Nanofibrous membranes loaded with bupivacaine and carica papaya extract for pain management and wound healing in postoperative wounds

PCL (Mw 80000), PVA, Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), lipopolysaccharide (LPS), MTT, Bupivacaine hydrochloride, were purchased from Sigma Aldrich, USA. Ethanolic extract of carica papaya was purchased from Barij Essence company located at Tehran, Iran. L929 fibroblast and RAW 264.7 cell lines were purchased from Pasteur Institute, Tehran, Iran. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), RPMI-1640 medium, and penicillin-streptomycin were purchased from Invitrogen, USA. Dimethyl sulfoxide (DMSO), phosphate buffer solution (PBS), acetic acid, H₂O₂, methanol were purchased from Merck, Germany.

Firstly, PCL was dissolved in acetic acid at 14 wt.% for 12 hours. Then, PVA was separately dissolved in distilled water at 12 wt.% for 12 hours. Next, 250 mg of Bupivacaine hydrochloride was added to 5 ml of the PVA solution and mixed for 6 hours. Subsequently, carica papaya extract was added to the PVA/bupivacaine solution at 5 v/v% and mixed for 6 hours. Finally, PCL and PVA/bupivacaine/carica papaya extract solutions were loaded into two separate syringes and fixed in a double-nozzle electrospinning device. The PCL solution was then electrospun at 18 kV, a polymer feeding rate of 0.5 ml/hour, a needle-to-collector distance of 15 cm, and a mandrel turning rate of 500 rpm, while the PVA/bupivacaine/carica papaya extract solution was electrospun at 18 kV, a polymer feeding rate of 1.2 ml/hour, and a needle-to-collector distance of 16 cm on the same collector. Figure 1 shows the schematic illustration of our fabrication method.

Fig. 1.

Schematic illustration representing the fabrication process of BUPCPEPCLPVA and PCLPVA scaffolds

BUPCPEPCLPVA and PCLPVA scaffolds were coated with gold for 250 seconds followed by imaging under 25 kV accelerating high voltage.

In the assessment of ultimate tensile strength for BUPCPEPCLPVA and PCLPVA scaffolds, a standardized testing procedure based on ASTM guidelines was followed. Rectangular samples of both scaffold types were prepared with dimensions of 40 mm length and 15 mm width. Then, the sample thickness was measured (250–300 μm). Tensile testing was carried out using a universal testing machine (UTM) with a load cell capacity of 500 N. The samples were clamped securely into the UTM. A constant crosshead speed of 1 mm/min was chosen to maintain a consistent deformation rate during testing. As the axial load was applied to the samples, load and displacement data were continuously recorded until the scaffolds failed. Finally, ultimate tensile strength was calculated using the acquired data.

The viability of L929 fibroblast cells cultured on BUPCPEPCLPVA and PCLPVA scaffolds was assessed using the MTT assay on days 1, 3, and 5. The MTT assay is a widely used colorimetric method that measures the activity of mitochondrial enzymes in viable cells, providing an indirect measure of cell viability. Prior to cell seeding, scaffolds were immersed in 70 v/v% ethanol for 10 minutes and allowed to dry for 3–4 hours. Then, the constructs were irradiated with UV for 30 minutes. L929 fibroblast cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen, USA). Cells were expanded to reach 80% confluence. On each assessment day, 1 × 10⁴ L929 fibroblast cells were seeded onto each scaffold in a 96-well plate. The cell-seeded scaffolds were cultured for 5 days. For the MTT assay, on days 1, 3, and 5, the culture medium was removed from the wells, and 200 μl of MTT solution (0.5 mg/ml in PBS) was added to each well. The scaffolds were incubated with the MTT solution for 4 hours at 37°C to allow the formazan crystals to form. Following this, the MTT solution was aspirated, and 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The microplates were agitated for 10 minutes on a shaker to ensure complete dissolution. The resulting solution was transferred to a microplate reader, and the absorbance was measured at a wavelength of 570 nm. The absorbance measurement was proportional to the number of viable cells on the scaffolds.

BUPCPEPCLPVA and PCLPVA scaffolds were sterilized as described in sections 2–4. The RAW 264.7 murine macrophage cell line was cultivated in RPMI-1640 medium, which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen, USA). For the evaluation process, the scaffolds were positioned within cell culture inserts inside 24-well plates. RAW 264.7 macrophages were introduced onto the scaffolds at a density of 2 × 10⁵ cells per scaffold and allowed to firmly attach for a period of 24 hours. Subsequently, an inflammatory response was induced by introducing lipopolysaccharide (LPS) into the culture medium at a concentration of 100 ng/ml. Following 24 hours of LPS stimulation, the culture medium was gathered from each well. The concentration of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), present in the medium, was determined using enzyme-linked immunosorbent assay (ELISA) kits (Abcam, USA) as per the guidelines provided by the manufacturer. To evaluate the anti-inflammatory potential, the levels of TNF-α and IL-6 in the culture medium from macrophages cultured on BUPCPEPCLPVA and PCLPVA scaffolds were contrasted with those from macrophages grown on a tissue culture plate.

