Fabrication and Characterization of Composite Meshes Loaded with

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24609−24617

Fabrication and Characterization of Composite Meshes Loaded with Antimicrobial Peptides Pengbi Liu,†,‡ Kun Fu,‡,§ Xiaomei Zeng,‡ Nanliang Chen,*,† and Xuejun Wen*,‡,∥ †

College of Textiles, Donghua University, Shanghai 201620, P. R. China Department of Chemical and Life Science Engineering, School of Engineering, Virginia Commonwealth University, Richmond, Virginia 23284, United States § Department of Stomatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P. R. China ∥ Beijing Ditan Hospital, Capital Medical University, Beijing 100015, P. R. China Downloaded via BUFFALO STATE on July 31, 2019 at 05:02:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Biomaterials-centered infection or implant-associated infection plays critical roles in all areas of medicine with implantable devices. The widespread over use of antibiotics has caused severe bacterial resistance and even super bugs. Therefore, the development of anti-infection implantable devices with non-antibiotic-based new antimicrobial agents is indeed a priority for all of us. In this study, antimicrobial composite meshes were fabricated with broad-spectrum antimicrobial peptides (AMPs). Macroporous polypropylene meshes with poly-caprolactone electrospun nanosheets were utilized as a substrate to load AMPs and gellan gum presented as a media to gel with AMPs. Different amounts of AMPs were loaded onto gellan gum to determine the appropriate dose. The surface morphologies, Fourier-transform infrared spectroscopy spectra, in vitro release profiles, mechanical performances, in vitro antimicrobial properties, and cytocompatibility of composite scaffolds were evaluated. Results showed that AMPs were loaded into the meshes successfully, the in vitro release of AMPs in phosphatebuffered saline was prolonged, and less than 60% peptides were released in 10 days. The mechanical properties of composite meshes were also within the scope of several commercial surgical meshes. Composite meshes with the AMP loading amount of over 3 mg/cm2 showed inhibition against both Gram-negative and Gram-positive bacteria effectively, while they presented no toxicity to mammalian cells even at a loading amount of 10 mg/cm2. These results demonstrate a new simple and practicable method to offer antimicrobial properties to medical devices for hernia repair. KEYWORDS: implant associated infection, antimicrobial peptides, antimicrobial composite mesh, cytocompatibility, slow release

1. INTRODUCTION Biomaterials-centered infection or implant-associated infection is a serious surgical complication and it is reported that about 1−15% hernia repair surgeries result in infectious complications.1−3 Over 1 million Americans are affected by hernia each year4 and nearly 14% of the population develops a hernia in the UK every year,5 and therefore, the percentage of infection relates to large numbers of cases every year. Furthermore, infection in hernia repair is a surgical disaster to patients, with severe pain and significant costs.6 While bacterial infection is the predominant cause of these infections, the most common bacteria involved in infections are Staphylococcus aureus (SA), Staphylococcus epidermidis, Escherichia coli (EC), Pseudomonas aeruginosa (PA), Proteus mirabilis, and Streptococcus pyogenes.7−10 There are many meshes with antibacterial efficacy appeared in the market, and many new meshes with anti-infection property are investigated. Researchers observed that meshes © 2019 American Chemical Society

knitted with monofilament presented a higher rate of bacterial clearance than with multifilament or composite meshes.11 ́ Diaz-Godoy et al. also suggested that meshes with large porosity (3.6 mm × 2.8 mm) and light weight (48 g/m2) resulted in no clinical infection or positive microbiologic cultures.12 Thus, mesh textile parameters turn out to be a crucial factor in the mesh design for antibacterial purpose, and large-pore, light-weight mesh knitted with monofilament is favored.13 Another effective way to treat the infection of surgical implants is to prevent the colonization of microorganisms by utilizing meshes with antibacterial performance.14 Antibiotics, such as gentamicin,15,16 ampicillin,17 and ciprofloxacin, are mostly used in these research studies.8 However, it is known Received: April 25, 2019 Accepted: June 14, 2019 Published: June 14, 2019 24609

DOI: 10.1021/acsami.9b07246 ACS Appl. Mater. Interfaces 2019, 11, 24609−24617

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ACS Applied Materials & Interfaces Scheme 1. Schematic Representation of Fabrication of Antibacterial Composite Meshes

cytocompatibility and is biodegradable with no toxicity.33−35 Both GG and PCL were approved by the United States Food and Drug Administration as biomaterials.36,37 The composite meshes were subsequently characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), AMPs in vitro control release study, uniaxial tensile strength test, antibacterial studies, and in vitro cytotoxicity tests. The results proved that this is a simple and practicable method to produce biocompatible composite meshes with antibacterial properties.

