Fabrication, characterization and fibroblast proliferative activity of electrospun Achillea lycaonica-loaded nanofibrous mats

European Polymer Journal 120 (2019) 109239

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Fabrication, characterization and fibroblast proliferative activity of electrospun Achillea lycaonica-loaded nanofibrous mats

T

Muhammet Emin Cama,b,c, , Sumeyye Cesura,d, Turgut Taskine, Gokce Erdemirf, Durdane Serap Kurucag, Yesim Muge Sahinh,i, Levent Kabasakalc, Oguzhan Gunduza,d ⁎

a

Center for Nanotechnology and Biomaterials Research, Marmara University, Istanbul 34722, Turkey Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE, UK c Department of Pharmacology, Faculty of Pharmacy, Marmara University, Istanbul 34668, Turkey d Department of Metallurgy and Material Engineering, Faculty of Technology, Marmara University, Istanbul 34722, Turkey e Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Istanbul 34668, Turkey f Department of Molecular Medicine, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul 34093, Turkey g Department of Physiology, Faculty of Medicine, Istanbul University, Istanbul 34093, Turkey h Department of Biomedical Engineering, Faculty of Engineering and Architecture, Istanbul Arel University, Istanbul 34537, Turkey i ArelPOTKAM (Polymer Technologies and Composite Application and Research Center), Istanbul Arel University, 34537 Istanbul, Turkey b

ARTICLE INFO

ABSTRACT

Keywords: Electrospinning Achillea lycaonica Nanofibers Polylactic acid Fibroblast proliferation Skin tissue engineering

The use of natural compounds such as biocompatible and non-toxic plant extracts, without undesired side effects, in tissue engineering applications, is highly preferred compared to chemical drugs. In this study, the characterization and performance of electrospun Achillea lycaonica-loaded (0.125, 0.250 and 0.500, wt%) poly (lactic acid) (PLA) (8%, w/v) nanofibrous mats for skin tissue engineering were investigated. SEM, FTIR, DSC, and tensile strength test of the electrospun nanofibers have been investigated. Drug releasing test and cell culture study were also carried out. Achillea lycaonica-loaded nanofibrous mats in 0.250 (wt%) and 0.500 (wt%) demonstrated excellent cell compatibility and increased the viability of NIH/3T3 (mouse embryo fibroblast) cells within 72 h. According to the results, Achillea lycaonica-loaded PLA nanofibers have proper tensile strength and controlled release. The working temperature range enlarged for the composites having higher plant extract content. Consequently, Achillea lycaonica-loaded nanofibrous mats have a great potential in skin tissue engineering applications.

1. Introduction The skin, the largest organ of the body, is the first line of defense that protects the human body against external organisms [1]. Wound healing, a complex series of reactions, has been divided into four phases: (i) coagulation and hemostasis; (ii) inflammation; (iii) proliferation; and (iv) wound remodeling with scar tissue formation. Wound healing treatment emphasis on new therapeutic approaches and the advancement of technologies for acute and chronic wound management [2]. Pharmaceutical industry provides an enormous and complex treatment to struggle with wound-healing problems [3]. The annual cost in managing these wounds and associated comorbidities was estimated to be £5.3 billion in UK [4] and $20 billion in the USA [5].

Nanofibers are ideal for wound healing because their dimensions are similar to the components of native extracellular matrix and also they mimic its fibrillar structure by providing essential cues for cellular organization and survival function [6]. Electrospinning is the most popular and preferred technique to fabricate nanofibers, due to its flexibility, simplicity, cost-effectiveness, potential to scale up, and ability to spin a broad range of polymers and it provides the opportunity for direct encapsulation of drugs into the electrospun fibers. Fabrication parameters such as polymer solvent, polymer concentration, collector distance and voltage are varied to modify nanofiber and scaffold properties [7]. The use of synthetic polymers have several key advantages compared to natural polymers. For instance, synthetic polymers offer proper options for controlling shape, architecture and chemistry to create

Corresponding author at: Marmara University Faculty of Pharmacy Tıbbiye Street No: 49, Haydarpasa, 34668 Istanbul, Turkey. E-mail addresses: [email protected] (M.E. Cam), [email protected] (S. Cesur), [email protected] (T. Taskin), [email protected] (G. Erdemir), [email protected] (D.S. Kuruca), [email protected] (Y.M. Sahin), [email protected] (L. Kabasakal), [email protected] (O. Gunduz). ⁎

https://doi.org/10.1016/j.eurpolymj.2019.109239 Received 11 June 2019; Received in revised form 3 September 2019; Accepted 4 September 2019 Available online 10 September 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved.