The release of bupivacaine and carica papaya extract from the nanofibers made of BUPCPEPCLPVA was evaluated via UV-visible spectroscopy at 255 nm and 319 nm, correspondingly. To outline the process, 150 mg of the scaffolds were immersed in a 20 ml solution of phosphate buffer (PBS) and maintained at a temperature of 37 °C over the course of 48 hours. At various intervals, 0.5 ml of the released solution was collected, and an equivalent volume of fresh PBS was introduced into the release medium. Subsequently, the absorbance levels of the extracted samples were measured, and these values were then aligned with the standardized curves of bupivacaine and carica papaya extract within the PBS solution. The cumulative release of the two substances was eventually computed separately for each of the drug components.

The assessment of in vitro wound closure activity for BUPCPEPCLPVA and PCLPVA scaffolds was carried out using L929 fibroblast cells at two points: days 0 and 48. The objective of this study was to investigate the potential wound healing properties of these scaffold materials using a cellbased model. A predetermined weight (120 mg) of the BUPCPEPCLPVA and PCLPVA scaffolds was immersed in 5 ml DMEM media and kept for 7 days. Then, the media was filtered and kept at 4 °C until use. L929 fibroblast cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained in a controlled environment of 37°C, 5% CO₂, and 95% humidity. For the assessment, linear wounds were created on confluent monolayers of L929 cells cultured in 24-well plates. A sterile pipette tip was used to scrape the cell monolayer, resulting in a consistent wound area. The wounded monolayers were then washed gently with PBS to remove detached cells and debris. A subset of wells served as control groups with wounded cells but no scaffold. Then, the cells were cultured with the scaffolds’ extract for 48 hours. Images of the wounded areas were captured at both day 0 and day 48 using a phase-contrast microscope equipped with a camera. The images allowed for the measurement of wound closure by comparing the initial wound area (day 0) with the wound area at day 48. Image analysis software was used to measure the wound area and calculate the percentage of wound closure.

Besides cell viability assay under normal conditions, which was explained in sections 2–5, we wanted to see if our developed scaffolds can protect cells under oxidative stress. BUPCPEPCLPVA and PCLPVA scaffolds were sterilized and seeded with 10000 L929 fibroblast cells in 96-well plates and cultured for 48 hours. Then, the culture media in each well was supplemented with 1% v/v H₂O₂ and cells were incubated for 1 hour. Finally, the viability of cells in each group was assessed using MTT assay. For the MTT assay, the culture medium was removed from the wells, and 200 μl of MTT solution (0.5 mg/ml in PBS) was added to each well. The scaffolds were incubated with the MTT solution for 4 hours at 37°C to allow the formazan crystals to form. Following this, the MTT solution was aspirated, and 200 μl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The microplates were agitated for 10 minutes on a shaker to ensure complete dissolution. The resulting solution was transferred to a microplate reader, and the absorbance was measured at a wavelength of 570 nm. The absorbance measurement was proportional to the number of viable cells on the scaffolds.

To elucidate the mechanism by which the electrospun dressings protected L929 cells against oxidative stress, the radical scavenging activity of the samples was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, a widely accepted method for evaluating antioxidant properties. This assay aimed to determine the ability of these scaffold materials to neutralize free radicals and exhibit antioxidant behavior. The DPPH reagent was prepared by dissolving DPPH in methanol to create a 0.1 mM solution. A series of scaffold extract solutions were prepared by immersing different weights of each scaffold type in 10 ml of methanol and keeping them for 7 days. The solutions were kept in a shaker incubator to facilitate the release of potential antioxidant compounds from the scaffolds. To initiate the assay, 2 ml of each scaffold extract solution was mixed with 2 ml of the DPPH solution. The mixture was allowed to react in the dark for 30 minutes at room temperature. The absorbance of the resulting solutions was measured at 517 nm using a UV-visible spectrophotometer. Lower absorbance values indicated higher radical scavenging activity, reflecting the antioxidant potential of the scaffold extracts. As positive and negative controls, ascorbic acid (vitamin C) and methanol, respectively, were used. The percentage of DPPH scavenging activity was calculated using the formula: Scavengingactivity(%)=(Absorbanceofcontrol−Absorbanceofsample/Absorbanceofcontrol)×100 \[\text{Scavenging}\,\text{activity}\,\left( % \right)=\left( \text{Absorbance}\,\text{of}\,\text{control}-\text{Absorbance}\,\text{of}\,\text{sample/Absorbance}\,\text{of}\,\text{control} \right)\times \text{100}\]

