Bio-based Electrospun Nanofiber of Polyhydroxyalkanoate Modified

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Bio-Based Electrospun Nanofiber of Polyhydroxyalkanoate Modified with Black Soldier Fly’s Pupa Shell with Antibacterial and Cytocompatibility Properties Chin-San Wu* and Shan-Shue Wang

ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by CALIFORNIA STATE UNIV FRESNO on 11/29/18. For personal use only.

Department of Applied Cosmetology, Kao Yuan University, Kaohsiung County, Taiwan 82101, Republic of China S Supporting Information *

ABSTRACT: We report on the antibacterial and cytocompatibility properties of a bio-based electrospun polyhydroxyalkanoate (PHA) nanofiber modified with Black Soldier Fly (BSF) pupa shell. A 5−50 μm chitosan powder (CSP) was made by grinding BSF pupa shell in water, acid, alkali. CSP was combined with PHA in an electrospinning machine using a biaxial feed method and manufactured into a 50−500 nm antibacterial nanofiber. We studied the morphology, mechanical properties, water absorption, and antibacterial properties of the electrospun PHA/CSP nanofiber. To improve the fiber’s compatibility and functionality, acrylic acid (AA) was grafted onto PHA. The resulting tensile properties and morphological characterizations indicated enhanced adhesion between CSP and PHA-g-AA nanofiber, as well as an improvement in its water resistance and tensile strength, compared with the PHA/ CSP nanofiber. To study the cytocompatibility of the material, human foreskin fibroblasts were seeded onto the nanofiber specimens with 3.0 and 6.0 wt % CSP. Increasing the CSP content in PHA/CSP and PHA-g-AA/CSP nanofibers enhanced cell proliferation; additionally, the nanofibers with CSP showed strong inhibition of bacteria. The enhanced antibacterial and biodegradable properties of PHA-g-AA/CSP and PHA/CSP nanofibers demonstrate their potential for biomedical material applications. KEYWORDS: antibacterial activity, Black Soldier Fly, chitosan, electrospun nanofiber, polyhydroxyalkanoates Taylor cone and whipping motion principles, after curing.14,15 This nanofiber can be made into a thin film, which is characterized by good air and moisture permeability, water repellency, and antiviral properties, for tissue regeneration.16,17 As such, CS−polymer combinations have been used as biomedical materials for wound dressings, drug delivery, and tissue engineering scaffolds. However, when the CS solution is used in electrospinning, the poor adhesion of CS often results in nonuniform fibers or nonoptimal spray conditions during electrospinning, which impedes film quality. To remedy the aforementioned defects, some researchers have introduced complex additives to the CS−polymer solution during the fabrication process, mixing fully degradable polymers with the CS solution to create electrospun fiber. Liu et al.18 used CS and polylactic acid material as the main materials and glutaraldehyde as a cross-linking agent for an electrospun fiber; the fiber showed good mechanical strength and biocompatibility for applications involving heart tissue engineering scaffolds. Urbanek et al.19 used an electrospinning process to create a mixed solution of CS and poly-

1. INTRODUCTION With an increase in organic waste such as kitchen waste, poultry and animal manure, deteriorated vegetables and fruits, aged food, and food processing waste, the Black Soldier Fly (BSF) has been used as an insect biotreatment medium to generate ecological materials with circular economic value. BSF is a nonpest organism typically found in tropical, subtropical, and warm zones that offer optimal temperature (24−40 °C) and humidity (30−90%) conditions for BSF breeding and mass rearing.1−3 The life cycle of a BSF comprises four major stages: ovum, larva, pupa, and imago. After 49−56 days of pupa exuviation, the spent puparium contains abundant chitin. Chitin has excellent biocompatibility and biodegradability; thus, it has been used extensively as a biomedical material.4−6 However, the physicochemical properties of chitin, such as its solubility and absorbability, are lacking. Chitosan (CS), the deacetylated form of chitin, has better cytocompatibility, solubility, antibacterial, and film-forming properties.7−11 There is an increasing demand for CS, due to its softness, bulkiness, porosity, and pore diameter.12,13 Electrospinning techniques have been used to combine CS with polymers to create a solution state. Static electrification of the CS−polymer solution with high-voltage electric field application allows the polymer to be stretched and shaped into a nanofiber, based on © XXXX American Chemical Society