European Polymer Journal 120 (2019) 109239

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reasonable alternatives or mimic native extracellular matrice systems [8,9]. Poly(lactic acid) (PLA) is one of the most widely used synthetic polymers in the biomedical area. PLA is an ideal candidate for producing nanofiber through its cytocompatibility and biodegradability. In addition to that, nanofibrous PLA scaffolds hold great promise as drug delivery carriers, where fabrication parameters and drug-PLA compatibility greatly affect the drug release kinetics [10,11]. Green synthesis of nanofibers includes using plant products or extracts that are less harmful and less expensive to the environment than the standard physicochemical methods that are commonly used [12]. Herbal therapies for skin disorders have been the most commonly used traditional therapies for thousands of years. There are some plants that have been searched for wound-healing therapies and tissue engineering applications such as Aloe vera, Achillea biebersteinii, Achillea kellalensis, Achillea millefolium, Morinda citrifolia, Camellia sinensis, Rosmarinus officinalis L. [13–15]. The genus Achillea comprises of ~85 species, most of which are endemic to Europe and the Middle East. Turkish flora possesses 42 Achillea species and 23 of them are endemic. In literature data, various Achillea species have demonstrated several biological activities such as antioxidant, antibacterial, antispasmodic and anti-inflammatory [16–20]. Besides, the wound healing effects of Achillea biebersteinii and Achillea millefolium extracts was proved in animal models and cell culture studies [21–23]. Achillea lycaonica Boiss. et Heldr. is a member of the genus Achillea L. It belongs to the Asteracea family and is an endemic species to Turkey. This plant species shows valuable antioxidant, wound healing, antimicrobial and cytotoxic activities in the studies [24,25]. Many plants traditionally used in wound healing or have proved in wound-healing therapy, also have high level of antioxidant properties [26]. In our previous study, we evaluated the the highest α, α-diphenylβ-picrylhydrazyl (DPPH) antioxidant activity; one of the strongest 2 2′azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) radical cation scavenging activities and one of the highest ferric reducing activities in 1:1 ethanol-water Soxhlet extraction of Achillea lycaonica [27]. Therefore, we preferred 1:1 ethanol-water Soxhlet extract depending upon its high antioxidant activities. Dimetyl sulfoxide (DMSO) is a biocompatible solvent with low toxicity (FDA directive 67/548/ec). In this study, DMSO was used as the assistant solvents to increase the conductivity of the PLA solution; decrease viscosity in the present case also increase solution fluidity, enhance spinning rate as a consequence; increase the solubility of the plant extract. The bead-free fibers can also be obtained from the solutions with mixed solvents of chloroform/DMSO. Thus, the morphologies of the obtained fibers are enhanced clearly and more uniform appearance was created with significantly reduced diameters [28]. In this study, four different solutions were prepared in order to produce electrospun nanofibers for tissue engineering applications. Pure PLA and Achillea lycaonica plant extract in three different concentrations (0.125, 0.250 and 0.500, wt%) were added to mixed solvents. The physical parameters of solution and the morphology, physical and chemical composition, drug release behavior, and in-vitro cell proliferation effect of the electrospun nanofibers were performed.

2.2. Plant material and extract preparation The aerial parts of Achillea lycaonica were collected from Malatya, Turkey in blooming semester and the taxonomic identified by Assoc. Prof. Dr. Turan Arabacı, Inonu University, Faculty of Pharmacy, Pharmaceutical Botany Department. The voucher specimen (T. Arabacı 2969) was deposited in the herbarium of the Faculty of Pharmacy, Inonu University for future reference. The plant powder of Achillea lycaonica aerial part was extracted with ethanol-water (50:50, v/v) in a Soxhlet apparatus for 6 h. 2.3. Characterization of solutions In order to measure physical parameters such as viscosity, surface tension, electrical conductivity, and density of four different solutions, viscometer (DV-E, Brookfield AMETEK, USA), force tensiometer (Sigma 703D, Attension, Germany), electrical conductivity probe (Cond 3110 SET 1, WTW, Germany) and density bottle (10 mL specific density bottle, Boru Cam Inc., Turkey) were used. All the measurements were repeated three times at ambient temperature (25 °C). These equipments were calibrated prior to measurements. 2.4. Fabrication of nanofibers Firstly, PLA was dissolved in chloroform (CF) at the concentration of 8% (w/v) at the room temperature and mixed for almost 1 h. After mixing, Tween80 was added to the PLA solution at the ratio of 1% (w/ w) and the solution was gently stirred for a further 10 min at room temperature. The Achillea lycaonica extract was dissolved in three different concentrations (0.125%, 0.250% and 0.500% (w/w)) in dimetyl sulfoxide (DMSO) and stirred for 20 min, separately. After preparing PLA solution and plant extract solution separately, both were mixed and stirred for an hour. In conclusion, PLA was dissolved in mixed solvents of (10:1, v/v) (Chloroform: DMSO). Furthermore, two different solvent ratios (4:1 and 5:1 v/v) (Chloroform: DMSO) were tried and failed due to impaired surface morphology obtained by scanning electron microscopy (SEM). Therefore, it is not possible to use the plant extract more than 0.500 (wt %) depending on the volume of DMSO. 2.5. Electrospinning procedure of nanofibers The experimental setup consists of a single brass needle, a syringe pump containing the polymer solution (NE-300, New Era Pump Systems, Inc., USA), a high precision voltage generator connected to the needle and a laboratory scale electrospinning unit (NS24, Inovenso Co., Turkey). A 10 mL volume syringe was loaded to the syringe pump containing the pure PLA solution and three different plant extract solutions (0.125, 0.250 and 0.500, wt%) and silicone tubes were used to transfer it to the needle. The flow rate was set to a fixed value of 12, 11, 10 and 9 mL/h and the voltage was subjected at 29.3, 30.1, 30.2 and 24.1 kV for pure PLA and plant extract solutions (0.125, 0.250 and 0.500, wt%), respectively to optimize the morphology of the nanofibers. The working distance between the needle tip and the circular collector was set to 170 mm.

