Optimization and characterization of electrospun bilayered PVA/collagen nanofibers loaded with quercetin for tissue engineering applications

Abstract

Bilayered electrospun membranes for skin tissue engineering and wound dressings were developed using Poly vinyl alcohol (PVA), collagen (COLL), chitosan (CH), and a tamarind seed polysaccharide (TSP) are crosslinked with quercetin (QU). To maintain a sustained release of drug from the membrane we have developed a novel crosslinked two layered stable PVA fibrous mat, top layer composed of PVA/CH/TSP and quercetin and the bottom layer is composed of PVA/CH/COLL. Developed bilayered scaffolds were characterized using Field Emission Scanning Electron Microscope (FE-SEM) and their surface morphologies exhibited random fibrous orientation with porous morphology. Cross sectional images showed the junction of the bilayered scaffold with different fiber morphology between the layers. The results revealed fibrous bilayer separation confirming the bilayered scaffold with uniform porosity which is beneficial for tissue engineering application, moreover the PVA fibrous mats are stable after crosslinking it with quercetin, more than 24 h. Invitro biological studies results also showed desirable cell viability on fibroblast cells with increased proliferation and adhesion on the bilayer material making it potential for targeted tissue engineering application.

Keywords

Biomaterial PVA tamarind seed polysaccharide collagen chitosan drug delivery

Introduction

Nanofibrous materials represent a significant category of biomaterials extensively utilized in tissue engineering applications and employed with range of naturally derived or synthetic biodegradable polymers and their combinations, to encapsulate cells, growth factors, proteins, etc., for diverse tissue regenerative purposes.^1,2 Nanofibrous scaffolds composed of biodegradable polymers find primary application in wound healing,³ nerve tissue regeneration,⁴ blood vessels,⁵ and cardiac tissue regeneration.⁶ These biomaterials mimic the nanofibrous architecture of the extracellular matrix (ECM), enhancing cell attachment and interaction. Techniques such as electrospinning, phase separation, and self-assembly are utilized to create ECM-mimicking nanofiber architecture, allowing for the development of fibers with various diameters with randomized and ordered orientations.⁷

Electrospinning technique emerge as a crucial tool for fabricating nanostructured fibers for diverse polymers, holding immense potential in tissue engineering applications.⁸ Nanofibrous mat provides a bionic microenvironment to maintain the structural integrity of regenerated tissue, which has an important influence on cell behavior regeneration and tissue repair.⁹ For the past few decades electrospinning technique has attracted many researchers of different field for developing various scaffolds from micron (>1 mm) down to nano range (<1000 nm) for tissue engineering applications. Nanofibers are formed when high electric voltage is introduced into the polymeric solution on a metallic needle causing instability in the polymer solution. The charged polymer solution due to applied electric field and reciprocal repulsion exceeds the surface tension and the droplets ejecting out are transformed into cones on increasing the intensity of electric field. Finest nanofiber emerges from conical polymer droplets (Taylor cone) and get collected in metal plate collector kept at an optimized distance.¹⁰ In the field of biomaterials natural and synthetic polymers are employed in electrospinning process, among them the natural polymeric collagen scaffolds find a prominent role in tissue engineering and drug delivery therapeutic application.¹¹

Collagen is synthesized by the fibroblast cells in the connective tissue and plays a critical role in maintaining molecular and cellular interaction with ECM, as that of the native tissue.¹² Collagen is a multifunctional protein with superior biocompatibility,^13,14 low immunogenicity¹⁵ and biodegradability.^16,17 Nanofibrous collagen, mimicking ECM structure, has demonstrated improved cell attachment, proliferation, and differentiation, making it suitable as a scaffold.¹⁷ Due to its poor mechanical strength, it is combined with other polymeric components such as chitosan, gelatin and silk to improve its strength while exploring its excellent cytocompatibility and proliferation efficiency.¹⁸