Received: September 23, 2018 Accepted: November 19, 2018 Published: November 19, 2018 A

DOI: 10.1021/acsami.8b16606 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Diagram of a 5−50 μm-Sized Chitosan Powder (CSP) Made of Black Soldier Fly (BSF) Pupa Shell after Grinding in Water, Acid, and Alkali

water for 1 day to separate the impurities from the shell. The chitin was prepared by soaking with NaOH and HCl and rinsed with water.31 The extracted chitin was deacetylated to form CS. Specifically, the chitin was soaked in 30% NaOH and heated at 50 °C for 15 h for acetylation and then placed in a 70−80 °C oven to remove moisture for 1 day to obtain CS. The prepared CS was sieved through a 400− 500 screen mesh by an oscillating screen-mesh machine and dried in a 100−110 °C oven for 12 h to keep the moisture below 0.05%. The end product was 5−35 μm CSP (Scheme 1) with a molecular weight of 400 000−430 000 and a deacetylation degree of 82−86%. 2.3. Preparation of Electrospinning Solution and Nanofiber Specimens. Solution A sample: 2 g of the prepared CSP was dissolved in 2% acetic acid. The dissolved volume was about 100 mL in a 150 mL circular beaker. The 2 wt % CSP solution was colloidal and transparent when it was continuously mixed at 200 rpm and 30 °C for about 8 h. Solution B sample: about 20 g of PHA or PHA-g-AA was placed in a 150 mL circular beaker and dissolved by dichloromethane to a quantity of 100 mL. The solution was then stirred at 200 rpm at 30 °C until the PHA or PHA-g-AA was completely soluble in dichloromethane. After 6 h, we obtained 20 wt % PHA and PHA-gAA sample solutions. A and B solution samples (10 mL of each) were placed in an injection syringe to be fed into the spinneret. The flow velocity was adjusted by an injection pump (Cole-Parmer Instrument Co., Chicago, IL) controller. The two-orifice spray nozzle of the spinneret was connected to solution A via the machine spray nozzle core end; solution B was connected to the machine spray nozzle shell. To provide adequate power for the electrospinning field, a high potential was applied to the spinneret via a high-voltage power supply (205B; Bertan, Hicksville, NY). The positive voltage was set at 25 kV. The core flow velocity was maintained at 0.3 and 0.6 mL/h, and the shell flow velocity was held at 1 mL/h at 25 °C. Solutions A and B were delivered to the spinneret (inside and outside diameters: 0.51 and 1.02 mm, respectively) to create the nanofibers via electrospinning. One end of the fiber was connected to an aluminum revolving tray collector (diameter: 10 cm; length: 20 cm). The distance between spinneret tip and collector was fixed at 20 cm. The nanofibers were collected by the aluminum revolving tray at 50 rpm. A cone-jet electrospinning mode was maintained for several hours of spinning. The resulting thin film (length: 10 cm; width: 5 cm) was dried in an oven at 60 °C for 1 day and then made into standard samples for

(caprolactone) for nonwoven materials; a surface modification solution was introduced for surfaction to improve the wetting, mechanical, and crystalline properties of the nonwoven materials. Polyhydroxyalkanoate (PHA), a fully degradable polymer, is made from microorganisms and has excellent cytocompatibility, biocompatibility, biodegradability, hot workability, and mechanical properties.20−22 In this study, we fabricated a 50− 500 nm antibacterial electrospun PHA nanofiber from 5−50 μm chitosan powder (CSP) made from BSF pupa shell. PHA and CSP were mixed thoroughly by double-shaft feeding of the electrospinning head. Acrylic acid (AA) was grafted onto PHA to enhance the interface compatibility and mechanical properties between PHA and CS. The resulting tensile properties and morphological characterizations indicated enhanced adhesion between CSP and the PHA-g-AA nanofiber, as well as an improvement in its water resistance and tensile properties, compared with the PHA/CSP nanofiber. Collectively, the PHA-g-AA CSP nanofiber shows great potential for use in protein separation and purification membranes, regeneration membranes of guided tissue for dressing material in oral cavities, tissue engineering, biological protection materials, and air filtration membranes.23−25

