Antimicrobial properties and biocompatibility of electrospun poly-ε-caprolactone fibrous mats containing Gymnema sylvestre leaf extract

Materials Science & Engineering C 98 (2019) 503–514

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Antimicrobial properties and biocompatibility of electrospun poly-εcaprolactone fibrous mats containing Gymnema sylvestre leaf extract

T

Raghavendra Ramalingama,b,c, Chetna Dhandd, Chak Ming Leunge, Seow Theng Ongf, Sathesh Kumar Annamalaia, Mohammed Kamrudding, Navin Kumar Vermad,f, ⁎ ⁎ ⁎⁎ Seeram Ramakrishnac, , Rajamani Lakshminarayanand, , Kantha Deivi Arunachalama, a

Center for Environmental Nuclear Research, SRM Institute of Science and Technology, Kattankulathur Campus, Kancheepuram, Tamilnadu 603203, India Department of Physics and Nanotechnology, SRM Institute of Science and Technology, Kattankulathur Campus, Kancheepuram, Tamilnadu 603203, India c Center for Nanofibers and Nanotechnology, Department of Mechanical Engineering, Faculty of Engineering, 2 Engineering Drive 3, National University of Singapore, 117576, Singapore d Anti-Infectives Research Group, Singapore Eye Research Institute, The Academia, 20 College Road, Discovery Tower, 169856, Singapore e Department of Biomedical Engineering, National University of Singapore, 117581, Singapore f Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Experimental Medicine Building, 59 Nanyang Drive, 636921, Singapore g Materials Physics Division, Material Science Group, Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamilnadu 603102, India b

ARTICLE INFO

ABSTRACT

Keywords: Electrospinning Gymnema sylvestre leaf extracts Nanofiber Gymnemagenin Wound dressing

Wound care management presents one of the substantial and tenacious challenges to the healthcare systems worldwide. Microbial colonization and subsequent biofilm formation after injury have garnered much attention, as there is an appreciable correlation between biofilms formation and delayed healing in chronic wounds. Nanotechnology has emerged as a potential platform for the management of treating acute and chronic wounds. This study presents the utility of electrospun nanofiber mats containing a natural extract (Gymnema sylvestre) that averts biofilm formation but supports human dermal fibroblasts (hDFs) attachment. The scaffolds exhibited good wettability, enhanced mechanical properties and contact mediated inhibition of Gram-positive and Gramnegative bacteria. MTS viability assay and confocal imaging further confirmed that the natural extract loaded mats remained non-cytotoxic for hDFs. Overall these findings evidenced the suitability of the Gymnema sylvestre (GS) functionalized electrospun poly-ε-caprolactone (PCL) nanofibers, as an effective wound dressing with broad spectrum anti-bacterial properties.

1. Introduction Globally, over 6 million people are estimated to be affected by severe skin injuries every year as a consequence of fire, accident or surgery, and this figure is continue to raise in both industrialized and developing countries [1]. Microbial colonization and subsequent biofilm formation of the wound bed further exacerbate the conditions, delay wound healing and increase the number of hospital days [2–4]. Biofilm associated infections are hard to cure using a standard antibiotics regimen, hence requiring preventive as well as curative strategies [5]. In addition, the evolution of antimicrobial resistant pathogens that are refractory to the antibiotics of last resort confer significant economic burden [6]. Therefore, there is a need for new class of

antimicrobial agents which can effectively impede the microbial growth in hospitals and less likely to develop antimicrobial resistance while safe to use in humans [7]. Recent studies reported the use of natural products of traditional medicine origin such as garlic, Cleome droserifolia, plant extracts and manuka honey as alternative therapies against drug-resistant pathogens [8,9] Nano processing of natural products with long-term antimicrobial activities are considered as potential alternatives as cost-effective materials in combating antimicrobial resistance [10,11]. Electrospinning is one of the widely explored nano processing method for the design of nanofibrous scaffolds [12]. Electrospun nanofibers possess high surface to volume ratio, high porosity and the mechanical properties can be tuned to confer high drug loading efficiency, sustained release of

Corresponding authors. Correspondence to: K.D. Arunachalam, Center for Environmental Nuclear Research, SRM University, Kattankulathur Campus, Kancheepuram, Tamilnadu 603203, India. E-mail addresses: [email protected] (S. Ramakrishna), [email protected] (R. Lakshminarayanan), [email protected] (K.D. Arunachalam). ⁎

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https://doi.org/10.1016/j.msec.2018.12.135 Received 23 August 2018; Received in revised form 17 December 2018; Accepted 29 December 2018 Available online 03 January 2019 0928-4931/ © 2018 Published by Elsevier B.V.