Chitosan is a linear natural polysaccharide composed of β-(1,4)-2-amido-2-deoxy- D- glucan (glucosamine) and β-(1,4)-2-acetamido-2-deoxy-D-glucan (acetyl glucosamine) units¹⁹ and is obtained by deacetylation of chitin from natural resources like crustaceans.²⁰ Chitosan possesses exceptional biocompatibility, low toxicity, immunostimulatory, antibacterial, antitumor, antifungal activity and anti-coagulant activity.^21,22 Chitosan based material as drug delivery systems are known to have more efficiency and less side effects.²³ A polysaccharide galactoxyloglucan extracted from tamarind seed is used as a bioactive material for tissue engineering and it has the potential to act against the cancer cells.²⁴ Tamarind indica belonging to family Leguminosae and widely grown in India. TSP is a natural polysaccharide with a molecular weight of 700–880 kDa, tamarind seed is used as anti-helminthic and antidiarrheal agent apart from the other component of fruit.²⁵ PVA is a biodegradable polymer which is nonionic and semicrystalline in nature.²⁶ Its application was limited due to its hydrophilicity, but chemical crosslinking of PVA has increased the stability and mechanical behavior using the chemical crosslinking agent glutaraldehyde.²⁷

In our previous study we have utilized polyphenol (quercetin) as a crosslinking agent to increase the stability of PVA. Quercetin is a naturally available polyphenolic flavonoid, with pharmacological activity such as antioxidants, anti-inflammatory, antiatherosclerosis.²⁸ Quercetin belongs to a class of flavonoids that exists abundantly in onions, apples, berries, nuts, cauliflower, cabbage and red wine.²⁹ It is a hydrophobic drug with poor bioavailability,³⁰ and it limits its application as a drug delivery vehicle. This study focuses on developing a bilayered electrospun nanofibrous scaffold for biomedical applications, particularly in tissue engineering and drug delivery. One layer consists of PVA, collagen, and chitosan, while the other comprises PVA, chitosan, and TSP, crosslinked using quercetin. The significance of this work lies in employing quercetin as a natural crosslinker for PVA/CH/TSP nanofibrous matrices, enhancing mechanical strength and stability while preserving biocompatibility. This bilayered structure develop a bionic microenvironment that closely mimics the ECM, facilitating tissue regeneration and repair. This innovative approach represents a promising strategy for developing advanced biomaterial scaffolds.

Materials and Method

Materials

Rat tail tendons were collected from the animal house of CSIR-CLRI. Tamarind seed were collected from Kanchipuram, Tamil Nadu. Petroleum Ether, Ethanol, Chloroform, Ferric chloride, ninhydrin, sulphuric acid, chitosan low molecular weight, and dimethyl sulfoxide (DMSO) was procured from SRL chemicals (India). Acetic acid, sodium chloride, PVA (molecular weight 1, 60,000), dialysis membrane (molecular weight –10 kDa), (4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbeccos Modified Eagle Medium (DMEM), acridine orange, fetal bovine serum (FBS), were procured from Hi-media (India). Quercetin hydrate was procured from the Sigma-Aldrich (USA).

Extraction of Tamarind Seed Polysaccharide

TSP was extracted from Tamarindus indica seeds. The outer shells were removed by drying the seeds in a hot air oven at 40°C for 20 min, followed by crushing. The inner part of seed, kernels were crushed and ball-milled at 350 rpm for 20 min. To remove lipids, 10 g of powdered tamarind seed was mixed with 30 ml of petroleum ether and kept under stirring for 72 h, then dried. The 10ml defatted residue was mixed with 100 ml of double-distilled water and added to 400 ml of boiling water, continuous stirring at 260 rpm for 24 h without forming any lumps. The mixture was then centrifuged at 1000 rpm for 5 minutes. The supernatant was collected and stored at 4ºC for further use. To concentrate the polysaccharide, 400 ml of the supernatant was mixed with an equal volume of ethanol and stirred overnight at 4ºC. The precipitated solution was then combined with an equal volume of chloroform. This mixture was poured into a separation funnel and left undisturbed for 12 h. The protein-free samples were dialyzed against water for 4 days, and the dialyzed samples were lyophilized at −80ºC for 8 h. The resulting polysaccharide powder was then stored for further experiments.³¹