2. Materials and methods 2.1. Materials

2.6. Scanning electron microscopy

Poly (L-lactic acid) (PLA) 2003D was purchased from Nature Works LLC, Minnetonka, MN. Other chemicals and reagents, e.g., chloroform, dimetyl sulfoxide, Tween80 employed in the experiments were purchased from Sigma-Aldrich.

The size and morphologies of nanofibers were investigated with scanning electron microscopy (SEM) (EVO LS 10, ZEISS). The surface of samples was coated with gold for 60 s. The average fiber diameter and their distribution were determined by using image software (Olympus AnalySIS, USA). 2

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2.7. Fourier transform infrared spectroscopy (FTIR)

active cells and reduced in the mitochondria to an insoluble purple formazan granule [30]. Subsequently, supernatant was discarded, and the precipitated formazan was dissolved in dimethyl sulfoxide (100 µL per well), and optical density of the solution was evaluated using a microplate spectrophotometer (Kayto RT-2100C) at a wavelength of 570 nm. For SEM investigations, the specimens were placed in the wells of 6well cell culture plates and sterilized for 2 h. NIH/3T3 cells were seeded in these plastic dishes and incubated for 24 h in a humidified incubator at 37 °C with 95% air and 5%CO2. At the end of 24 h, the media were removed, and specimens were fixed with 3% volume fraction of glutaraldehyde, subjected to graded (30–100%) alcohol dehydration and kept at −20 °C. The confluent cells were observed on SEM investigations.

Fourier-transformed infrared (FT-IR) (Jasco, FT/IR 4700) was used for analyzing molecular contents of nanofibers. 2.8. Differential scanning calorimetry (DSC) DSC analysis was conducted using Perkin Elmer Jade DSC and Pyris software (PerkinElmer Inc., Mass., USA) at a heating rate of 10 °C min−1 between 0 and 200 °C under dynamic argon atmosphere (20 mL min−1) to determine thermal properties of the nanofibrous mats. Temperature calibration of DSC was performed according to the indium melting point and melting enthalpy. 2.9. Tensile test

2.12. Statistical analysis

The tensile strength of nanofiber samples were determined and evaluated using an Instron 4411 tensile test machine working at room temperature (23 °C). The results were analyzed using Bluehill 2 software (Elancourt, France). Six fibrous scaffolds (1x5 cm) specimens were tested for each set of samples and the thicknesses of the specimens were evaluated using a digital micrometer (Mitutoyo MTI Corp., USA). Both ends of each specimen were compressed by the top and bottom grip and subjected to a tensile test under conditions of 5 mm min−1 test speed and 1 cm distance between grips.

Values are presented as mean ± standard deviation in SEM analysis, mean ± standard error of mean in MTT assay. Statistical analysis was performed using analysis of variance (ANOVA) test and Bonferroni’s post hoc test. The value of p < 0.05 was considered statistically significant. 3. Results and discussion

2.10. In vitro Achillea lycaonica release

3.1. Physical properties of solutions

To study the drug release kinetics, Achillea lycaonica-loaded nanofibrous mats were removed from the aluminium foils and were cut into with an average weight of 10 mg each and then, immersed in 1 mL of phosphate-buffered saline (PBS) (pH 7.4 at 37 °C) with a constant stirring rate (200 rpm) for a period of 24 h in the thermal shaker (BIOSAN TS-100). At the defined time intervals (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h) PBS was removed from each sample and 1 mL of fresh PBS was added again to continue the release test. UV spectroscopy (Shimadzu UV-3600) was used for monitoring of Achillea lycaonica releasing profile at 780 nm [29].