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bioactive substances [13,14] with potential applications in regenerative medicine, antimicrobial packaging materials [15] and wound dressings [16–18]. In the context of wound management, the dimension and morphology of electrospun nanofibrous can be controlled to closely mimic the extracellular matrix (ECM) of the injured tissues to promote the cellular adhesion and proliferation thereby hastening the healing process [19]. The surface chemistry and topography of the nanofibrous scaffolds significantly influence the cell-material interactions, while sustained antimicrobial release characteristics is beneficial in the management of wound infections [20]. A number of previous studies reported the preparation of antimicrobial wound dressings by electrospinning of natural and synthetic polymers. The advanced wound dressings are impregnated with carbon nanotubes [21], honey [22,23], herbal extracts and phytochemicals [8,24–26], metal ions, metal oxide, and organic nanoparticles [27–29], antibiotics [30] and cationic polymers [31] to prevent microbial adhesion. In addition, plant-derived secondary metabolites (phytochemicals/medicinal plant extracts) are explored as a promising alternative either alone or in combination with antibiotics as they may overcome the evolution of antibacterial resistance with reduced side effects, improved availability, and cost effectiveness [32,33]. Jin et al. investigated the potential of four different leaf extracts namely Indigofera aspalathoides (IA), Azadirachta indica (AI), Memecylon edule (ME) and Myristica andamanica (MA) incorporated into PCL via electrospinning technique as a nanofibrous wound dressing. Their results suggest that the leaf extracts containing PCL scaffolds exhibited mechanical properties similar to that of human skin. Among all the mats, ME loaded PCL mats displayed least toxicity for hDFs, thus could be used as a substrate for skin tissue engineering [25]. Dai et al. engineered curcumin blended gelatin nanofibrous mats for promoting acute wound repair. The curcumin-loaded mats were biocompatible in vitro and in vivo and also could deliver the compound effectively at the wound sites, thus improving the wound healing process [34]. However, both the studies did not investigate the antibacterial activities of the natural extracts/compound, a key determinant of wound healing in tropical climates. PCL is a synthetic polyester polymer, widely used in tissue engineering and drug delivery applications due to its excellent biocompatibility, mechanical properties, process ability and, cost-effectiveness [35,36]. Gymnema sylvestre (Asclepiadaceae; GS) is a wellknown Ayurvedic medicinal plant, used as a remedy for the treatment of diabetes and also shown to exhibit broad therapeutic effects for the treatment of microbial infection, wound healing, inflammation, obesity, arthritis, constipation, and cancer [37–40]. Their phytoconstituents include gymnemic acids, gymnemasaponins, anthraquinones, flavones, phytin, lupeol, and stigmasterol. Gymnemagenin (GYM), a triterpene saponin which is one of the vital phytoconstituent of G. sylvestre [40]. The herbal preparations of G. sylvestre are presently used in tea bags, health tablets and supplements, beverages, and confectioneries. Only few reports investigated the antimicrobial properties of the G. sylvestre [41,42] and G. sylvestre in the form of electrospun nanofibers have not been reported yet. In the present manuscript, we report the preparation of PCL nanofiber mats containing G. sylvestre plant extracts and investigated their antimicrobial properties and biocompatibility for primary human dermal fibroblasts (hDFs). Scheme 1 shows the overall strategy adopted to design GS, GYM loaded nanofibrous PCL based wound dressings.