2. EXPERIMENTAL SECTION 2.1. Materials. The following materials including PHA,26 dicumyl peroxide (DCP) and AA,27 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolelium bromide (MTT), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS),28 minimum essential medium (MEM), nutrient broth, fetal bovine serum, and agar29 were commercially available. BSF was purchased from the Livestock Research Institute in Taiwan. PHA-g-AA was self-prepared. The loading quantity of AA to the dichloromethane-soluble polymer was measured by titration and shown as a grafting percentage,30 which was 5.86 wt %. The loadings of DCP and AA were kept at 0.3 and 10 wt %, respectively. 2.2. Chitosan Powder Preparation of Black Soldier Fly Pupa Shell. The BSF resource insect was fed with recovered kitchen waste at Kao Yuan University, Kaohsiung, Taiwan, for 49−56 days. BSF pupa shell derived from the sixth exuviation was soaked in distilled B


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Scheme 2. Fabrication of Electrospun Nanofiber Membranes from Acrylic Acid-Grafted Polyhydroxyalkanoate (PHA-g-AA) and CSP Solution

To observe the cell growth pattern, the 2.5 × 104 CCD-966SK cells were grown in a 6 cm diameter culture dish with PHA/CSP and PHA-g-AA/CSP electrospun nanofiber for 72 h, then the supernatant was removed, washed with 2 mL of PBS twice, detached the cells with 1.5 mL of trypsin/ethylenediaminetetraacetic acid in a 5% CO2 cell incubator for 5 min, and the cell suspension was obtained. The cell suspension was then centrifuged at 2400 rpm to remove the supernatant liquid, mixed with 3 mL of MEM, and placed in a cell culture box with the cells, before being placed in a cell incubator for 8 h. 2.7. Antimicrobial Assay. The antimicrobial behavior of the electrospun nanofiber membranes was assessed with Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria). The bacteria were grown in liquid medium.36 The microbes were spread by a sterilized triangular glass rod after 0.1 mL of microbial solution was dropped onto the culture dish. The nanofiber membrane specimen with 0.05 cm thickness and 1.20 cm diameter was washed with 75% alcohol twice, irradiated by UV light, and then placed in the middle of the culture dish.26 The culture dish was sealed with elastic adhesive tape and incubated at 37 °C for 18 h. The antimicrobial activity was evaluated by the inhibition zone method.37 The microorganism growth was determined by cell counting.37 2.8. Biodegradation Test. For the in-soil degradation study, the electrospun nanofiber samples (3 × 2 × 0.05 cm3) were carefully weighed after vacuum drying at room temperature for 1 day (W0). Then, they were buried into boxes containing soil to assess their biodegradation.27 The boxes environment was maintained at approximately 40−50% moisture and 28−36 °C for 18 days. At 3 day intervals, samples were weighed again after a deionized water rinse and vacuum drying for 2 days (Wt). Weight loss (%) is given by the following equation

measurement of various properties. The manufacturing process is shown in Scheme 2. 2.4. Electrospun Nanofiber Measurements. The solid-state carbon-13 nuclear magnetic resonance (13C NMR) spectra28 and Fourier transform infrared (FTIR) spectra27 were recorded. The samples were ground, mixed with KBr, and pressed into pellets. The fiber morphology was observed by scanning electron microscopy (SEM).32 The fiber diameters were measured based on a collection of ∼600 fibers, from which the average fiber diameter was determined. The surface elements of the gold-coated fiber were analyzed by energy-dispersive spectrometry (EDS; model EX-220; Horiba, Japan). In accordance with American Society for Testing and Materials standard D638, the tensile properties were measured33 by a mechanical tester.26 The mean values were obtained from six specimens. 2.5. Surface Wettability and Water Absorption Measurements. The wettability of the electrospun nanofiber membranes was performed27 and assessed34 by a goniometer.26 Water absorption was measured and evaluated.35 The percentage weight increase from water uptake at predefined time intervals, wf, can be calculated as follows %wf =

w1 − w0 × 100 w0

(1)

where w0 is the dried sample weight before soaking water treatment and w1 is the weight of the treated sample after excess water elimination. 2.6. Cytotoxicity Assay. The cytotoxicity study of CCD-966SK human skin fibroblast (FB) was grown in culture medium at 37 °C and 5% CO2.26 The cytotoxicity of electrospun nanofiber samples against CCD-966SK was assessed by MTT analysis. The 2 × 104 CCD-966SK cells, were seeded into each well of a 96-well plate. The cells were obtained at 90% confluence and then incubated with various concentrations of electrospun nanofiber samples for 1, 3, and 7 days. The cells with no treatment are acted as a positive control. The 50 μL MTT was added into each well plate for 4 h. Then, the 100 μL DMSO was added to dissolve the formazan completely after supernatant was removed. The solution absorbance was measured at 570 nm with a microplate reader (BioTek Instruments, Winooski, VT). The survival rate (%) is expressed as (viable cells of treatment/ viable cells of control group) × 100%.