Many processing parameters affect the nanofiber formation and homogeneity [31]. The solution density, viscosity, electrical conductivity and surface tension were investigated for all solutions in Fig. 1. It is observed that increasing the plant extract ratio caused the rise in electrical conductivity and the decrease in surface tension [32]. 3.2. Morphological evaluation of nanofibers The change in fiber size and morphology of nanofibrous mats were analyzed using SEM. The most of obtained nanofibers were homogeneously dispersed and distributed within the polymer matrices. They were smooth and randomly oriented without visible beads. Consequently, plant extract-loaded nanofibrous mats (ϕ = 1094.96 ± 124.34 nm for 0.125 (wt%); ϕ = 890.12 ± 145.05 nm for 0.250 (wt%); ϕ = 571.89 ± 74.62 nm for 0.500 (wt%)) exhibited smaller diameters when compared with PLA nanofibers (ϕ = 1452.17 ± 154.40 nm) as shown in Fig. 2.

2.11. Methylthiazolydiphenyl-tetrazolium bromide (MTT) assay NIH/3T3 (mouse embryo fibroblast) cell line was purchased from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) with 10% Fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin in a 5% CO2 humidified air incubator, maintained at 37 °C. When the cells reached 80% of confluence they were washed with PBS and trypsinized with 0.25% Trypsin-EDTA for passaging and seeding each time. The confluent cells were utilized in cytotoxicity tests and SEM investigations. At the beginning, the conditioned medium was prepared to understand any possible toxic effect induced by possible ionic leach-out product from the samples into the medium. For this purpose, 5 mL fresh medium was added in tubes with a piece (≅0,05 g) of tested material (plant extract loaded nanofibers), which were kept in the incubator. One week after the conditioned medium was extracted, and later used in cytotoxicity tests. MTT assays were performed in 96-well plates. NIH/3T3 cells (about 105 cells per well) were seeded onto the 2 h UV sterilized polymers and incubated for 3 days. Cell viability was measured by determining mitochondrial NADH/NADHP–dependent dehydrogenase activity, which resulted in the cellular conversion of the 3-(4, 5-dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulfophenyl-2H) tetrazolium salt into a soluble formazan dye. After 3 days, supernatants were removed, and 10 µL 3-(4, 5-dimethylthiazol-2yl)-2,5diphenyl-2H-tetrazolium-bromide (MTT- 5 mg/ml- Sigma) solution was added to each well. Following incubation at 37 °C for 3.5 h and kept dark in humidified atmosphere at 5% CO2 in air. MTT was taken up by

Fig. 1. Physical parameters of all solutions used in the experiment: (a) the density, (b) viscosity, (c) electrical conductivity and (d) surface tension. 3

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3.3. Analysis of fiber composition The results of FTIR analysis of the molecular structure for the nanofibrous mats were indicated in Fig. 3. For pure PLA, the characteristic absorption bands were observed CH3 and C-H stretching vibrations at 2994 and 2947 cm−1, C]O vibration peak at 1748 cm−1, CH3 asymmetrical scissoring at 1452 cm−1, CeO asymmetrical stretching and CH3 twisting at 1181 cm−1, CeCH3 stretching at 1042 cm−1 and CeCOO stretching at 868 cm−1 [33]. The FT-IR spectrum of Achillea lycaonica presents prominent absorption bands at 3259 cm−1 for eOH stretching vibration, 2919 cm−1 for CeH stretching vibration of methylene group, 1581 cm−1 for CeO vibration, 1395 cm−1 for eCH3 symmetrical deformation vibration, 1028 cm−1 for CeO stretching [34,35]. Similar type of peaks in different intensities was obtained in the spectra for the plant extract-loaded nanofibers. These results indicate successful formulation and drug encapsulation. 3.4. Thermal properties The main thermal transitions of nanofibrous mats were analyzed by DSC, as shown in Table 1 and Fig. 4, respectively. The melting temperature (Tm), glass transition temperature (Tg) and crystallization temperature (Tc) for all composite fibers were studied. It is clearly shown that Tm and Tg decreased as the plant extract ratio in the composite mats increased compared to pure PLA nanofibers. More remarkable decrease in Tg values was detected whereas a slight decrease in the Tm values was obtained for the composite mats. Thus, the working temperature range between Tg and Tm enlarged for the composites having higher plant extract content. As plant extract enriched in the composite solution, physical properties of solutions, especially the electrical conductivity, increased as mentioned previously. In the electrospinning process, smaller diameters were obtained for more plant extract incorporated mats as presented in SEM images. Electrospun nanocomposite mats indicated lower Tg values when smaller fiber diameters are obtained. The proposed size-dependent glass transition behavior of electrospun mats can be atributed to polymer chain confinement. Similar results have been obtained in the study of Wang et al. [36]. On the other hand, the decrement in Tc of nanofibrous mats may be due to chain alignment that is suggested up to a certain level in electrospinning process [37]. DSC results indicate that while cold-crystallization is in a highly amorphous form in pure PLA, the decreased cold-crystallinity is associated with the increase of the plant extract ratio [38]. Thus, the material structure becomes more ordered. The ΔHc values decreased with the increment of plant extract; this behaviour can be associated with cold-crystallinity increase for these nanocomposite mats. Considering all DSC data, loading Achillea lycaonica to PLA nanofibrous mats influence the molecular chain mobility and resulted in a change of the material's thermal behavior. This output is found to be in good agreement with experimental results obtained for morphological and mechanical tests of the mats.