from Gibco®. All other cell culture reagents were obtained from Life Technologies Corporation, Singapore. Fresh Gymnema sylvestre leaves were collected from the herbal garden, Tamil University, Tamilnadu, India. The plant specimen was identified and authenticated as Gymnema sylvestre R. Br. by Dr. G.V.S Murthy, Botanical Survey of India (BSI/SRC/5/23/2016/Tech/215). 2.1.1. Phytochemical and plant extracts used in the study i) Gymnemagenin (GYM, purity > 95%) was purchased from Natural Remedies (Bangalore, India) ii) Ultrasound assisted Gymnema sylvestre extracts (USE). iii) Cold maceration assisted Gymnema sylvestre extracts (CME). 2.1.2. Microbial strains used in the study ATCC strains used: Methicillin-Resistant Staphylococcus aureus (MRSA 700699), Staphylococcus aureus (SA 29213), Pseudomonas aeruginosa (PA 9027), Staphylococcus epidermidis (SE 12228), Escherichia coli (E. coli 8739). Clinical Strains used: Methicillin-Resistant Staphylococcus aureus (MRSA 9808), Staphylococcus aureus (SA 4400), Pseudomonas aeruginosa (PA 4877). 2.2. Processing of plant materials to extract the active components The freshly collected Gymnema sylvestre leaves were rinsed thoroughly with distilled water to remove the unwanted adhered dust/ debris. The washed leaves were drained and shade dried at room temperature for 2 to 4 weeks. Once the leaves were dried completely, they were powdered in a blender, sieved using a 20-μm mesh. The sieved powder was defatted with petroleum ether for 8 h in Soxhlet apparatus, air dried at 25 ± 2 °C and stored in an airtight container for carrying out the further processing. 2.2.1. Cold maceration extraction (CME) 20 g of the defatted Gymnema sylvestre powder was extracted with 500 mL of 70% methanol at 25 ± 2 °C for 24 h in a rotary shaker at 100 rpm. This step was repeated thrice and the solvent was filtered, concentrated in a rotary vacuum at 40 °C and made into fine powders using lyophilizer [43]. 2.2.2. Ultrasound-assisted extraction (USE) 20 g of the defatted Gymnema sylvestre powder was soaked with 70% methanol for 3 h, followed by exposure to the ultrasound waves of 40 kHz frequency for 50 min at 50 °C in a digital ultrasonic bath (Wensar, India) for extraction. The extracted solvent was filtered, concentrated in a rotary vacuum at 40 °C and lyophilized into fine powder [44]. 2.3. Electrospinning of Gymnema sylvestre extract and gymnemagenin containing nanofibers For electrospinning, PCL, CME/USE/GYM were dissolved in chloroform: methanol (3:1 v/v) to form a homogenous solution. The concentration of the PCL was kept constant as 10%, whereas the concentration of the CME/USE compounds was varied as 5, 10 and 25% (w/w of PCL). Above 25% of USE/CME, it was difficult to spin the fibers. For GYM sample electrospinning, 0.5% of gymnemagenin was added to the PCL solution. The polymer solutions were then stirred overnight and then loaded into a 5 mL standard polypropylene syringe attached to a 27G blunted stainless steel needle for spinning. To generate electrospun mats, the polymer solutions was pumped at the feeding rate of 1 mL h−1 using the syringe pump (KDS 100, KD Scientific, Holliston, MA, USA) and the polymer droplet generated at the orifice of the needle were stretched and drawn into continuous nanofibers by applying high voltage of 13 kV (Gamma High Voltage

2. Experimental section 2.1. Materials Poly Caprolactone (PCL, Mw 80,000), chloroform, methanol, Hoechst and Flouromount™ were purchased from Sigma Aldrich (Singapore). Alexa Fluor 647 was procured form Thermo Fisher Scientific (Singapore). Mueller-Hinton agar (MHA) was obtained from BD (USA). Dulbecco's Modified Eagle's Medium (DMEM) was purchased 504

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Scheme 1. Schematic showing electrospinning set-up used to prepare GS and GYM loaded PCL nanofiber mats.

Research, USA). The nanofibers were collected on aluminum foil wrapped receiver kept at a distance of 12 cm away from the needle tip. The experiments were conducted at the room temperature maintaining the relative humidity of 60%. The collected nanofibers were vacuum dried overnight, stored in dry cabinets and used for various characterizations, antimicrobial assays, and cell culture experiments [25]. Hereafter, in this manuscript PCL fibers containing USE 5%, USE 10%, USE 25%, CME 5%, CME 10%, CME 25% and gymnemagenin will be represented as USE 5, USE 10, USE 25, CME 5, CME 10, CME 25 and GYM for simplicity.

2.7. Mechanical studies The mechanical properties of the electrospun mats were evaluated using Instron uniaxial tensile tester (Instron 5345, USA). The load cell of 10 N capacity was used for the study. The nanofiber mats were cut into rectangular strips with dimensions 40 × 10 mm and an average thickness of 80 μm. The rectangular pieces were stretched at a crosshead speed of 10 mm min−1. Minimum of 5 samples was tested for each group, and the mean value was reported. 2.8. Water contact angle studies

2.4. Field Emission Scanning Electron Microscopy (FESEM) analysis

The hydrophilicity/hydrophobicity properties of the electrospun fibrous mats were evaluated by sessile drop water contact angle method using VCA Optima Surface Analysis system (AST products, Billerica, MA). 1 μL of distilled water was used for drop formation on the nanofibers coated coverslips. The dynamic contact angle measurement was taken at different time points for 60 s. The experiment was conducted in triplicates, and the mean value was recorded.

The surface features of the nanofibers were analyzed using Field Emission Scanning Electron Microscope (FESEM) FEI-QUANTA 200F, (Netherlands) using an accelerating voltage of 10 kV. The electrospun mats were sputter coated with platinum using sputter coater (JEOL JSC1200 fine coater, Japan) prior FESEM analysis. Using the Image Analysis Software (Image J, National Institute of Health, USA) the average diameter of the nanofibers were calculated from the SEM images by selecting approximately around ~50 random fibers.