that the use of aminoglycosides will increase the risk of bacterial resistance.18,19 Another antibiotic investigated widely is vancomycin,9,20,21 which is mainly used in treating methicillin-resistant SA (MRSA) infections and showed complete bacterial eradication of SA and MRSA.20,22,23 However, vancomycin has little efficacy on Gram-negative bacteria such as EC or PA.24 Heavy metal-based antimicrobials of silver or gold coating or nanoparticles are frequently used in the medical devices, too. Muzio et al. fabricated a composite mesh by coating polypropylene (PP) prostheses with thin silica and silver layer. It turned out that this composite mesh afforded cell growth and had antibacterial activity.25 However, the silver content in human body is very limited and the American Conference of Governmental Industrial Hygienists (ACGIH) has established separate threshold limit values for metallic silver (0.1 mg/m3) and soluble compounds of silver (0.01 mg/ m3), while the recommended exposure limit set by the National Institute for Occupational Safety and Health is 0.01 mg/m3 for all forms of silver.26 Scoccianti et al. studied the levels of silver ions in body fluids of patients implanted with silver coating medical devices and found the mean levels of silver ranging from 0.41 to 5.33 μg/L in blood and from 0.28 to 0.86 μg/L in urine at 24 h to 36 months after surgery.27 This value (5.33 μg/L) is over 500 times higher than the ACGIH limitation required (0.01 mg/m3). Hence, the use of heavy metals in implantable medical devices is not a permanent solution. Some government agencies have banned heavy metals in medical devices. Therefore, meshes with antimicrobials which can be used in the implantable medical devices with broad spectrum antimicrobial property and excellent biocompatibility are urgently needed. This study describes a new composite mesh with antibacterial property by loading antimicrobial peptides (AMPs) in gellan gum (GG) and a large-pore mesh knitted with PP monofilament used as a matrix. AMPs are deemed to be a new promising class of antibacterial agents because of their broad spectrum and rapid bactericidal performances.28,29 The AMP we used in this study is a synthetic cationic peptide produced and purified in our lab. In order to load the AMP into meshes effectively, electrospinning poly(ε-caprolactone) (PCL) film and GG solution were selected. PCL is a biodegradable and biocompatible polymer which has been applied in many fields of medical uses,30,31 and the PCL nanofiber layer was used to improve the adhesion of GG and AMP to meshes in this study. While in a previous report, a natural polysaccharide gel, guar gum, was proved to be an alternative, practical and safe delivery system for nisin.32 Herein, GG is an anionic polysaccharide with high

2. MATERIALS AND METHODS 2.1. Materials. The large-pore light-weight PP mesh was knitted and heat-set as previously described,38 and mesh with round pore (R) was used in this study. PCL (Mn = 80 000), GG (Phytagel), dimethyl sulfoxide (DMSO), and glutaraldehyde were supplied by SigmaAldrich (St. Louis, MO, USA). Hexafluoro-2-propanol (HFIP, Oakwood, West Columbia, SC, USA) was used as a solvent to dissolve PCL. Phosphate buffered saline (PBS) tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved and filtered following the instructions. AMP PEP-1 (RRRGRRRGPPGRRRGRRR) was synthesized and purified in our laboratory as described in the supplementary. Microorganisms SA (CGMCC 1.2465, Gram positive) and EC (ATCC 25922, Gram negative) were obtained from the Department of Microbiology and Immunology at Virginia Commonwealth University. LB-broth and LB-agar were purchased from Fisher Scientific, USA. Human dermal fibroblasts (HDFs) were purchased from ScienCell, catalog #2310. Hank’s balanced salt solution (HBSS) was obtained from Gibco, USA. HDFs cell culture medium was consisted of 10% fetal bovine serum, 1% penicillin/streptomycin [both from SigmaAldrich (St. Louis, MO, USA)], and 89% DMEM/F12 + GlutaMAX1 (Gibco, USA). alamarBlue reagent was obtained from BIO-RAD, USA. LIVE/DEAD Reduced Biohazard Cell Viability Kit was provided by Invitrogen (Carlsbad, CA, USA). 2.2. Electrospinning of PCL Film. PCL (1 g) was dissolved in 10 mL of HFIP and magnetically stirred at room temperature overnight to obtain homogenous solution. PP mesh after heat-setting was cut and placed on aluminum foil to set as a collector. PCL solution was pumped into a 3 mL syringe fitted with a 23 G blunt-tipped needle. To obtain a bead-free nanofiber, voltage to the needle was adjusted as 13 kV and the flow rate of PCL solution was 25 μL/min. The tip-tocollector distance was set at 10 cm. PCL nanofiber sheet was yielded after 30 min electrospinning. Fabricated sheets were vacuumed for 7 days to eliminate the residues of solvent. 2.3. Fabrication of Composite Meshes. GG (0.8 g) powder was dissolved in 100 mL of deionized water and autoclaved for 30 min at 120 °C to obtain sterile GG solution. The PP and PCL composite sheets were sterilized under ultraviolet light for 1 h on both sides, and GG solution was used as a binding agent to reinforce the adhesion between PP and PCL substrates. 24610