Fig. 2. SEM images and fiber diameter distribution of pure PLA (a) and Achillea lycaonica-loaded nanofibers at 0.125 (b), 0.250 (c), and 0.500 (d) (wt%) concentrations.

Fig. 3. FTIR spectrums of pure Achillea lycaonica, pure PLA nanofiber and Achillea lycaonica-loaded nanofibers in three different concentrations (0.125, 0.250, and 0.500, wt%). Table 1 Main thermal transitions and crystallinity of nanofibers. Achillea lycaonica (wt%)

Tg (°C)

Tm1 (°C)

ΔH1 (mj/mg)

Tm3 (°C)

ΔH3 (mj/mg)

Tc (°C)

ΔHcrystallization (mj/mg)

0 0.125 0.250 0.500

62.8 64.4 54.0 45.1

154.3 152.7 152.7 152.4

3.18 3.34 2.18 1.68

151.8 150.4 150.5 150.2

3.42 2.10 3.23 1.27

109.2 84.7 82.5 84.8

2.83 1.15 1.81 0.98

4

European Polymer Journal 120 (2019) 109239 0.4

a

PLA

0.125%

0.250%

0.500%

0.3 0.2 0.1

100

Strain at break (%)

Tensile strenght (MPa)

M.E. Cam, et al.

80

b

PLA

0.125%

0.250%

0.500%

60 40 20

0.0

0

Achillea lycaonica (wt%)

Achillea lycaonica (wt%)

1,6

2

a

1 mg/ml 0,8 mg/ml 0,6 mg/ml 0,4 mg/ml 0,2 mg/ml

1,2 0,8 0,4 0 200

Absorbance (a.u)

Absorbance (a.u)

Fig. 5. Physical parameters of nanofiber mats: (a) tensile strenght and (b) strain at break.

1,6

300

350

400

Wavelength (nm)

450 500

y = 1,6065x R² = 0,96684

1,2 0,8 0,4 0

250

b

0

0,2

0,4

0,6

0,8

1

1,2

Concentration of Achillea Lycaonica

Fig. 6. In vitro drug release profiles of nanofibrous mats: (a) Absorption spectra of different concentrations of Achillea lycaonica, (b) Achillea lycaonica calibration curve, and (c) Achillea lycaonica releasing profiles of Achillea lycaonicaloaded nanofibrous mats in three different concentrations (0.125, 0.250 and 0.500, wt%) according to first-order model and (d) Higuchi model. All the values were obtained from the averages of three experiments, and the errors were < 5%.

Fig. 4. DSC thermogram of pure PLA and Achillea lycaonica-loaded nanofibrous mats in three different concentrations (0.125, 0.250 and 0.500, wt%).

0.189 MPa for pure fiber to 0.273 MPa for 0.500 (wt%) concentration. As a result, it has been shown that the tensile strength increases as the diameter decreases [39].

3.5. Mechanical strength of nanofibers

3.6. Release of Achillea lycaonica from nanofibers

Tensile strength and strain at break were indicated for each samples (Fig. 5a and b). To load Achillea lycaonica into PLA increased the mechanical properties such as tensile strenght of fiber enhanced from

Drug releasing behaviors of Achillea lycaonica-loaded nanofibrous mats were analyzed according to first-order and Higuchi model. Firstly, the UV sprectra obtained with the concentration range of Achillea 5

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M.E. Cam, et al.

whereas 0.500 (wt%) concentration showed the burst release properties in 24 h (93.84%). For Achillea lycaonica-loaded nanofiber in 0.500 (wt %) concentration, 78.22% of the plant extract was released within 0.5 h. Thus, it has been shown that Achillea lycaonica was released from nanofibers in a controlled manner. In Higuchi model, the regression (R2) values were calculated independently for each samples. Calculation of kH was done by using Eq. (1) and kH was found as 5,809; 3,467 and 6,855 h−1 for three different concentrations (0.125, 0.250 and 0.500, wt%), respectively. Higuchi model was applied to data obtained for the final fiber formulation (Fig. 6d). The Higuchi model can be represented by below equation;