2.9. Cytocompatibility of electrospun nanofibers

2.5. Rheology and conductivity of the spinning solution

As dermal fibroblasts play a key role in wound healing process, we used hDFs for determining the cytocompatibility of the electrospun mats [34]. Cells were cultured in DMEM (Gibco®) supplemented with a mix of fetal bovine serum 10% (v/v), 50 U mL−1 penicillin and 50 μg mL−1 streptomycin at 37 °C and 5% CO2 in a humidified incubator. For biocompatibility experiments, cells (8000 cells well−1) were seeded onto nanofiber coated coverslips placed in 24-well plates (Nunc®) and allowed to grow for 24 h before analysis as described earlier [45]. The viability of the hDFs on the nanofibrous mats was determined using the colorimetric CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS assay) (Promega) according to manufacturer's

The viscosity and conductivity of the electrospinning solution were measured using Brookfield Cap 2000+ digital viscometer (USA) and conductivity meter ES-14 series (Horiba Scientific, Singapore), respectively. 2.6. Fourier Transform Infra-red Spectroscopy The functional group analysis of the electrospun scaffolds was performed in Alpha FTIR spectrometer (Bruker instrument, Germany) over the range of 500–4000 cm−1 at a resolution of 4 cm−1. The spectrum was plotted using Origin 8 software. 505

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3. Results and discussion

Table 1 Viscosity and conductivity measurements for different PCL solutions. Sample

Viscosity (cP)

Conductivity (μS/cm)

PCL USE 5 USE 10 USE 25 CME 5 CME 10 CME 25 GYM

251.6 ± 1.5 202.3 ± 2.5 173.6 ± 1.5 135.6 ± 4.1 197 ± 1 174.3 ± 3.2 141.6 ± 1.5 248.3 ± 2

0.48 ± 0.04 1.53 ± 0.06 2.37 ± 0.2 3.46 ± 0.15 1.87 ± 0.05 3.1 ± 0.14 4.03 ± 0.21 1.31 ± 0.12

3.1. Surface morphology and diameter distribution analysis using Field Emission Scanning Electron Microscopy (FE-SEM)

Fiber diameter (nm) 452 328 282 206 316 250 190 380

± ± ± ± ± ± ± ±

92 42 33 46 55 36 50 96

Prior to the electrospinning, we determined the antimicrobial properties of USE/CME. The MIC values were 4–8 mg/mL against Gram-positive strains and 16 mg/mL against Gram-negative strains. These results indicate that MIC range of USE/CME against pathogenic strains was similar to that of other plant extracts [8]. Next, electrospinning of PCL dope solution containing three different concentration of extracts obtained after ultrasonication and cold maceration were carried out (5%–25% w/w of PCL). However, due to poor solubility, we used a final concentration of 0.5% (w/w) Gymnemagenin powder for the preparation of GYM mats. The solution properties of the dope solution are shown in Table 1. The results indicated USE/CME concentrationdependent decrease in viscosity and increase in conductivity of the dope solution. However, addition of GYM did not affect the viscosity but resulted in > 2 fold increase in conductivity of the dope solution. PCL solution showed the lowest electrical conductivity, accounted for the nonpolar nature of PCL molecules [47]. Electrospinning of the dope solutions was carried out after optimizing the instrumental parameters that confer bead-free nanofibers. SEM images of the electrospun samples showed the presence of smooth nanofibrous morphology without any marks of morphological defects (Fig. 1). This demonstrates no phase separation of the plant extract during electrospinning process which leads to uniform distribution of the compounds throughout the mats. The average fiber diameters of PCL, USE 5, USE 10, USE 25, CME 5, CME 10, CME 25 and GYM were in the range of 452 ± 92, 328 ± 42, 282 ± 33, 206 ± 46, 316 ± 55, 250 ± 36, 190 ± 50 and 380 ± 96 nm respectively. The addition of both types of Gymnema sylvestre plant extracts, viz. ultrasound assisted, and cold macerated resulted in the reduction of fiber diameter significantly compared to that of pristine PCL mats. It has been reported that the diameter of the electrospun PCL nanofibers decreased by decreasing the viscosity and increasing the conductivity of dope solution [48]. It is likely that the presence of phytochemicals namely gymnemic acid, gymnemagenin, lupeol, anthraquinones, and flavones in the GS extract are responsible for enhanced conductivity and a reduced viscosity of the resultant solution, leading to a reduction in the fiber diameter of the composite mats. Similar observations were also reported by others where the fiber diameter of the electrospun mat was highly affected by its solution parameters [49]. Arslan et al. fabricated PET nanofibrous scaffold loaded with different concentration of honey ranging from 10 to 40 wt %. The authors documented a gradual decrease in the fiber diameter on increasing honey concentration due to increase in the conductivity of electrospinning solution [22]. Maleki et al. also observed a decrease in the fiber diameter of PVA nanofibers with increasing honey concentration (0–40 wt%) due to the decrease in viscosity and increase in conductivity of the electrospinning solution upon adding honey [23]. Hadisi et al. also reported a reduction in the diameter of electrospun gelatin/oxidized starch hybrid nanofibers with an increase in the henna concentration due to the reduction in its solution viscosity [50].