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ACS Applied Materials & Interfaces Then, GG solution was spread evenly onto the PCL film, and a certain amount of PEP-1 in water solution was sprayed onto the GG and PCL film. All of these were processed under sterile environment and the meshes were dried before aseptic packaging. According to the amount of PEP-1 loaded per square centimeter of mesh, samples were recorded as CM-1, CM-3, CM-5, and CM-10, respectively. Composite meshes without PEP-1 were set as negative control (NC). The total manufacturing process is illustrated in Scheme 1. 2.4. Scanning Electron Microscopy. The morphology of the composite meshes was examined by a field-emission scanning electron microscopy (FE-SEM) system (Hitachi SU-70, Japan). Samples were coated with a mixture of palladium and gold (Pd/Au = 40:60). 2.5. FTIR Spectroscopy. Composite meshes, PEP-1, PP mesh, and the composite mesh without peptide were characterized by a FTIR spectroscopy system (Nicolet iS10, Thermo Scientific). All spectra were recorded in the frequency region 500−4000 cm−1. 2.6. In Vitro Release of AMPs. The in vitro release characteristic of peptides was determined following the references with a little modification.37,39−42 Briefly, the composite meshes CM-1 (10 × 10 mm) were incubated in 1 mL of PBS on a shaker at a speed of 100 rpm, 37 °C. Then, 100 μl solution was collected at predetermined time (2, 4, 6, and 24 h and then every day) for 10 days. After each collection, 100 μL of fresh PBS was added to the incubation solution. In order to examine the AMP concentration more accurately, two arginine residues (R1, R2) in the PEP-1 peptide sequence were replaced by tryptophan residues, which shows strong UV−vis signal at 280 nm.39,43 Peptide concentrations were calculated using a standard curve produced by the absorption value of different concentrations of peptides at 280 nm by NanoDrop 2000 Spectrophotometers (Thermo Scientific, USA). This test was carried out three times and 10 absorbance values were collected at each time point. 2.7. Mechanical Properties. Three commercially available surgical meshes, PROLENE, PROLENE Soft, and PROCEED Surgical mesh from Ethicon, Inc (Somerville, NJ), were used to evaluate the composite meshes we fabricated in this study. PP mesh R was also used as control. All samples were cut into 10 × 45 mm pieces in the transverse (weft) or longitudinal (warp) direction for the uniaxial tensile strength test. Samples were gripped to a Shimadzu EZ Graph tensile tester (Nakagyoku, Kyoto, Japan) with an initial gauge length of 15 mm and stretched at a speed of 25 mm/min. Trapezium 2.32 software was utilized for max stress and elastic modulus data acquisition (n = 5). 2.8. Antibacterial Tests. Antibacterial properties of the composite meshes were measured according to an agar diffusion method with slight modification.41,43−45 SA and EC were cultured separately in LB-broth for two passages before tests and samples were cut into 1 × 1 cm pieces (four pieces for one type of bacteria) under sterile conditions. Bacteria density was adjusted to 1−5 × 106 CFU/ mL and 100 μL of bacteria solution was spread evenly onto LB-agar plate. Then, samples were put on the agar plate carefully and incubated at 37 °C for 48 h. The zone of inhibition was determined by measuring the distance between the mesh edge to the inhibition zone edge. Scaffolds without peptides were utilized as control. 2.9. In Vitro Cytotoxicity Evaluation. In vitro cell culture was performed with HDFs on the samples according to the International Standards (ISO 10993-5).46 Five 1 × 1 cm pieces of each sample were cut under sterile condition and placed in HDFs cell culture medium for 24 h at 37 °C in a 5% CO2 incubator to obtain leaching liquid. The ratio of the surface of the samples to the volume of leaching liquid was 1 cm2/mL. HDFs were collected and counted when 90% confluency reached. DMSO (10%) in cell culture medium was set as positive control (PC). The leaching liquid of composite meshes without peptide was used as NC and cell culture medium was also used as control. HDFs were seeded in a 48-well cell culture plate (Cellstar, Greiner Bio-One) with a density of 1.5 × 104 cells/well and 200 μL medium per well. After 24 h culture, medium was replaced by equivalent sample leaching liquid or control medium and incubated for another 24 h. To evaluate cell viability of each group, 20 μL alamarBlue was added to each well and incubated for 4 h before fluorescence reading by a micro plate reader (Synergy H1 Hybrid