Mt = kHx t1/2

(1)

where kH is the Higuchi constant, Mt is the quantity of cumulative drug released at time t [40]. 3.7. In vitro cytotoxicity assays Achillea lycaonica-loaded nanofibers were cultured and analyzed on the NIH/3T3 (mouse embryo fibroblast) cell for 24 h and then SEM images were obtained (Fig. 7). In the SEM image, it is clearly observed that the cell clusters in three different concentrations of Achillea lycaonica-loaded nanofibers are proliferating, especially in 0.250 (wt%). Thus, it has been verified that Achillea lycaonica possess significant fibroblast proliferation properties as expected in literature data and traditional use [21–25]. The cytotoxicity of the electrospun nanofibrous mats was evaluated by the MTT assay. The cytotoxicity (MTT) tests indicated that Achillea lycaonica-loaded nanofibers had no cytotoxic effect compared to control. The Achillea lycaonica-loaded nanofibers in 0.250 and 0.500 (wt%) concentration showed excellent cell compatibility and increased the viability of cells compared to control group within 72 h (p < 0.05) (Fig. 8). Thus, the biocompatibility tests demonstrate that all composites have suitable cytocompatibility, and can be recommended for the further development of biomedical applications. Besides, the SEM images of the nanofibers are compatible with the MTT test results.

Fig. 7. SEM images of proliferated cells on (a, b) Achillea lycaonica-loaded nanofiber in 0.125 (wt%), (c, d) 0.250 (wt%), (e, f) 0.500 (wt%) concentration and (g, h) pure PLA nanofiber.

4. Conclusion In summary, different concentrations of Achillea lycaonica extracts were successfully loaded into PLA nanofiber mats via electrospinning method. Addition of the plant extract reduced the size of electrospun nanofiber diameters, which could be attributed to the increase in electrical conductivity. Among the nanofibers, Achillea lycaonica-loaded nanofiber in 0.500 (wt%) concentration had the lowest fiber average (571.89 nm) diameter. The cytotoxicity (MTT) tests indicated nanofibers had no cytotoxic effect and exhibited excellent cell compatibility and increased the viability of cells compared to control group within 72 h (p < 0.05), especially in 0.250 (wt%) and 0.500 (wt%) concentration but the most effective concentration was 0.250 (wt%). Loading Achillea lycaonica into PLA fibers increased the tensile strength and also Achillea lycaonica can be successfully released from nanofibers. 0.125 and 0.250 (wt%) concentrations exhibited sustained release, whereas 0.500 (wt%) concentration showed burst release in 24 h. The working temperature range between Tg and Tm enlarged for the composites having higher plant extract content. Consequently, Achillea lycaonica-loaded PLA nanofibers have promising potential for cell proliferation activity that cause faster and better wound healing.

Fig. 8. NIH/3T3 (mouse embryo fibroblast) cell proliferation and viability of pure nanofiber and Achillea lycaonica-loaded nanofibrous mats in three different concentrations (0.125, 0.250 and 0.500, wt%) at the end of 72 h. All data were presented as mean ± standard error of the mean. *p < 0.05 versus control group (3 T3).

lycaonica from 0,2 to 1 mg/mL and a linear standard calibration curve drawn from Achillea lycaonica absorption values (R2 = 0.9668) from these spectra were obtained for quantitative determination for drug releasing (Fig. 6a-b). The releasing profiles of Achillea lycaonica-loaded nanofibers were measured in phosphate-buffered saline (PBS) of pH 7.4 and controlled temperature of 37 °C to mimic the physiological conditions in the living organisms. As shown in Fig. 6c, Achillea lycaonica can be successfully released from nanofibers over 24 h according to firstorder model. Achillea lycaonica in 0.125 and 0.250 (wt%) concentrations exhibited more sustained release in 24 h (39.80% and 41.80%),

Acknowledgements Dr. Muhammet E. Cam was supported by a TUBITAK 2219 Research Programme Grant (Scientific and Technological Research Council of Turkey-TUBITAK) and thanks UCL Mechanical Engineering for hosting his post-doctoral research in the UK. 6