instructions. Briefly, cells grown on the nanofiber-coated coverslips were incubated with 400 μL of DMEM for 24 h and 100 μL of MTS reagent were added for 2 h at 37 °C before analysis. Subsequently, the absorbance of the medium was measured at 490 nm using a microplate reader (Infinite M200 Pro, Tecan, and Mannedorf, Switzerland). Each experiment was performed in triplicates. To visualize the cell phenotype, hDFs cultured on different electrospun mats for 24 h were washed with PBS and then fixed with 4% paraformaldehyde (Sigma, Singapore) at room temperature for 30 min. Cells were stained with phalloidin for 1 h at room temperature to visualize the cell morphologies and Hoechst to visualize the nuclei. Coverslips were washed with PBS, mounted on glass slides using Flouromount™ and visualized under a laser scanning confocal microscope (Zeiss LSM800, Carl Zeiss Microimaging Inc., NY, USA) using a 40× oil immersion objective lens. At least 5 different microscopic fields were analyzed for each sample. 2.10. Antimicrobial studies The antibacterial activity of Gymnema sylvestre containing mats was evaluated using Kirby-Bauer radial disc diffusion method. The experiment was carried out in accordance with Clinical and Laboratory Standards Institute (CLSI). The bacterial cultures (concentration adjusted to 0.5 McFarland standards) were spread onto the sterile Muller Hinton Agar (MHA) plates using a cotton spreader. The USE 25, CME 25 and GYM containing mats (10 × 10 mm) were placed on the swabbed bacterial cultures and incubated at 35 ± 2 °C for 24 h. The antibacterial activity was assessed as a zone of inhibition in millimeters. The assay was performed in duplicates [17]. 2.11. In vitro drug release and weight loss studies The release of extracts (USE 25 and CME 25) from the electrospun mats was measured by incubating 1 × 1 cm mats weighing approximately 2.8 ± 0.6 mg in 2 mL of phosphate buffer saline (pH - 7.4) at 37 °C. At fixed time points, PBS was completely removed and replaced by fresh PBS. The amount of extract released was analyzed by UV spectrometer at λ298nm. The experiment was conducted in triplicates [46]. The weight loss for various mats was examined by incubating 5 × 5 cm of each mat type in PBS at 37 °C. The mats were removed at 24 h and 48 h, rinsed in deionised water and air dried. The weight loss was determined by measuring the dry weight of the mats before and after incubation and the experiment was conducted in triplicates.

3.2. Bonding analysis of the mats using Fourier Transform Infra-red Spectroscopy To affirm the incorporation of Gymnema sylvestre (CME/USE) and gymnemagenin (GYM), the ATR-FTIR spectrum of electrospun PCL, GYM, USE 25 and CME 25 was recorded as shown in Fig. 2. Pristine PCL mat revealed eCH2 (symmetric) and eCH2 (asymmetric) vibrations for eCH2 groups at 2865 and 2946 cm−1 respectively; C]O stretching vibrations of the ester group at 1724 cm−1; the CH2 bending vibrations at 1365 and 1468 cm−1and eCeOeC symmetric and asymmetric stretching vibrations at 1046, and 1240 cm−1 respectively [51]. The

2.12. Statistical analysis For statistical analysis, all the quantitative data were expressed as mean ± standard deviations with at least two individual experiments. Significant differences among each group were determined through one way ANOVA followed by Tukey's post hoc test using the software GraphPad Prism version 7 (GraphPad Software Inc., La Jolla, CA, USA). p values < 0.05 were considered to be statistically significant. 506

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Fig. 1. SEM images of electrospun nanofibers a) PCL, b) USE 5, c) USE 10, d) USE 25, e) CME 5, f) CME 10, g) CME 25 and h) GYM. Scale bar = 1 μm.

USE and CME extracts had following characteristic peak at 3316 cm−1 corresponding to eOH stretching in the - alcoholic/phenolic groups; at 2925 cm−1 due to CeH stretching vibrations of alkanes; at 1711 cm−1 related to C]O stretching of ketones; at 878 cm−1 and 1516 cm−1 assigned to eNeH bending (primary amines) and NeO asymmetric stretching (nitro compounds) respectively, and at 1034 and 1160 cm−1 corresponds to the stretching vibration of eCeO of primary and tertiary alcohols (Fig. S1). In gymnemagenin, peak at 1550 cm−1 corresponds to the eCeCe stretching of the aromatic rings. The presence of both PCL and the plant extracts related peaks in GYM, USE 25 and CME 25 samples confirmed incorporation/loading of these extracts inside the PCL mats. 3.3. Mechanical studies The wound dressings should be mechanically strong enough to withstand the physiological forces during the wound repair process and to support tissue growth [51]. Fig. 3 shows the stress-strain curves of the electrospun Gymnema sylvestre and gymnemagenin loaded PCL nanofibers under tensile loading. Young's modulus, ultimate tensile strength, tensile strain and energy to break for the various nanofiber mats were compared against PCL mats to infer the effect of plant extracts on the mechanical properties of the mats. Table 2 summarizes the tensile properties of the electrospun nanofiber mats obtained from five independent samples. Pristine PCL mats displayed the lowest tensile strength and maximum tensile strain, confirming plastic deformation of the mats. With the addition of the USE and CME extracts, the mats lose its plasticity, which was clearly seen by the significant reduction (P < 0.001) in the tensile strain compared to that of pristine PCL mats.