%Wloss =

W0 − Wt × 100 W0

(2)

2.9. Statistical Analysis. Unless otherwise indicated, all experiments were duplicated at least five times. Quantitative data were processed by t-tests and SigmaPlot 10.0 software (Systat Software Inc., San Jose, CA). The number was expressed as mean ± standard error (SE). It is considered statistically significant if the p-value is less than 0.05. C


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peaks at 1717 and 1746 cm−1. The peak at 1717 cm−1 was assigned to −CO of N(COR)2; the peaks at 1746 cm−1 were attributed to the absorption of −CO in OCOR.42 Ester and imide functional groups formed via the reaction between −OH of PHA-g-AA and −NH of CSP when the two polymers were blended.43 The chemical reaction between PHA-g-AA and CSP is shown in the Scheme S1. On the other hand, Figure 1c shows that this reaction did not occur with PHA and CSP. 3.2. Morphology and Tensile Properties of Electrospun Nanofiber. Figure 2 shows typical SEM images of the fractured surfaces of PHA/CSP and PHA-g-AA/CSP electrospun nanofiber specimens. The SEM images of the PHA/CSP nanofiber (3 and 6 wt %, respectively) in Figure 2a,c revealed that the CSP was nonuniformly dispersed throughout the PHA matrix, leading to poor adhesion between the fiber and matrix, as well as poor wettability. EDS results of Figure S2 show that the material on the nodes of PHA/CSP is composed of C, O, and N. Because PHA/CSP is thicker and has more nodes than PHA-g-AA/CSP, as can be seen in Figure 2A,C, it is deduced that the CSP was nonuniformly dispersed throughout the PHA matrix. Work of Kaewboonruang et al. shows similar results.44 SEM images of PHA-g-AA/CSP (3 and 6 wt % in Figure 2b,d, respectively), showed better coverage and adhesion of CSP in the PHA-g-AA matrix and a more uniform structure throughout; the improved interfacial adhesion was attributed to the chemical reaction leading to improved bonding between PHA-g-AA and CSP. The stress−strain curves, tensile strength, and elongation at failure of PHA/CSP and PHA-g-AA/CSP electrospun nanofiber specimens are illustrated in Figure 3a−c, respectively, as a function of CSP content. The tensile strength at failure of pure PHA (1.64 MPa) electrospun nanofiber decreased slightly after grafting with AA (1.59 MPa), and the strength of the PHA/ CSP electrospun nanofiber specimens reduced obviously and successively with increasing CSP content (from 1.64 to 0.78 MPa). This behavior was owing to poor adhesion of CSP to the PHA matrix. This incompatibility effect on the tensile properties of the electrospun nanofiber was substantial. The tensile strength at failure of the PHA-g-AA/CSP specimens (Figure 3b) displayed a distinct behavior, as it decreased slightly with increasing CSP content. In addition, the tensile strength at failure of PHA-g-AA/CSP was roughly 0.3−0.7 MPa higher than that of PHA/CSP. It also indicates that lower elongation at failure values of the PHA/CSP specimens when compared to the PHA-g-AA/CSP specimens shown in Figure 3a,c. For PHA/CSP, the poor compatibility between the two phases is demonstrated in Figure 2a,c, as they are separated. The elongation at failure of the PHA-g-AA/CSP specimens, shown in Figure 3a,c, also decreased with increasing CSP concentration, but they showed greater elongation at failure values than those of the PHA/CSP specimens. However, these values were still lower compared to those of pure PHA-g-AA. Figure 3 indicates that the tensile strength and elongation at failure of nanofiber specimens were improved with the grafting reaction of PHA-g-AA and CSP. 3.3. Surface Hydrophilicity and Water Absorption Measurement. The hydrophilic properties of PHA, PHA-gAA, and its composite electrospun nanofiber membranes were determined by water contact angle measurements, as shown in Figure 4. The contact angles of the pure PHA and PHA-g-AA electrospun nanofiber membrane surfaces were 116.43 and 114.92°, respectively (Figure 4a,b, respectively). With 3 and 6 wt % hydrophilic CSP, the contact angles of PHA/CSP