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References

[21] E.K. Akkol, U. Koca, I. Pesin, D. Yilmazer, Evaluation of the Wound Healing Potential of Achillea biebersteinii Afan. (Asteraceae) by In Vivo Excision and Incision Models, Evid. Based Complement, Alternat. Med. 2011 (2011) 474026. [22] Z. Ghobadian, M.R.H. Ahmadi, L. Rezazadeh, E. Hosseini, T. Kokhazadeh, S. Ghavam, In Vitro Evaluation of Achillea Millefolium on the Production and Stimulation of Human Skin Fibroblast Cells (HFS-PI-16), Med. Arch. 69 (2015) 212–217. [23] H. Tajik, F.S.S. Jajali, Influence of aqueous extract of yarrow on healing process of experimental burn wound in rabbit: clinical and microbiological study, J. Anim. Vet. Adv. 6 (2007) 1464–1468. [24] O.T. Agar, M. Dikmen, N. Ozturk, M.A. Yilmaz, H. Temel, F.P. Turkmenoglu, Comparative studies on phenolic composition, antioxidant, wound healing and cytotoxic activities of selected Achillea L Species Growing in Turkey, Molecules 20 (2015) 7976–8000. [25] A.T. Azaz, T. Arabaci, M.K. Sangun, B. Yildiz, Composition and the in vitro antimicrobial activities of the essential oils of Achillea wilhelmsii C. Koch. and Achillea lycaonica Boiss. & Heldr, Asian J. Chem. 20 (2008) 1238–1244. [26] I. Süntar, E.K. Akkol, L. Nahar, S.D. Sarker, Wound healing and antioxidant properties: do they coexist in plants? Free Rad. Antiox. 2 (2012) 1–7. [27] T. Taskin, D. Taskin, E. Rayaman, T. Dikpınar, S. Suzgec-Selcuk, T. Arabaci, Characterization of the Biological Activity and Phenolics in Achillea lycaonica, Anal. Lett. 51 (2018) 33–48. [28] T. Yang, D. Wu, L. Lu, W. Zhou, M. Zhang, Electrospinning of polylactide and its composites with carbon nanotubes, Polym. Composite. 32 (2011) 1280–1288. [29] A. Schönbächler, G. Olfa, J. Huwyler, M. Frenz, U. Pieles, Indocyanine green loaded biocompatible nanoparticles: Stabilization of indocyanine green (ICG) using biocompatible silica-poly(ε-caprolactone) grafted nanocomposites, J. Photochem. Photobiol. 261 (2013) 12–19. [30] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [31] C. Angammana, S. Jayaram, Analysis of the effects of solution conductivity on electrospinning process and fiber morphology, IEEE T. Ind. Appl. 47 (2011) 1109–1117. [32] K. Garg, G.L. Bowlin, Electrospinning jets and nanofibrous structures, Biomicrofluidics 5 (2011) 013403. [33] Z.R. Ege, A. Akan, F.N. Oktar, C.-C. Lin, B. Karademir, O. Gunduz, Encapsulation of indocyanine green in poly(lactic acid) nanofibers for using as a nanoprobe in biomedical diagnostics, Mater. Lett. 228 (2018) 148–151. [34] I. Petrov, B. Šoptrajanov, Infrared spectrum of whewellite, Spectrochim. Acta. A Mol. Biomol Spectrosc. 31 (1975) 309–316. [35] J. Mejuto, J. Rakmai, B. Cheirsilp, A. Torrado, J. Simal-Gándara, Encapsulation of yarrow essential oil in hydroxypropyl-beta- cyclodextrin: Physiochemical characterization and evaluation of bio-efficacies, CYTA-J. Food 15 (2017) 409–417. [36] W. Wang, A.H. Barber, Measurement of size-dependent glass transition temperature in electrospun polymer fibers using AFM nanomechanical testing, J. Polym. Sci. B 50 (2012) 546–551. [37] J.E. Oliveira, L.H.C. Mattoso, W.J. Orts, E.S. Medeiros, Structural and Morphological Characterization of Micro and Nanofibers Produced by Electrospinning and Solution Blow Spinning: A Comparative Study, Adv. Mater. Sci. Eng. 2013 (2013) 1–14. [38] H. Li, Q. Wang, Y. Xiao, C. Bao, W. Li, 25-Hydroxyvitamin D(3)-loaded PLA microspheres vitro characterization and application in diabetic periodontitis models, AAPS PharmSciTech 14 (2) (2013) 880–889. [39] S.-C. Wong, A. Baji, S. Leng, Effect of fiber diameter on tensile properties of electrospun poly(ɛ-caprolactone), Polymer 49 (21) (2008) 4713–4722. [40] A. Topsakal, M. Uzun, G. Ugar, A. Ozcan, E. Altun, F.N. Oktar, F. Ikram, O. Ozkan, H.T. Sasmazel, O. Gunduz, Development of amoxicillin-loaded electrospun polyurethane/chitosan/β-tricalcium phosphate scaffold for bone tissue regeneration, IEEE Trans. NanoBiosci. 17 (3) (2018) 321–328.