reader, BioTek, USA) at 540 nm excitation wavelength and 570 nm emission wavelength. AlamarBlue (20 μL) in 200 μL cell culture medium without cells was set as blank control. To observe the cell morphology of each group, HDFs in each well were processed with the LIVE/DEAD Reduced Biohazard Cell Viability Kit following the instructions from Invitrogen. Briefly, the cells were washed with HBSS and then covered with a mixture of live/ dead reagents for 15 min at room temperature. Finally, 4% freshly prepared glutaraldehyde in HBSS was used to fix cells for 1 h. The fluorescence images were observed under a confocal laser scanning microscope (Olympus IX81, Japan). 2.10. Statistical Analysis. A one-way analysis of variance (ANOVA) was performed using Minitab software (version 17), followed by Tukey’s post-test. The statistical significance was set at the p < 0.05 level, and all results were reported as the mean ± standard error of the mean.

3. RESULTS 3.1. Composite Mesh Morphology. Figure 1 shows FESEM images of electrospun PCL and AMP-loaded composite

Figure 1. SEM images of (a) electrospun PCL and (b−d) composite mesh. (b) Surface of composite mesh; (c) interface of PP mesh and GG incorporated with electrospinning PCL; and (d) edge of composite mesh.

meshes. There are no obvious beads on the PCL fibers and the average diameter of them is 569.83 ± 46.89 nm (Figure 1a). All the PCL fibers were covered by GG and AMP cross-linked gel (Figure 2b). Also, the GG between PP filament and PCL fibers was used to increase the cohesive force between PP and PCL. 3.2. FTIR Spectroscopy. FTIR spectra of control and test samples are shown in Figure 2. Absorbance bands at 1374, 1454, 2914, and 2958 cm−1 represent the −CH2 or −CH3 groups on PP mesh.44 The broad peak near 3352 cm−1 on the NC spectrum indicates stretching vibrations of hydrogenbonded OH groups.47,48 Also, absorbance bands at 1723 cm−1 are the characteristic peak of PCL.49,50 The peaks appearing at 1607 cm−1 are due to the presence of carboxylate groups.47 The absorbance bands at 3182 and 3282 cm−1 on the PEP-1 spectrum attributed due to the NH stretching vibration (ν NH) of amide groups. Peaks at 1658, 1536, and 1248 cm−1 indicate amide I (CO stretching), amide II (CN stretching, NH bending), and amide III (CN stretching, NH bending) bands, respectively.51 While to the four test samples, amide I, amide II, amide III, and (ν NH) absorbance bands (at 3182 24611