Extraction of Type I Collagen

Type I collagen was extracted from RTT. Rat tails were collected, washed with cold water, and the tendons were isolated. The tendon fibrils were washed and incubated in 0.9% saline. These fibrils were then immersed in 0.5 M acetic acid and stirred at 4ºC until completely dissolved. The solution was centrifuged at 6000 rpm for 10 min at 4ºC, and the pellet obtained was dissolved in 0.25 M acetic acid. After centrifugation, the supernatant was collected and subjected to salt precipitation by slowly adding sodium chloride. The precipitated collagen was thoroughly washed with distilled water to residual salt. The pellet was then dissolved in 0.05 M acetic acid, treated with pepsin to remove the telopeptide regions, and dialyzed against 20 mM disodium hydrogen phosphate, 50 mM acetic acid, and distilled water at 4ºC for 3 days. The dialyzed collagen was freeze-dried at −80ºC and stored at 4ºC for future use.³²

Sample Preparation

Sample I (PVA/CH/TSP/QU)

A PVA solution (molecular weight ∼160,000) was prepared by dissolving 10% (w/v) of PVA in double-distilled water and stirring it overnight on a magnetic stirrer at 400 rpm. CH was dissolved in 0.5 M acetic acid and stirred overnight on a magnetic stirrer at 600 rpm. A 0.5% (w/v) solution of chitosan and a 0.5% (w/v) solution of tamarind seed polysaccharide were both dissolved in 0.5 M acetic acid and stirred overnight to achieve a homogeneous mixture. A composite solution was then prepared by blending PVA, chitosan, and tamarind seed polysaccharide in a ratio of 7:1.5:1.5. Once the composite solution was homogeneous, it was placed in an ultrasonic bath to remove any air bubbles. Quercetin drug solution was prepared by dissolving 0.05% (w/v) quercetin powder in 100 µl of DMSO. This drug solution was then added dropwise to 10 ml of the composite solution and stirred for 1 h, with the mixture completely covered with aluminum foil to prevent light interference.

Sample II (PVA/COLL/CH)

PVA solution was made by dissolving 10% (w/v) PVA in double-distilled water and stirring overnight at 400 rpm. CH at 0.5% (w/v) and a 0.3% (w/v) RTT collagen solution were prepared using 0.05 M acetic acid and stirred cold on a magnetic stirrer at 900 rpm for 48 h. A composite solution was then prepared in a ratio of 7:1.5:1.5 and stirred cold for 2 h.

Electrospinning process

A nanofibrous bilayer scaffold was developed using electrospinning technique. The electrospinning setup was composed of a syringe pump, a high voltage power supply and a collector. To develop a nanofibrous mat, a syringe (22 gauge) was filled with 5 ml of each prepared polymeric solution and was mounted on to the syringe pump, and the positive electrode of the high voltage power supply was connected to the needle. Upon adjusting the applied voltage, fiber was collected on to the collector wrapped with aluminum foil and applied voltage, distance between collector and syringe pump as mentioned in Table 1.

Table 1.

A applied voltage, flow rate and distance between spinneret tip and collector.

Polymeric solution	Flow rate	Applied voltage	Distance between spinneret tip & collector
PVA	1 ml/h	22 kV	13 cm
PVA/QU	1 ml/h	22.5 kV	13 cm
PVA/QU/CH/TSP	1 ml/h	23.7 kV	13 cm
PVA/CH/COLL	1 ml/h	24 kV	13 cm

Bilayer scaffold preparation

Bilayer scaffold was prepared using electrospinning PVA/COLL/CH fibers directly onto a layer of PVA/CH/TSP/QU nanofibers, resulting in a PVA/CH/TSP/QU–PVA/COLL/CH bilayer scaffold. All electrospun fibrous membranes were then stored in a desiccator for further characterization.