Fig. 2. FT-IR spectrum of PCL, GYM, USE 25 and CME 25 nanofibers.

As the concentration of the plant extract in the mats increases, the maximum tensile strain decreased significantly. The ultimate tensile strength, toughness and tensile modulus of the USE and CME mats were significantly higher (P < 0.001) compared to that of PCL mats. As the concentration of the plant extracts increases, there was a gradual increase in the maximum bearable stress, toughness and tensile modulus under applied tension. Among all the mats, USE 25 showed the highest tensile strength, toughness, whereas, CME 25 mats showed the 507

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extracts and gymnemagenin over the surface wettability of the electrospun PCL mats, the dynamic water contact angle (WCA) measurements were performed. The images captured for WCA measurements on various PCL mats at 0 s and after 60 s of dropping the water drop were shown in Fig. 4. For pristine PCL mats, the contact angle value remained constant at 139.2 ± 5.2° over 60 s, indicating typical hydrophobic nature of the PCL mats (Fig. 4a). Incorporation of USE 5 and CME 5 did not contribute much to alter the wettability of PCL mats with WCA values of 134.5 ± 4.1° (Fig. 4b) and 133.8 ± 2.4° (Fig. 4e), respectively. However, the addition of USE 10, USE 25, CME 10 and CME 25 significantly reduced (P < 0.001) the WCA values which reached 0° within 30 s. The presence of polar phytochemicals in the Gymnema sylvestre enhanced the wettability of the PCL mats. There was no statistical significance (P > 0.05) in the contact angle for GYM mats. Fig. 4i shows the time-dependent changes in the dynamic contact angle plot for various PCL mats revealing the variation in WCA values with time with and without incorporating the plant extract. Mary et al. reported that with the increasing concentration of the Aloe vera extract (5%, 10%, and 15%), the wettability of PCL nanofibrous composite increased mainly due to the presence of polar phytochemicals in the Aloe vera [56]. The hydrophilic surfaces were reported to enhance the adsorption of ECM components such as collagen, fibronectin, and laminin which promotes the regeneration of various cell types especially epithelial, fibroblasts, and endothelial cells [57]. To infer if increased wettability had any effect on weight loss, we determined the dry weight of the USE 25 and CME 25 mats after immersion in PBS at 37 °C for 24 and 48 h. A weight loss of 3.4% and 4.1% was observed for USE 25 mats after 24 and 48 h, respectively and, similarly, CME 25 mats immersed in PBS displayed very low weight loss (4.0% at 24 h and 4.5% at 48 h). Taken together, these observations indicate increased wettability caused minimal weight loss of PCL mats containing plant extracts.

Fig. 3. Stress-strain curve of PCL, USE 5, USE 10, USE 25, CME 5, CME 10, CME 25 and GYM nanofibers.

maximum tensile modulus. However, we didn't observe statistically significant (P > 0.05) changes in the tensile modulus, ultimate tensile strength, and toughness for the GYM mats. Chuysinuan et al. studied the effect of inclusion of different concentration of Garcinia mangostana extract over the mechanical properties of as-spun PAN fibrous mats. They found that with an increase in the mangostana extract between 10 and 30 wt% based on the PAN solution, the mechanical properties of the G. mangostana-loaded fiber mats increased monotonically [24]. In general, the tensile modulus and strength of the fibers increase as the fiber diameter decreases whereas failure strain increase with increase in fiber diameter [52,53]. In the case of USE 25/CME 25, the fiber diameter (206 ± 46 and 190 ± 50 nm) was smaller, displayed densely packed fibrillar structures with many junctions and higher entanglement compared to that of pristine PCL fibers (452 ± 92 nm) as shown in Fig. 1. Lim et al. suggested that densely packed lamellae and fibrillar structures in smaller diameter fibers has high molecular orientation and align themselves to the applied tensile force, thereby enhancing its mechanical properties. As the fiber diameter increases, the alignment of the fibrillar structure decreases resulting in reduced mechanical properties [54]. Taken together, these observations suggest a marked improvement in the mechanical properties of PCL mats upon addition of USE and CME which highlights the excellent reinforcing ability of the extracts.