3. RESULTS AND DISCUSSION 3.1. Structural Analysis of Chitosan and Composite Electrospun Nanofibers. Solid-state 13C NMR spectra of CS from BSF pupa shell were measured to determine the structural makeup of the nanofibers; these spectra are demonstrated in Figure S1. Three peaks were observed corresponding to carbon atoms in the CS (C1: δ = 97.23; C1′: δ = 100.12; C2, C2′: δ = 51.21; C3, C3′: δ = 71.06; C4, C4′δ = 66.81; C5: δ = 75.02; C5′: δ = 69.26; C6, C6′: δ = 56.96; a: δ = 19.08; b: δ = 175.12 ppm). The spectrum of free CS was in good agreement with that previously reported by Gartner et al.38 Figure 1a,b shows the FTIR spectra of unmodified PHA and PHA-g-AA, respectively: the peak assignment of PHA is as

Figure 1. Fourier transform infrared (FTIR) spectra of (a) PHA, (b) PHA-g-AA, (c) PHA/CSP (3 wt %), (d) PHA-g-AA/CSP (3 wt %), and (e) CSP.

follows (cm−1): 3300−3700 (O−H stretching), 2980−2850 (C−H stretching of methyl and methine), 1746 (C−O stretching), 1453 (C−H bending of methyl), 1350−1390 (C−H bending of methine), and 1050−1250 (C−O stretching).39 The characteristic absorption peaks of PHA appeared in the spectra of both polymers, with two extra shoulders observed at 1710 and 3281 cm−1 in the PHA-g-AA spectrum. These features are characteristic of AA carboxyl groups. Similar results have been reported in the literature.40 The shoulders correspond to AAs in the modified polymer and, therefore, denote the grafting of AA onto PHA. FTIR spectra of PHA/CSP (3 wt %) and PHA-g-AA/CSP (3 wt %) are shown in Figure 1c,d: the peak assignment of CSP in Figure 1e is as follows (cm−1): 3300−3400 (O−H stretching overlapped with N−H stretching), 2850−2950 (C− H stretching), 1652 (amide I, secondary amide) and 1590 (nonacylated primary amide), 1350−1460 (C−H bending), and 1050−1200 (C−O stretching).41 The FTIR spectrum of the PHA-g-AA/CSP (3 wt %) membranes in Figure 1d shows D


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Figure 2. Distribution and wetting of various electrospun nanofibers are shown in scanning electron microscopy (SEM) images as (a) PHA/CSP (3 wt %), (b) PHA-g-AA (3 wt %), (c) PHA/CSP (6 wt %), and (d) PHA-g-AA/CSP (6 wt %).

viability. After 3 days, the cell survival percentages of PHA/ CSP (6.0 wt %) and PHA-g-AA/CSP (6.0 wt %) samples were 176.29 ± 6.33 and 168.44 ± 6.28%, respectively. The aforementioned phenomenon may result from the following two conditions. First, after the electrospinning nanofiber fabrication process, PHA/CSP and PHA-g-AA/ CSP samples had a higher specific surface area, leading to a larger amount of cell adhesion.45 Second, the amino groups of CSP generate an interaction, increasing cell viability.46 Figure 5a shows that the PHA/CSP sample had higher cell survival than the PHA-g-AA/CSP sample, perhaps because the AAgrafted PHA shields the CSP, resulting in loss of contact of CSP and CCD-966SK cells. Hence, the cell viability of the PHA-g-AA/CSP sample was lower. Figure 5b shows that the cell growth dispersion conditions in PHA, PHA/CSP, and PHA-g-AA/CSP nanofiber membranes were observed through an inverted microscope. The cell growth layers of CCD-966SK cells in the PHA and PHA-gAA nanofiber membranes without CSP were relatively sparse, likely because the PHA and PHA-g-AA nanofiber membranes had higher hydrophobicity, and the cell proliferation was limited.34,47 However, the cell growth was dense in PHA/CSP and PHA-g-AA/CSP nanofiber membranes, as the addition of CSP improves the surface structure hydrophilicity, which is favorable for cell proliferation. To sum up the results, PHA/ CSP and PHA-g-AA/CSP nanofiber membranes provided excellent cell-growing environments and proliferation effects for CCD-966SK cells. 3.5. Antibacterial Activity and Biodegradation Behavior Test. E. coli (Gram-negative) and S. aureus (Grampositive) are standard bacteria of common biological indices for pollution and frequently used to determine the inhibitory activity of materials.48,49 Figure 6a shows that PHA and PHAg-AA nanofiber membranes had no antibacterial effect of E. coli