Characterization of tamarind seed polysaccharide and type I collagen

Phytochemical characterization

Tamarind seed Polysaccharide solution was prepared by dissolving 0.1% (w/v) of lyophilized TSP in double distilled water and stirring it overnight on a magnetic stirrer at 700 rpm. The phytochemical characterization of the extract was followed as described below,

Test for carbohydrate: 5% phenol solution was prepared and sulphuric acid was added to the test solution, yellow orange color indicates the presence of carbohydrate.

Test for tannin: 5% (w/v) ferric chloride solution was prepared, few drops of this solution was added to the test solution, Dark green color indicates presence of tannin.

Test for protein: 2% (w/v) ninhydrin solution was prepared, few drops of this solution was added to the test solution and test tubes were kept in warm water for 5 min, Deep blue indicates presence of amino acid.

Test for saponin: The test solution, 20 ml of water was added to this 1 ml of alcohol was added and kept in a shaker for 15 min, 1 cm layer of foam indicates presence of saponin.

The total amount of carbohydrates was estimated by the phenol sulphuric acid method. Carbohydrates react with phenol and sulphuric acid to give yellow-orange color.^33,34 Due to the presence of sulphuric acid, it dehydrates polysaccharide and carbohydrates which converts nonreducing sugars to reducing sugars. The amount of reducing sugar was measured using a UV spectrophotometer and the absorbance was read at 490 nm.

Fourier transform infrared spectroscopy (FTIR) and circular dichroism studies

The FTIR spectral analysis of tamarind seed polysaccharide was performed under attenuated total reflectance (ATR) mode Jasco FTIR 4000 instrument. The spectrum was recorded in transmittance mode and scanned over a range of 4000-400 cm^-1. Collagen solution extracted from RTT was analyzed for conformational changes using Circular Dichroism (CD). Conformation of type I collagen (0.3 mg/ml) in 50 mM acetic acid was recorded in the far UV region Jasco 715 CD spectropolarimeter. CD spectrum was recorded in a range of 190–250 nm.³⁵

Characterization of Nanofiber

The morphology of PVA, PVA/QU, PVA/CH/TSP/QU, PVA/CH/COLL and the bilayered scaffolds were observed under FESEM [TESCAN S9000]. Samples were mounted on stubs and sputter coated with Au grid to make the scaffold conductive. To determine the hydrophilicity of scaffolds, static contact angle was measured using sessile drop method using a goniometer (VCA Optima Pvt, Ltd). During measurement, electrospun mats were cut into 1 cm² size and they were placed on the testing plate. A 10 µl drop of distilled water was dropped on the surface of the nanofibrous mat and the images were recorded immediately using a high-resolution camera. Five different samples were measured to obtain concordant results.³⁶ Mechanical properties of the electro spun scaffolds were determined using a tabletop uniaxial tensile tester (INSTRON Corp). Nanofibrous samples were cut into dumbbell shape with a dimension of 45 mm length and 5 mm breadth, the thickness of the sample was around 60 µm.³⁴ Triplicates of samples were tested for the data, tensile strength and elongation at break were calculated. X-Ray diffraction (XRD) study was used to find out the crystallinity of the developed scaffold. XRD was registered in the 2.5Ө range of 5–80º using X-Ray diffractometer (Bruker D8 Focus). The applied voltage was 30 kV and the current was 15 mA with a step size 4º per minute.³⁷ Quercetin released from the nanofibrous mat was determined using phosphate buffer saline (PBS) pH 7.4 as the release medium. Nanofibrous mats were suspended in 10 ml of the PBS and kept under stirring at 500 rpm. At specified time interval, 100 µl from the medium was taken out and replaced with 100 µl fresh PBS. The concentration of quercetin in the release medium was measured by microplate reader (synergy, Biotek) at 370 nm.³⁸ To evaluate the cytotoxicity of the electrospun mats, an MTT assay (extraction method) was performed. L929 cells were cultured in complete DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a 96-well plate for 24 h at 37°C and 5% CO₂. Sterilized 1 cm² sections of the mats were incubated with the complete media for 24 h. Subsequently, 200 µl of the media containing the extract was transferred to L929 cells, with a control group receiving only media. After 24 h and 72 h of exposure, the cells were incubated with MTT dye for 4 h at 37°C. The resulting formazan crystals were dissolved in DMSO, and the optical density of each well was measured at 570 nm using a microplate reader. Cell viability data were expressed as a percentage relative to the control group, reflecting the potential cytotoxic effects of the electrospun mats. To further assess cell proliferation and viability, cells were exposed to the test extract for 72 h, then washed with PBS. They were stained with 100 µg/ml acridine orange in PBS (pH 7.4) for 10–12 min in the dark, rinsed with PBS, and examined with an inverted phase contrast epi-fluorescence microscope (Leica DMi8).³⁹