3.5. Cytocompatibility of electrospun nanofibers Next, we investigated the in vitro biocompatibility of the USE/CME and GYM loaded PCL nanofibers by MTS viability assay and cytoskeleton staining after 24 h post seeding of hDFs. Cells cultured on coverslip or pristine PCL mats served as control. MTS assay indicated that none of the mats displayed any cytotoxic effect for hDFs, suggesting that cells retained their metabolic activity (Fig. 5a). However, when compared to pristine PCL, the hDFs growth was seen to be significantly higher for USE 25 (P < 0.001), USE 10 (P < 0.01), CME 10 (P ≤ 0.05) and CME 25 (P < 0.01) nanofibrous scaffolds. Thus, the MTS results indicated higher hDFs metabolic activity and proliferation on PCL mats containing higher amounts of USE and CME. The hDFs phenotype was observed by visualizing cell cytoskeleton under confocal microscopy. Fig. 5(b–j) showed that fibroblasts exposed to both pristine PCL and mats containing plant extracts displayed intact cytoplasmic filamentous distributions (green color) of F-actin with a prominent nucleus (blue color) and lack of any structural abnormalities. Interestingly, a greater density of cells was observed when seeded on to USE 25 and CME 25 nanofiber mats, corroborating the results derived

3.4. Contact angle measurement The wettability of the biomaterial surface determines its affinity towards the cell attachment and spreading. In general, the hydrophilic surface exerts better affinity to cells, promotes cell spreading and absorbs excess wound exudates [55], resulting in better wound healing compared to that of hydrophobic surfaces. To infer the effect of plant

Table 2 Mechanical properties of electrospun nanofibers. Significant difference against mechanical properties on different electrospun fibers compared against PCL at *p ≤ 0.05;**p < 0.01;***p < 0.001;****p < 0.0001 and ns p > 0.05 by 1-way ANOVA. Sample

Tensile modulus (MPa)

Ultimate tensile stress (MPa)

Ultimate tensile strain (%)

Toughness (MJ m−3)

PCL USE 5 USE 10 USE 25 CME 5 CME 10 CME 25 GYM

6.68 ± 0.9 17.38 ± 1.76**** 28.66 ± 1.46**** 37.98 ± 2.41**** 31.64 ± 2.89**** 37.82 ± 3.61**** 39.66 ± 3.36**** 10.3 ± 2.35ns

1.8 ± 0.38 8.85 ± 1.58** 13.59 ± 2.82**** 19.31 ± 2.84**** 6.04 ± 1.58* 8.71 ± 1.79** 11.88 ± 2.45**** 2.8 ± 0.4ns

110.57 ± 15.5 39.16 ± 5.38**** 44.04 ± 5.46**** 50.08 ± 4.9**** 28.62 ± 4.1**** 32.29 ± 3.5**** 46.2 ± 5.5**** 80.2 ± 5.5****

12.95 ± 2.1 29.01 ± 2.22**** 60.62 ± 5.18**** 63.05 ± 4.23**** 10.42 ± 1.67ns 29.01 ± 1.02**** 39.88 ± 3.2**** 17.6 ± 2.2ns

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Fig. 4. Photographs showing the water contact angle at 0 s and 60s on a) PCL, b) USE 5, c) USE 10, d) USE 25, e) CME 5, f) CME 10, g) CME 25, h) GYM and i) Dynamic water contact angle measurement for various electrospun mats against time.

from MTS assay. The hDFs were attached on all the electrospun mats and displayed normal cell morphology. These observations demonstrated that the GS and gymnemagenin loaded PCL mats were non-cytotoxic for hDFs and promoted cell proliferation. The above findings could be related to the presence of bioactive constituents in Gymnema sylvestre, such as triterpene saponin (gymnemagenin), oleanane saponin (gymnemic acids), flavonoid (kaempferol) and sterol (stigmasterol) which decreases the hydrophobicity of PCL mats, thus promoting higher cell adhesion and proliferation. The diameter of the electrospun fibers also influences the cellular activity. It has been reported that nanofibers with smaller diameters encourage fibroblasts attachment, spreading and proliferation [58]. As shown in

Fig. 1, the addition of GS extract to the PCL matrix decreased the diameter of electrospun fibers, which could have a positive impact on the growth of dermal fibroblasts. Thus, both the reduction in average diameter of the nanofibers and the enhanced wettability could contribute significantly to the observed higher cell proliferation in USE/CME mats. 3.6. Antimicrobial activity of electrospun GS nanofibers One of the essential requirements for the advanced wound dressings is to prevent the bacterial adhesion and colonization at the wound sites [59]. It is important to ascertain if the extracts retained the antimicrobial properties after electrospinning. Thus, the antimicrobial 509