electrospun nanofiber membranes decreased to 109.84 and 105.41°, respectively. This decrease in contact angle was due to the existence of hydrophilic matters. Moreover, comparing the contact angles of PHA/CSP and PHA-g-AA/CSP electrospun nanofiber membranes with the same wt % of CSP, the contact angle of PHA/CSP was lower by ca. 1.88° at 3 wt % and 1.85° at 6 wt % than that of PHA-g-AA/CSP. The hydrophilic polar groups of CSP are effectively eliminated by bonding of the PHA-g-AA with CSP. A comparison of the water absorption rate of PHA/CSP and PHA-g-AA/CSP electrospun nanofiber membranes after various immersion times is shown in Figure S3. It shows that the water absorption rates of the PHA/CSP and PHA-g-AA/ CSP electrospun nanofiber membranes are in positive relationship with CSP content and immersion time. Water was easily absorbed because of the hydrophilic property of CSP. The more biodegradation is with higher water absorption, which is beneficial for potential application. In addition, the PHA-g-AA/CSP membranes had lower water absorption than the PHA/CSP membranes under the same CSP content and immersion time. The contact angle test results demonstrated that PHA-g-AA/CSP was more hydrophobic. The hydrophobicity of CSP disturbs permeation of water molecules. Hence, PHA-g-AA/CSP showed more water resistance. 3.4. Cell Proliferation and Cell Dispersion Assessment. The cell viability is shown in Figure 5 for day 1 after CCD-966SK cells co-culturing with PHA/CSP and PHA-gAA/CSP nanofiber membranes. There was no significant difference between the control group and various composite samples in cell viability. However, the cell viabilities in PHA and PHA-g-AA without CSP were slightly lower than those of the control group on days 2 and 3; in contrast, the PHA/CSP and PHA-g-AA/CSP samples showed a slight increase in cell E


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Figure 4. Contact angle profile for (a) PHA, (b) PHA-g-AA, (c, e) PHA/CSP (3 and 6 wt %, respectively), and (d, f) PHA-g-AA/CSP (3 and 6 wt %, respectively) electrospun nanofibers.

control effect. Additionally, the PHA/CSP and PHA-g-AA/ CSP nanofiber membranes showed reinforced bacterial inhibition zones for E. coli (Gram-negative bacteria), but less in S. aureus (Gram-positive bacteria) in Figure 6. The cell wall of Gram-positive bacteria is stronger and thicker than that of Gram-negative bacteria. The more affected the cell wall, the greater is the inhibition.52 Figure S4 shows the SEM morphology and decomposition rates of the PHA, PHA/CSP, and PHA-g-AA/CSP nanofiber membrane samples after the soil burial test. The decomposition rates increased as the burial duration increased. Figure S4a−f shows the morphology before burial, in which the morphology was complete and uncracked. After 9 and 18 days in the soil, the decomposition and hole size of the nanofiber membranes became markedly increased, in the order of PHA/ CSP (3 wt %) > PHA-g-AA/CSP (3 wt %) > PHA. Figure S4j shows the percent weight change over time for PHA/CSP and PHA-g-AA/CSP nanofiber membranes buried in soil. The PHA/CSP and PHA-g-AA/CSP nanofiber membranes show sharp increase of biodegradation rates before day 9; after day 9, the biodegradation rate gradually increased. In this case, the CSP constituents have likely been degraded and thus lost. Without CSP, the nanofiber membranes lose weight more slowly. Our results also showed that the biodegradation rate of PHA/CSP was higher than that of PHA-g-AA/CSP. A covalent bond formation between PHA-g-AA and CSP resistant to biodegradation in rational explanation. The more tight covering of CSP by PHA-g-AA can be more preventive for PHA-g-AA/CSP reacted with water and microorganisms in the soil environment.