Results and discussion

Characterization of tamarind seed polysaccharide and type I collagen

Galactoxyloglucan is comprised of three sugars glucose, xylose and galactose an oligopoly saccharide capable of gel formation and can be used in various applications. In the present work extraction of TSP was carried out using aqueous solution and concentrated by ethanol precipitation. The results of phytochemical tests for the isolated tamarind seed polysaccharide were confirmed using phenol-sulfuric acid method. Phenol and sulfuric acid react with TSP and formation of yellow orange color was observed which indicates the presence of carbohydrate.⁴⁰ Tests for the presence of tannin, protein and saponin confirmed their absence in the TSP. The FTIR spectrum of the extract was provided in Figure 1(a). FTIR spectrum of extract shows its characteristic frequency of vibration a broad and intense peak appearing at 3,311 cm^-1 which is attributed due to the -OH stretching because of the strong intra and intermolecular hydrogen bonding, a narrow peak at 2,590 cm^-1 is attributed due to the asymmetric and symmetric stretching vibration of -CH₂ group and peak appearing at 1,623 cm^-1 is attributed to the stretching vibration of C=O groups. The peak at 1,050 cm^-1 is due to the stretching vibration of C-O-C. The characteristic triple helical structure of collagen (3 mg/ml) was confirmed through circular dichroism studies,⁴¹ as illustrated in Figure 1(b). Typically, native collagen displays a distinct CD spectrum with a positive peak at 219 nm and a negative peak at 199 nm. These peaks are indicative of the triple helical configuration of collagen. The presence of these spectral features confirms that the collagen maintains its native helical conformation, which is essential for its biological functions and structural integrity. The 219 nm peak reflects the ordered arrangement of the helical strands, while the 199 nm negative peak is associated with the unique secondary structure of collagen, further validating the integrity of the collagen triple helix.