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Fig. 5. a) Cytotoxicity assessment of hDFs on different electrospun mats by MTS assay. Laser confocal microscopy images of hDFs seeded on b) coverslip, c) PCL, d) GYM, e) USE 5, f) USE 10, g) USE 25, h) CME 5, i) CME 10, and j) CME 25 nanofibers. F-Actin stained green and the nucleus was stained blue. Scale bar = 20 μm. Significant difference against cell viability on different electrospun fibers compared against PCL at *p ≤ 0.05;**p < 0.01;***p < 0.001 and ns p > 0.05 by 1-way ANOVA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Disc diffusion images of GS loaded PCL nanofibers against the microorganisms. Scale bar = 10 mm.

activities of USE 25, CME 25 and GYM loaded electrospun PCL mats was determined by Kirby-Bauer disc diffusion assay. As anticipated, colonies of Gram-positive and Gram-negative bacteria were observed on pristine electrospun PCL mats whereas no clear zone of inhibition was found around the USE 25, CME 25 and GYM loaded electrospun PCL mats (Fig. 6). However, there was no microbial growth seen below or above the USE 25 and CME 25 mats. To confirm our observations, SEM imaging was performed for the pristine PCL, USE 25, CME 25 and GYM mats detached from disc diffusion assay (Fig. 7). Interestingly, the PCL mats contained micro colonies of cocci species spread across various areas of the mats and aggregates of P. aeruginosa/E. coli strains were visible. These results indicate the formation of biofilm-like structures by Gram-positive and Gram-negative bacteria on the pristine electrospun mats. However, USE 25 and CME 25 mats did not contain any traces of microbial colonies, although GYM mats contained few microbial colonies than pristine PCL mats. G. sylvestre extracts is composed of gymnemic acid, gymnemagenin, gymnemasaponins, flavones, lupeol and sterols which act synergistically to inhibit the growth of microorganisms. However, gymnemagenin alone displayed weak inhibitory action, possibly due to the low abundance of antimicrobial components in the electrospun mats. These results demonstrated that the USE/CME loaded mats displayed contact-mediated microbicidal

activity by the extracts present at the surface of the mats. The absence of a clear zone of inhibition around the mats suggest the possible confinement of the extracts within the PCL nanofibrous mats that prevent the release of active ingredients. As a result, bacterial growth was prevented above and below USE25/CME 25 mats suggesting contactinhibition. To confirm this, we monitored the release of aromatic products released from the mats by UV spectrometry (OD298nm) after incubating the mats in PBS for 48 h. The results indicated lack of release products from the USE 25 and CME 25 mats (data not shown), suggesting strong interactions between the polymer and extracts. The lack of release of antimicrobial components from the mats is advantageous since the process minimizes bacterial colonization for an extended period of time while allowing the migration of mammalian cells at the injured sites and promotes wound healing. 4. Conclusion In the present study, we have prepared Gymnema sylvestre, and gymnemagenin loaded PCL wound dressing by electrospinning technique. Our results revealed that Gymnema sylvestre incorporated nanofiber mats has combined advantages of high biocompatibility, antibacterial activity, superior mechanical properties, and wettability. By

Fig. 7. SEM images of PCL, USE 25, CME 25 and GYM nanofibers after 24 h incubation against the microorganisms. Scale bar = 1 μm. 512

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localizing and constraining the plant extract (GS) within the dressings, we can control the undesired interference of the extracts with the wound healing process and at the same time decrease the risk of developing resistant bacterial strains. Overall, we presented here a simple approach to design a mechanically competent integrated system that supports mammalian cells while inhibiting microbial colonization. Such cell-discriminative scaffolds could potentially be utilized as antimicrobial wound dressings or sterile skin substitutes where microbial colonization delays wound healing. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2018.12.135.

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Acknowledgments

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The authors gratefully acknowledge the financial support from UGC-DAE Consortium for Scientific Research (CSR-KN/CRS-70/201516/811) Kalpakkam, Tamilnadu, India, Translational and Clinical Research Flagship Program of the Singapore National Research Foundation (NMRC/TCR/008-SERI/2013), administered by the National Medical Research Council of the Singapore Ministry of Health. This work was supported by Co-operative Basic Research Grant from the Singapore National Medical Research Council (NMRC/CBRG/0048/ 2013) and SNEC Ophthalmic Technologies Incubator Program grant (Project no. R1181/83/2014) awarded to RL. NKV acknowledges funding support from Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant (L0412290), Strategic Academic Initiative Grant (SAI-L0494003), the Ministry of Education - Singapore under its Singapore Ministry of Education Academic (AcRF) Research Fund Tier I (2015-T1-001-082) and Industry Alignment Fund Pre-Positioning Grant (IAF-PP H17/01/a0/0K9).

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Conflict of interest

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The authors declare no conflict of interest.

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