Figure 3. Tensile properties of PHA/CSP and PHA-g-AA/CSP electrospun nanofibers: (a) representative stress−strain curves, (b) tensile strength, and (c) elongation at failure.

and S. aureus due to no bacterial inhibition zone formation. However, in the microbial control test for E. coli and S. aureus cells in PHA/CSP (3 wt %) and PHA-g-AA/CSP (3 wt %) nanofiber membranes, there were 0.50/0.42 and 0.44/0.36 cm bacterial inhibition zones, respectively. For PHA/CSP (6 wt %) and PHA-g-AA/CSP (6 wt %) nanofiber membranes, the bacterial inhibition zones were 0.60/0.53 and 0.56/0.48 cm, respectively. An electrostatic interaction between the positively charged CSP and negatively charged surface of the microorganism is possibly rational for bacteria inhibition. When the penetrability of the cell membrane is changed or compromised, the intracellular components could be lost or the nutrient delivery be obstructed, leading to the microorganism death.50,51 Therefore, higher CSP content in PHA/CSP and PHA-g-AA/CSP nanofiber membranes enhances the microbial F


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the structural, antibacterial, and biological properties of electrospun nanofibers made from PHA/CSP and PHA-gAA/CSP were investigated. FTIR analyses revealed condensation reactions between CSP and AA groups during PHA-g-AA formation. The morphology of the PHA-g-AA/CSP nanofiber was uniform, with good adhesion between the CSP and PHAg-AA matrix. Tensile properties showed that the improved adhesion between CSP and PHA-g-AA enhanced the tensile strength and elongation at failure. The water resistance of PHA-g-AA/CSP was higher than that of PHA/CSP. Moreover, increasing the CSP content in the PHA/CSP and PHA-g-AA/ CSP nanofibers enhanced cell proliferation. The antibacterial activity was assessed using E. coli and S. aureus bacteria; a strong inhibition effect was observed with both the PHA/CSP and PHA-g-AA/CSP electrospun nanofibers. Above all, this study offers the means to manufacture PHA/CSP and PHA-gAA/CSP electrospun nanofiber for future development of tissue engineering and bioprotective materials and that of air filtration membranes having promising antibacterial properties and cytocompatibility.

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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.8b16606.

Figure 5. (a) Cell viability of CCD-966SK normal fibroblasts (FBs) seeded on PHA/CSP and PHA-g-AA/SSS composite membranes. The cell viability was quantified using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolelium bromide (MTT) assay. (b) Inverted microscope images of CCD-966SK cells on membranes after 72 h of culture. All data are expressed as mean ± standard error (SE) of at least three separate experiments. Statistical analyses were performed using t-tests, with significant differences (*) determined at the p < 0.05 level for each treatment vs the control group.

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Additional structural analysis of the solid-state 13C NMR spectra of CS from BSF pupa shell, as well as additional information on the water absorption and biodegradable properties of PHA/CSP and PHA-g-AA/CSP electrospun nanofiber membranes (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +886-7-6077685. Fax: +886-7-6077788. Notes

The authors declare no competing financial interest.

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4. CONCLUSIONS To our knowledge, this is the first report of CSP manufactured from BSF pupa shell being combined with PHA to create an antibacterial nanofiber via electrospinning. The fabrication and

ACKNOWLEDGMENTS The author acknowledges the Ministry of Science and Technology (Taipei City, Taiwan, R.O.C.) and Green

Figure 6. Exposure time course of inhibition zones of E. coli and S. aureus cells during exposure to PHA or PHA-g-AA and its composite electrospun nanofiber membrane surfaces. 1: PHA, 2: PHA/CSP (3 wt %), 3: PHA/CSP (6 wt %), 4: PHA-g-AA, 5: PHA-g-AA (3 wt %), and 6: PHA-g-AA (6 wt %). G


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Engineering Technology Research (Kao Yuan University) for financial support (MOST-106-2622-E-244-002-CC3) and Black Soldier Fly’s.

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Bio-based Electrospun Nanofiber of Polyhydroxyalkanoate Modified

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