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illustrated in Figure 4. The CM showed no significant difference of max stress with PP mesh at either direction. In addition, the max stress of CM in both directions was between that of PROCEED and PROLENE. Both the max stress and the elastic modulus of CM at orthogonal directions had no statistically significant difference with PROLENE Soft, while the addition of PCL film and GG peptide cross-linked gel increased the elastic modulus of the weft direction. Moreover, it seems that CM and PROLENE Soft represent less anisotropic than PROCEED and PROLENE. 3.5. In Vitro Antibacterial Tests. To evaluate the efficacy of composite meshes we fabricated in this study to clear bacteria in vitro, EC and SA were used and the outcomes are shown in Figure 5. Obviously, peptides can diffuse from the meshes and the zone of inhibition increased along with the amount of AMP in the meshes. There was a small circle (3.42 ± 0.67 mm) around CM-1 to EC and no inhibition zone was observed to SA, and CM-3 showed inhibitory to SA of 1.01 ± 0.43 mm, while the inhibition zone of CM-10 was 8.5 ± 1.3 mm to EC and 4.41 ± 0.72 mm to SA. Additionally, composite meshes were found more effective to EC than SA, which is consistent with the antibacterial properties of PEP-1 (shown in the supplementary). 3.6. In Vitro Cytotoxicity Evaluation. Figure 6 shows the in vitro cytotoxicity test results of each sample. Figure 6a demonstrated that all the cells of composite meshes groups were alive and exhibited normal morphology as with the control group. Moreover, the mean fluorescence value of sample groups (except CM-1) was slightly higher than that of the control group, so was the NC group (Figure 6b), suggesting that the composite scaffolds improved cell viability to some degree. Also, cells incubated in CM-5 leaching liquid displayed the highest fluorescence value. Obviously, these four composite meshes all had no cytotoxicity to HDFs and exhibited no significant difference with the control group.

Figure 2. FTIR spectra of test samples, AMP PEP-1, NC (GG + PCL + PP), and PP samples.

cm−1) can be observed at the same wavenumbers with PEP-1, which confirmed the presence of PEP-1 on the composite meshes. The broad peaks at 3346 cm−1 on the test samples are due to the presence of −OH groups (peaks at 3352 cm−1) and NH stretching vibration absorbance bands at 3282 cm−1. As to the four composite meshes, the corresponding absorbance bands are slightly different with each other. Peaks of CM-10 are a little stronger than CM-5, followed by CM-3 and CM-1. 3.3. In Vitro Release of AMPs. The in vitro release characteristic of AMPs from composite meshes (CM-1) is illustrated in Figure 3. About 15.1 % AMP was released for the

4. DISCUSSION This study was conducted to fabricate new composite meshes with antibacterial property and excellent biocompatibility for hernia repair, as well as to provide a new strategy for AMPs application and antibacterial meshes design. Electrospinning nanofibers or microfibers are widely used in tissue engineering because of their special architecture. Plencner et al. suggested that resorbable nanofiber layers in direct contact with hernia site presented better mechanical properties of the tissue compared to PP mesh alone because the nanofiber layers would promote collagen deposition and remodeling.52 However, most mechanical properties of electrospun layers alone are not strong enough for soft tissue repair.53 While PCL is a biomaterial that will gradually degrade in months following implantation, which will eventually decrease the amount of foreign bodies.54,55 Hence, large-pore and light-weight PP mesh is utilized with electrospun PCL sheets in this research. The biocompatible GG solution was used as resorbable adhesive between them to prevent delamination. Natural AMPs are secreted by innate immune system of various organisms as a defense mechanism against competing pathogenic microbes.56 Most AMPs are short in length, generally 10−50 amino acids, and they normally target the microbials by interacting with the bacterial membranes.57,58 What’s more, because of the difference of bacterial and mammalian cells membranes, AMPs show selective toxicity to pathogen but not host cells.59

Figure 3. AMPs accumulative release in vitro from CM-1 in 10 days.

first 6 h and 15.64% AMP was released from CM-1 for 24 h culture, suggesting that the AMP showed a burst release at the first 6 h due to the loosely bound AMPs. From day 3 to day 5, the AMP gradually and linearly released from CM-1 and accumulative release reached 27.64% at day 5. It had a burst release from day 6 to day 7, during which almost 30% of the total AMPs was released. On day 10, the accumulative release reached nearly 60%, demonstrating that peptide in this scaffold could be sustainedly released over 10 days. 3.4. Uniaxial Tensile Strength Tests. The max stress and elastic modulus in warp and weft directions of each sample are 24612


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Figure 4. Uniaxial tensile strength test. (a) Max stress at break, (b) elastic modulus of each sample. CM represents CM-1. Different letters, if any, represent statistically significant differences.