Morphological characterization of nanofibrous mat

Structural morphology of the PVA and PVA blends were studied using FE-SEM analysis and the FE-SEM micrographs are given in Figure 2. The concentration and viscosity of the PVA solutions is an important parameter in determining the morphology of nanofiber. PVA/QU nanofibers exhibited higher diameter compared to PVA/QU/CH/TSP and PVA/COLL nanofibers. Addition of bioactive compounds resulted in a decrease in diameter of nanofiber which may be attributed due to addition of additives and the solvent evaporation rate during fiber formation. Concentration of PVA is also an important parameter determining the electrospinnability of a solution 10% PVA solution resulted in better fiber formation with addition of CH and other bioactive components no solution clogging or bead formation was observed with 10% PVA concentration along with the additives. The surface morphology of nanofibrous matrices were uniform, highly interconnected fiber alignment and are randomly orientated with high porosity. Surface morphology of PVA/QU fiber showed a higher diameter and merging of fiber, which may be due to less solvent evaporation in case of PVA/QU/TSP/CH and collagen loaded polymeric sample. The morphological observation also revealed that blending of PVA/QU/CH and TSP has resulted in better fiber morphology with 8% and 10% PVA concentration without any beads. However, further increasing the concentration of polymer and additives resulted in increased fiber diameter and increase in spinnability of the polymer solution. One of the crucial factors influencing the biological property of an implanted material is the surface wettability, which is a measurement of surface energy that determines the degree of implant material in contact with the physiologic environment.⁴² Protein absorption, platelet adhesion/activation, blood coagulation, and cell bacterial adhesion depends on the surface wettability property of the biomaterial.⁴³ Hydrophobic surface has poor cellular attachment, and the hydrophilic surface have moderate and better cell attachments property depending on the polymeric substrate.⁴⁴ The highest contact angle of the developed electro spun PVA/QU/CH/TSP composite based nanofiber mats was observed to be 63.84º indicating a typical hydrophilic feature of nanofiber. Contact angle of pure PVA nanofiber mats were found to be 0º and there is an increase in the contact angle of PVA mats after the incorporation or blending with quercetin, chitosan and collagen. By the incorporation of chitosan, TSP and quercetin hydrophilicity of nanofiber mats were found to be increased as compared with collagen, and chitosan. Contact angle with respective PVA composite was shown in Figure 3. XRD diffraction of PVA and PVA blends were shown in Figure (4a). The XRD pattern of pure PVA mats showed a significant crystalline peak 2Ө values at 19.8° and 20.8° respectively, representing the reflection from (101) and (101) with high intensity, whereas in case of blended PVA sample tend to lose the crystalline nature of PVA by the incorporation of additives a significantly manifested angle of diffraction. By incorporating QU, CH, TSP, COLL there is no significant change in the 2Ө value as the 70% of mat comprises parent polymer PVA. It was observed that electrospinning of PVA begin loses its crystalline nature, leading to a more amorphous morphology upon addition of bioactive components. Low crystallinity of PVA nanofiber may be attributed due to the solvent entrapment in the polymer solution.⁴⁴ If two polymers are miscible, chemical interaction between the individual polymer chains causes shifts in wavenumber and bands in the spectra of the blends. Therefore, no major shift in peak was observed for bilayered scaffold. The FTIR spectrum of PVA and its blend are illustrated in Figure 4(b). The FTIR spectrum of PVA shows its characteristic frequency of vibration of PVA nanofiber, the broad and intense peak appearing at 3,370 cm^-1 is attributed to- OH and -NH₂ stretching vibration because of the strong intra- and intermolecular hydrogen bonding. Narrow peak at 2,986 cm^-1 is attributed to the asymmetric and symmetric stretching vibration of the -CH₂ group. Residual vinyl acetate repeats in PVA due to residual peak appearing at 1,733 cm^-1 is attributed to stretching vibration of C=O groups. Peak at 1,090 cm^-1 is due to stretching vibration of acetyl linkage (C-O-C). Comparing with quercetin loaded PVA fiber, absorbance peak intensity at 3,370 cm^-1 of PVA/QU nanofiber decrease with decrease in concentration of PVA whereas PVA/QU/CH/TSP nanofiber absorbance peak was further as compare with previous nanofiber this happen due to the addition chitosan and tamarind seed polysaccharide decreases the concentration of PVA. This could be due to the reduction of hydroxyl groups. PVA/QU and PVA/QU/CH/TSP nanofibers have the same intensity at 2,986 and 1,090 cm^-1 where PVA nanofiber intensity is observed. Each application in tissue regeneration and drug delivery requires a different acceptability range for mechanical properties, as it is desirable for scaffolds to have mechanical properties close to those of native tissues or organs.⁴⁵ Table 2 shows the value of tensile strength, load, elongation at break, and maximum extension of PVA and QU, CH, TSP, COLL incorporated samples. It was observed that PVA nanofibrous mat showed a higher tensile strength but lower elongation at break compared to the blended nanofibrous mat. The cumulative in vitro release of quercetin is shown in Figure 5. These releasing patterns shown an initial burst release of 55% (of total drug) within 10 h followed by a slow and steady release of quercetin. Initial burst release of quercetin from nanofibrous mat may be due to the agents loosely bonded external drug molecule and also due to the hydrolysis of PVA upon starts to losing its stability with water. The study investigated the biocompatibility of various electrospun bilayered mats using the MTT assay with L929 cells. Figure 6 (a) and (b) The cell line morphology and in vitro cytotoxicity revealed excellent biocompatibility for all electrospun mats, with viability exceeding 80% after 24 and 72 h respectively. Interestingly, the composition of the mats influenced cell growth and better proliferation. Collagen in PVA/CH/COLL and bilayer mats promoted the highest viability, suggesting its potential to enhance cell proliferation. PVA/QU/CH/TSP exhibited increased viability compared to PVA/QU, possibly due to the combined effects of tamarind polysaccharide and chitosan. This finding aligns with the morphological analysis and live cell population observed using acridine orange staining after 72 h of exposure. Both results support that the prepared electro spun mats are not cytotoxic to the L929 cells. These findings highlight the possibility of tailoring the materials composition to optimize cell proliferation for specific biomedical applications.