[1] J.R. Dias, S. Baptista-Silva, C.M.T.D. Oliveira, A. Sousa, A.L. Oliveira, P.J. Bártolo, P.L. Granja, In situ crosslinked electrospun gelatin nanofibers for skin regeneration, Eur. Polym. J. 95 (2017) 161–173. [2] T. Velnar, T. Bailey, V. Smrkolj, The wound healing process: an overview of the cellular and molecular mechanisms, J. Int. Med. Res. 37 (2009) 1528–1542. [3] G.I. Broughton, J.E. Janis, C.E. Attinger, Wound Healing: An Overview, Plast. Reconstr. Surg. 117 (2006) 1–32. [4] J.F. Guest, K. Vowden, P. Vowden, The health economic burden that acute and chronic wounds impose on an average clinical commissioning group/health board in the UK, J. Wound Care 26 (2017) 292–303. [5] K. Järbrink, G. Ni, H. Sönnergren, A. Schmidtchen, C. Pang, R. Bajpai, J. Car, The humanistic and economic burden of chronic wounds: a protocol for a systematic review, Syst. Rev. 6 (2017) 15. [6] L.J. Gould, Topical collagen-based biomaterials for chronic wounds: rationale and clinical application, Adv. Wound Care 5 (2016) 19–31. [7] O. Suwantong, U. Ruktanonchai, P. Supaphol, Electrospun cellulose acetate fiber mats containing asiaticoside or Centella asiatica crude extract and the release characteristics of asiaticoside, Polymer 49 (2008) 4239–4247. [8] F.-M. Chen, X. Liu, Advancing biomaterials of human origin for tissue engineering, Prog. Polym. Sci. 53 (2016) 86–168. [9] T. Sango, G. Stoclet, N. Joly, A. Marin, A.M. Cheumani Yona, L. Duchatel, M. Kor Ndikontar, J.M. Lefebvre, Water–soluble extracts from banana pseudo–stem as functional additives for polylactic acid: Thermal and mechanical investigations, Eur. Poly. J. 112 (2019) 466–476. [10] M. Santoro, S.R. Shah, J.L. Walker, A.G. Mikos, Poly(lactic acid) nanofibrous scaffolds for tissue engineering, Adv. Drug Deliv. Rev. 107 (2016) 206–212. [11] M.P. Arrieta, C. López de Dicastillo, L. Garrido, K. Roa, M.J. Galotto, Electrospun PVA fibers loaded with antioxidant fillers extracted from Durvillaea antarctica algae and their effect on plasticized PLA bionanocomposites, Eur. Poly. J. 103 (2018) 145–157. [12] S. Das, A.B. Baker, Biomaterials and Nanotherapeutics for Enhancing Skin Wound Healing, Front. Bioeng. Biotechnol. 4 (2016) 82. [13] E. Nemeth, J. Bernath, Biological activities of yarrow species (Achillea spp.), Curr. Pharm. Des. 14 (2008) 3151–3167. [14] A.G. Pirbalouti, A. Koohpayeh, I. Karimi, The wound healing activity of flower extracts of Punica granatum and Achillea kellalensis in Wistar rats, Acta Pol. Pharm. 67 (2010) 107–110. [15] F. Jaffary, M.A. Nilforoushzadeh, N. Tavakoli, B. Zolfaghari, F. Shahbazi, The efficacy of Achilles millefolium topical gel along with intralesional injection of glucantime in the treatment of acute cutaneous leishmaniasis major, Adv. Biomed. Res. 3 (2014) 111. [16] F. Candan, M. Unlu, B. Tepe, D. Daferera, M. Polissiou, A. Sokmen, H.A. Akpulat, Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae), J. Ethnopharmacol. 87 (2003) 215–220. [17] M. Skocibusic, N. Bezic, V. Dunkic, A. Radonic, Antibacterial activity of Achillea clavennae essential oil against respiratory tract pathogens, Fitoterapia 75 (2004) 733–736. [18] M.K. Al-Hindawi, I.H. Al-Deen, M.H. Nabi, M.A. Ismail, Anti-inflammatory activity of some Iraqi plants using intact rats, J. Ethnopharmacol. 26 (1989) 163–168. [19] C. Karamenderes, S. Apaydin, Antispasmodic effect of Achillea nobilis L. subsp. sipylea (O. Schwarz) Bassler on the rat isolated duodenum, J. Ethnopharmacol. 84 (2003) 175–179. [20] R. Vazirinejad, F. Ayoobi, M.K. Arababadi, M.M. Eftekharian, A. Darekordi, M. Goudarzvand, G. Hassanshahi, M.M. Taghavi, B.N. Ahmadabadi, D. Kennedy, A. Shamsizadeh, Effect of aqueous extract of Achillea millefolium on the development of experimental autoimmune encephalomyelitis in C57BL/6 mice, Indian, J. Pharmacol. 46 (2014) 303–308.

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