To date, AMPs have been applied in many fields together with plenty of materials. Poelstra et al. investigated the outcomes of PP mesh with carboxymethylcellulose (cmc) gel containing 10 mg/cm2 commercial human pooled polyclonal human immunoglobulin G (IgG) in a mice-infection model and found that mice survival from PA infection was enhanced.60 Cao et al. studied the composites that bound antibacterial peptides G(IIKK)4I−NH2 onto graphene oxide and found that the composites showed long-term antibacterial property for up to 10 days.39 Thus, AMPs are promising agents in hernia repair applications to provide long lasting antibacterial property and reduce the rate of mesh infections. The AMPs PEP-1 in this research are cationic short peptides and have efficacy on Gram-positive, Gram-negative, and fungal microorganisms. Moreover, they present no toxicity to HDFs in vitro at the bactericidal concentration. To load this AMP to PP mesh effectively, GG and PCL electrospinning sheets were used. GG will become gel by enzymatic linkages, by adding salt, by treating with heat, or by applying pressure.61 In this study, GG gelled with AMPs instantly they met each other. Because GG is a negatively charged polysaccharide and the AMP here is cationic, they cross-link quickly through electrostatic and hydrogen-bonding interactions between the carboxyl groups of GG polymer chains, in random coil conformation, and the amine groups and guanidino groups of peptides. Also, the illustration of this process is presented as Scheme 2.

Figure 5. Composite meshes antibacterial test results after 48 h: (a) EC and (b) SA, and (c) inhibition zone of each sample against SA and EC.

Figure 6. In vitro cytotoxicity test of composite meshes to HDFs for 24 h. (a) Cell morphology observed by laser scanning confocal microscopy from the live/dead viability test. Scale bars represent 100 μm. (b) Fluorescence value of each sample from alamarBlue test. Different letters, if any, represent statistically significant differences. 24613


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very complex organic structure. It has many layers with different properties. Many reports figured out that human linea alba and anterior and posterior rectus sheaths have heterogeneous, nonlinear behavior.65,66 Uniaxial stress of linear alba and rectus sheath differed in the transverse and longitudinal directions. The linear alba transverse stress was about 9.2 MPa and longitudinal stress was around 4.3 MPa, which means that the transverse stress of linear alba is 2−3 times larger than the longitudinal stress. Also, the posterior rectus sheath stress and anterior rectus sheath also showed the same phenomena.66,67 Nevertheless, the composite layers of human abdominal wall present different characteristics. The average longitudinal and transverse strains exhibit almost the same value.68 Therefore, as described before,38 different PP meshes with appropriate textile structures could be used as the matrix to simulate the properties of different sites or layers of human abdominal wall. There is another problem that should be considered, which is the effective amount of antimicrobials loaded in the meshes. Too many antimicrobials loaded may result in high cost, bacterial resistance, or even toxicity to host, while too much less antibacterial compound may result in failure to resist bacteria.2 How to load the antimicrobials is also very crucial because the antibacterial compound must be able to diffuse from materials.69 To determine the appropriate amount of peptides loaded in the mesh, composite meshes with four different concentrations were made, and the in vitro antibacterial properties and cytotoxicity characteristics were tested and illustrated. Obviously, CM-1 contains too much less peptides to inhibit SA and all these four samples showed no toxicity to HDFs, while CM-3, CM-5, and CM-10 all exhibited inhibition against EC and SA, proving that peptides could diffuse from scaffolds here. What’s more, the composite film added to PP mesh may improve cell proliferation because cells incubated in CM-5 leaching liquid presented the highest cell viability and all samples except CM-1 and PC exhibited higher fluorescence value than control group (Figure 6b). This may be due to the addition of GG and PEP-1. In a previous report, GG hydrogel was proved noncytotoxic and we found that the cell viability of GG group was also slightly higher than that of the tissue culture polystyrene control group.70 The differences were also not statistically significant, while PEP-1 at certain concentrations may also improve the cell proliferation, just like the cell adhesive RGD peptides.71 Therefore, the addition of GG and PEP-1 may play a role in the enhancement of cell proliferation, and this should be further investigated specifically. In conclusion, this simple method is practicable to fabricate antibacterial meshes and the AMP amount of 10 mg/cm2 is appropriate to resist SA and EC without harming mammalian cells. To inhibit SA effectively, at least 3 mg/cm2 PEP-1 should be loaded. This method could also be used in other fields that require antibacterial performances, while the inhibition tests to other microorganisms in vitro and animal infection model need to be done for further study. Also, AMPs could be replaced by any other cationic peptides with better antimicrobial properties or growth-promoting abilities and the amount of peptide loaded should be adjusted along with the properties of loaded agents.