Table 2.

Tensile strength profile of nanofibrous mat.

Sample	Load (N)	Tensile strength (MPa)	Elongation at Break (MPa)	Maximum Extension (mm)
PVA	4.98	7.70	54.56	16.37
PVA/QU	5.32	3.95	55.8	20.57
PVA/QU/CH/TSP	6.59	4.25	62.97	22.98
PVA/CH/COLL	5.45	4.1	61.4	21.5

Conclusion

In the present study, TSP and Type I collagen were characterized to evaluate their feasibility for various biomedical applications. TSP was extracted and confirmed using the phenol-sulfuric acid method and FTIR analysis, revealing the presence of carbohydrates absence of tannin, protein, or saponin. Circular dichroism analysis confirmed the triple-helical structure of Type I collagen, ensuring its native form, which is essential for biological functionality. Bilayered electrospun membranes composed of PVA, collagen, chitosan, TSP, and quercetin were developed, demonstrating significantly enhanced properties for tissue engineering. Morphological characterization through FE-SEM illustrated that the incorporation of quercetin, chitosan, and TSP to PVA blends resulted in reduced fiber diameters, thereby improving mechanical properties and flexibility. The electrospun membranes exhibited a contact angle of 63.84º for the PVA/QU/CH/TSP composite, indicating the stability PVA acquired due to chitosan, collagen and quercetin which is crucial for cellular interactions. XRD analysis indicated a reduction in crystallinity due to the presence of bioactive additives, which may enhance the material’s biocompatibility. PVA mats exhibited higher tensile strength but lower elongation at break compared to blended nanofibers. The addition of bioactive additives reduced tensile strength while enhancing elongation, due to the incorporation of bioactive compounds. The cumulative release profile of quercetin from the electrospun fibers demonstrated an initial burst release of 55% within the first 10 h, followed by a sustained release, indicating potential for controlled drug delivery. The biocompatibility of the electrospun nanofibers was confirmed through MTT assay, revealing cell viability exceeding 80% after 24 and 72 h of exposure, with PVA/CH/COLL mats showing the highest viability of 90%. Additionally, the mechanical properties under physiological conditions require further investigation to ensure suitability for specific tissue engineering applications. Future work should focus on comprehensive in vivo studies to evaluate the long-term performance of these materials in biological systems. Overall, these electrospun mats exhibit strong potential for applications in tissue engineering and drug delivery, with opportunities for further optimization tailored to specific biomedical uses.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors would like to acknowledge ICMR DHR (Department of Health Research), Govt. of India, Ministry of Health and Family Welfare Project No: 12014/31/2020 & CATERS analytical facility utilized for the present research work. CSIR-CLRI Communication Number: A/2023/BIC/MLP/1922.

ORCID iD

Balaraman Madhan

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