hydrophilicities, either in the hydrophobic core of nanocapsules, as the amphiphilic surfactant on capsules’ surface, or dispersed in the hydrophilic polymer matrix of nanofibers. Moreover, the multicompartment nanofibers allow a flexible combination of the properties of nanocontainers and nanofibers, thereby providing a platform for the synthesis of multifunctional materials. As examples of the potential for the multifunctional use of the material, synergistic antibacterial effect against E. coli K-12 and B. subtilis and anti-UV properties are demonstrated. The specific synergistic effects of nanofibers NF-PO/OCT, NFPO/CHG, NF-OCT/CHG, and NF-PO/OCT/CHG are 67%, 38%, 46%, and 42%, respectively, in comparison with the sum of their individual counterparts.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b09165. Calibration curves for the determination of OCT and CHG in water (PDF)

■

Figure 7. Inactivation efficiency of E. coli K-12 and B. subtilis in the presence of (a) SiNC-OMC/OCT (0.28 mg·mL−1) and (b) NFOMC/OCT. Data represent the results of three independent experiments (mean ± SE of the mean). The control sample contains neither capsules nor fibers.

AUTHOR INFORMATION

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These authors contributed equally. DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922

Research Article

ACS Applied Materials & Interfaces Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.J. acknowledges the financial support from “MPG-CAS Joint Doctoral Promotion Program (DPP)”. B.C.M. acknowledges the financial support from DAAD, CAPES, and CNPq. S.J. and D.C. thank Dr. Volker Mailänder for the discussion about cell culture and Dr. Michael Kappl, Andreas Hanewald, and Regina Fuchs for the tensile tests.

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DOI: 10.1021/acsami.6b09165 ACS Appl. Mater. Interfaces 2016, 8, 29915−29922