Scheme 2. Schematic Illustration of Gelling Process between GG and AMPs

The FTIR is an effective and easy method to analysis substrates. In this study, the FTIR spectra confirmed the presence of PEP-1 in composite meshes through the specific absorbance bands. While in the in vitro release assay, the release profile relates to the interaction forces between GG and antimicrobial agents in solution. In other words, a strong interaction force will prevent the drugs from rapid release. Peptides PEP-1 released from CM-1 gradually because of the continuous formation and rupture of hydrogen bonds and electrostatic interactions between peptides and GG in PBS. On the other hand, GG may also release from the composite meshes with time, along with peptides. The forces between GG and PEP-1 were not broken in this situation. Some AMPs were also released along with the loss of GG because GG will degrade slightly in PBS for the first 28 days.62 As Yu et al. described,63 the ratio of surface area to mass of GG affects its degradation rate. Therefore, it could be the release of GG from composite meshes and the slight degradation of GG that caused the burst release of peptides from day 6 to day 7 in Figure 3, which is an issue that needs further study. The in vitro release of antimicrobials is evaluated in many research studies. Zhang et al. used the coaxial electrospinning method to load vancomycin into the core−shell nanofiber and it turned out that over 50% vancomycin was released for 6 h in the in vitro release test.64 While in Poelstra et al.’s study, about 85% of the loaded IgG was released from the cmc gel in PBS at the first 6 h and nearly 90% IgG is released by 9 h.60 However, only 15.1% PEP-1 was released for the first 6 h and only less than 60% AMPs was released from CM-1 for 10 days in this study, which clearly shows the excellent sustained release ability of the composite meshes fabricated through this method. In order to repair the hernia successfully, the mechanical property of meshes is a crucial factor. Composite meshes we fabricated here exhibited no significant difference with the commercial available product PROLENE Soft in the uniaxial tensile strength test (Figure 4), suggesting that their mechanical properties meet the requirements. Also, they almost maintained the same mechanical performance with the matrix PP mesh except a little increase in the elastic modulus of weft direction. We may figure out that the mechanical properties of these composite meshes depend mainly on the PP meshes used. The human abdominal wall is a 24614