The rat model of excisional wound injury was utilized to investigate the efficacy of various treatment groups in promoting healing. The research protocol received ethical approval from the university’s ethics committee and was conducted following established guidelines. The study involved the use of nine adult male Sprague-Dawley rats, each weighing around 250–300 grams. The rats were housed under standard laboratory conditions that maintained controlled temperature, humidity, and a 12-hour light-dark cycle. A one-week acclimatization period was observed before commencing the experiment.

Before initiating the surgical procedure, the rats were subjected to anesthesia via an intraperitoneal injection of a blend containing ketamine (80 mg/kg) and xylazine (10 mg/kg). The anesthesia’s adequacy was verified by monitoring the absence of reflex responses to noxious stimuli. Pre-surgery preparations included shaving the dorsal region of each rat and performing thorough disinfection using a povidone-iodine solution. Employing a sterile scalpel blade, a full-thickness excisional wound was meticulously created on the dorsum of each rat. A circular area, demarcated by a sterile template, exhibited a 1.5 cm diameter. Subsequently, this marked region was excised down to the underlying fascia, with meticulous removal of the overlying skin and subcutaneous tissue. Hemostasis was accomplished using sterile gauze pads and gentle pressure.

Following the wound creation phase, the rats were randomly categorized into these groups, ensuring an unbiased distribution. Each group comprised three rats, allowing for statistical analysis and comparison between the different treatments. The groups were as follows:

The “BUPCPEPCLPVA” group involved rats treated with BUPCPEPCLPVA scaffolds.

On the 7th and 14th days post-injury, the wounds were visually evaluated using a digital camera, and the percentage of wound size reduction was quantified utilizing the formula: Wound Size Reduction (%) = ((Initial Wound Size – Current Wound Size) / Initial Wound Size) × 100.

After the completion of the experiment on the 14th day post-injury, the animals were euthanized to collect wound tissues for histopathological examinations. The sampling process involved several steps to ensure the preservation and proper analysis of the tissues. First, following euthanasia, the wound tissues were meticulously collected from the dorsal region of each rat. Care was taken to include the entire area of the excisional wounds to capture the full extent of tissue regeneration and repair. The collected tissues were then fixed in a 3.7% formalin solution, which helps preserve the tissue structure by preventing degradation. Subsequently, the fixed tissues were embedded in paraffin wax. The embedded tissues were sliced into thin sections, around 5 μm in thickness, using a microtome. These tissue sections were then mounted onto glass slides for further processing and staining. Both H&E (Hematoxylin and Eosin) staining and Masson’s trichrome staining techniques were employed to visualize different aspects of tissue morphology and composition. Throughout the sampling process, strict adherence to sterile techniques was maintained to minimize contamination and ensure the integrity of the tissue samples. An independent histopathologist, who was blinded to the study details to prevent bias, was responsible for analyzing the stained tissue slides.

To evaluate reactions to thermal stimuli, every rat was positioned within a transparent acrylic enclosure (measuring 20 × 9.5 × 12.5 cm) on days 7 and 14. A period of 15 minutes was allowed for the rats to adapt within the testing enclosure before the application of focused (4 × 6 mm) radiant heat originating from a 50-W light bulb. This heat was directed onto the skin injury area, and a total of 4 trials were conducted. A 20-second limit was imposed to avoid any potential harm to the tissues. The time elapsed from the start of the light bulb exposure till the animal’s response via screaming or sudden movement was recorded. As a point of reference, the healthy shaved skin of each rat was employed as a control for comparison.

On day 14th, the animals were sacrificed the wound tissues were harvested for detecting the tissue concentrations of (tumor necrosis factor-a) TNF-a, (interleukin-6) IL-6, Glutathione peroxidase (GPx), and Transforming growth factor (TGF-β) using enzyme-linked immunosorbent assay (ELISA). Briefly, tissue samples were collected and stored at -80°C until analysis. ELISA kits specific for TNF-a, IL-6, GPx, and TGF-β were obtained from Abcam, USA and used for the experiment according to the instructions provided by the manufacturer.

The data were analyzed using GraphPad Prism version 5, employing the student’s t-test and oneway ANOVA techniques.