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(5) Song, Z.; Ma, Y.; Xia, G.; Wang, Y.; Kapadia, W.; Sun, Z.; Wu, W.; Gu, H.; Cui, W.; Huang, X. In vitro and in vivo combined antibacterial effect of levofloxacin/silver co-loaded electrospun fibrous membranes. J. Mater. Chem. B 2017, 5, 7632−7643. (6) Falagas, M. E.; Kasiakou, S. K. Mesh-related infections after hernia repair surgery. Clin. Microbiol. Infect. 2010, 11, 3−8. (7) Cobb, W. S.; Harris, J. B.; Lokey, J. S.; McGill, E. S.; Klove, K. L. Incisional herniorrhaphy with intraperitoneal composite mesh: a report of 95 cases. Am. Surg. 2003, 69, 784−787. (8) Laurent, T.; Kacem, I.; Blanchemain, N.; Cazaux, F.; Neut, C.; Hildebrand, H. F.; Martel, B. Cyclodextrin and maltodextrin finishing of a polypropylene abdominal wall implant for the prolonged delivery of ciprofloxacin. Acta Biomater. 2011, 7, 3141−3149. (9) Fernandez-Gutierrez, M.; Olivares, E.; Pascual, G.; Bellon, J. M.; Román, J. S. Low-density polypropylene meshes coated with resorbable and biocompatible hydrophilic polymers as controlled release agents of antibiotics. Acta Biomater. 2013, 9, 6006−6018. (10) Guillaume, O.; Garric, X.; Lavigne, J.-P.; Van Den Berghe, H.; Coudane, J. Multilayer, degradable coating as a carrier for the sustained release of antibiotics: preparation and antimicrobial efficacy in vitro. J. Controlled Release 2012, 162, 492−501. (11) Engelsman, A. F.; van Dam, G. M.; van der Mei, H. C.; Busscher, H. J.; Ploeg, R. J. In vivo evaluation of bacterial infection involving morphologically different surgical meshes. Ann. Surg. 2010, 251, 133−137. (12) Díaz-Godoy, A.; García-Ureña, M. Á .; López-Monclús, J.; Ruiz, V. V.; Montes, D. M.; Agurto, N. E. Searching for the best polypropylene mesh to be used in bowel contamination. Hernia 2011, 15, 173−179. (13) Zhu, L.-M.; Schuster, P.; Klinge, U. Mesh implants: An overview of crucial mesh parameters. World J. Gastrointest. Surg. 2015, 7, 226−236. (14) Engelsman, A. F.; Krom, B. P. Antimicrobial effects of an NOreleasing poly(ethylene vinylacetate) coating on soft-tissue implants in vitro and in a murine model. Acta Biomater. 2009, 5, 1905−1910. (15) Junge, K.; Rosch, R.; Klinge, U.; Krones, C.; Klosterhalfen, B.; Mertens, P. R.; Lynen, P.; Kunz, D.; Preiß, A.; PeltrocheLlacsahuanga, H. Gentamicin supplementation of polyvinylidenfluoride mesh materials for infection prophylaxis. Biomaterials 2005, 26, 787−793. (16) Klink, C. D.; Binnebösel, M.; Lambertz, A.; Alizai, H. P.; Roeth, A.; Otto, J.; Klinge, U.; Neumann, U. P.; Junge, K. In vitro and in vivo characteristics of gentamicin-supplemented polyvinylidenfluoride mesh materials. J. Biomed. Mater. Res., Part A 2012, 100, 1195−1202. (17) Labay, C.; Canal, J. M.; Modic, M.; Cvelbar, U.; Quiles, M.; Armengol, M.; Arbos, M. A.; Gil, F. J.; Canal, C. Antibiotic-loaded polypropylene surgical meshes with suitable biological behaviour by plasma functionalization and polymerization. Biomaterials 2015, 71, 132−144. (18) Leclercq, R.; Courvalin, P. Intrinsic and unusual resistance to macrolide, lincosamide, and streptogramin antibiotics in bacteria. Antimicrob. Agents Chemother. 1991, 35, 1273−1276. (19) van der Waaij, D.; Nord, C. E. Development and persistence of multi-resistance to antibiotics in bacteria; an analysis and a new approach to this urgent problem. Int. J. Antimicrob. Agents 2000, 16, 191−197. (20) Blatnik, J. A.; Thatiparti, T. R.; Krpata, D. M.; Zuckerman, S. T.; Rosen, M. J.; von Recum, H. A. Infection prevention using affinity polymer-coated, synthetic meshes in a pig hernia model. J. Surg. Res. 2017, 219, 5−10. (21) Shukla, A.; Avadhany, S. N.; Fang, J. C.; Hammond, P. T. Tunable vancomycin releasing surfaces for biomedical applications. Small 2010, 6, 2392−2404. (22) Grafmiller, K. T.; Zuckerman, S. T.; Petro, C.; Liu, L.; von Recum, H. A.; Rosen, M. J.; Korley, J. N. Antibiotic-releasing microspheres prevent mesh infection in vivo. J. Surg. Res. 2016, 206, 41−47. (23) Harth, K. C.; Rosen, M. J.; Thatiparti, T. R.; Jacobs, M. R.; Halaweish, I.; Bajaksouzian, S.; Furlan, J.; von Recum, H. A.

5. CONCLUSIONS This study developed antibacterial composite meshes with a new method. A gel film made by loading antibacterial peptides we synthesized in our lab into GG solution was integrated with PCL electrospun sheets onto macroporous light-weight PP meshes. The results proved that the mechanical properties of the composite meshes were enough for hernia repair. They also exhibit inhibition efficacy against Gram-negative bacteria EC and Gram-positive bacteria SA at a AMPs loading amount of over 3 mg/cm2. For safety concern, an amount of 10 mg/cm2 is nontoxic. What’s more, this method enables peptides of PEP-1 to sustained release for over 10 days. The integration of AMPs and GG developed a new approach for effective therapeutics of bacterial-infected tissue repairs. Investigations of the antimicrobial studies of composite meshes on other bacteria or fungi and the animal infection model could be done furtherly.

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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.9b07246. Peptides synthesis; reverse-phase HPLC analytical and purification; and antimicrobial activities of the synthesized peptides (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (N.C.). *E-mail: [email protected] (X.W.). ORCID

Pengbi Liu: 0000-0002-1230-5034 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China, grant number 2016YFB0303300; the China Scholarship Council [grant numbers 201706630077], the international visiting program from Donghua University; and the Alice T. and William H. Goodwin Jr. Endowment. We would like to thank Prof. Dennis Ohman (Department of Microbiology and Immunology, Virginia Commonwealth University) for providing assistance with the antibacterial studies.

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REFERENCES

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