Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine for diabetic foot ulcer healing: in vitro and in vivo studies

Functional dressing with tailored physicochemical and biological properties is vital for diabetic foot ulcer (DFU) treatment. Our main objective in the current study was to fabricate Cellulose Acetate/Gelatin (CA/Gel) electrospun nanofibrous mat loaded with berberine (Beri) as the DFU dressing. The results demonstrated that the diameter of the nanofibers was around 502 nm, the tensile strength, contact angle, porosity, water vapor permeability, and water uptake ratio of CA/Gel nanofibers were around 2.83 MPa, 58.07, 78.17 %, 11.23 mg/cm2 hr, and 12.78 respectively, while these values for CA/Gel/Beri nanofibers were 2.69 ± 0.05 MPa, 56.93 ± 1, 76.17 ± 0.76 %, 10.17 ± 0.21 mg/cm2 hr, 14.37 ± 0.42 respectively. The bacterial evaluations demonstrated that the dressings are an excellent barrier against bacterial penetration with potent antibacterial activity. The animal studies depicted that the collagen density and angiogenesis score in the CA/Gel/Beri treated group were 88.8±6.7 % and 19.8±3.8, respectively. These findings implied that the incorporation of berberine did not compromise the physical properties of dressing, while improving the biological activates. In conclusion, our findings implied that the prepared mat is a proper wound dressing for DFU management and treatment.

Mechanical property. The mechanical properties of the nanofibers were measured by the tensile strength method based on ISO 5270:1999 standard test methods. The results showed that the incorporation of berberine compromised the mechanical property and slightly reduced the tensile strength from 2.83 ± 0.08 to 2.69 ± 0.05 MPa; however, the observed difference was not statistically significant (p < 0.32). Our previous study 47 showed that the addition of taurine to electrospun Poly (ɛ-caprolactone)/Gelatin nanofibers reduced the mechanical property. The obtained results can be attributed to the berberine-induced reduction in the physical interaction between the polymer chains. Berberine weakened interchains and intrachain physical interaction and forces such as van der Waals forces and hydrogen bonding 47,48 . However, the obtained tensile strengths are in the acceptable range. Vatankhah et al. 45 reported the tensile strength in the range of 15 to 3 Mpa for CA/Gel nanofibers fabricated from different weight ratios of CA and Gel. In another study, Waghmare et al. 49

fabricated
Porosity. The liquid displacement technique was used to measure the porosity of the fabricated nanofibers.
The results showed that the porosity of CA/Gel and CA/Gel/Beri nanofibers were 78.17 ± 1.04 and 76.17 ± 0.76%, respectively. Chong et al. 52 concluded that the porosity in the range of 60-90% is preferred for tissue engineering applications. They also reported the porosity range of 60-70% for their fabricated electrospun PCL/Gel nanofibrous scaffolds. In another study, Shan et al. 53 reported a porosity value of around 87% for the fabricated silk fibroin/Gel electrospun nanofibrous dressing. Although the porosity of the fabricated CA/Gel/Beri nanofibers are in the acceptable range, Water vapor permeability (WVP) should be measured along to conclude the efficacy of the prepared CA/Gel/Beri nanofibers.
Wettability. A suitable dressing should be able to absorb the wound exudates and maintain the moisture of the wound site. These criteria are under the influence of the surface wettability and hydrophilicity of the dressing. The wettability of the fabricated dressings was measured based on the water contact angle method, and the results are presented in Table 1. The water contact angle of CA/Gel and CA/Gel/Beri nanofibers were 58.07 ± 2.35° and 56.93 ± 1°, respectively, which indicated that the fabricated dressings are hydrophilic and proper for absorbing exudates and maintaining the moisture of the wound bed. Liu et al. 54 demonstrated that the incorporation of Gel could increase the surface wettability of cellulose acetate due to its hydrophilic nature.
Water vapor permeability and water-uptake capacity. The vapor exchange through the dressing is a critical property determining the efficacy of the dressing. High WVP value dehydrates the wound and induces scar formation, while the low WVP value delays the wound healing process due to the deposited exudates. Therefore, a proper dressing should exhibit an optimum value of WVP. The results showed that the WVP value for CA/Gel dressing was 11.23 ± 1.05 mg/cm 2 /hr, while the addition of berberine reduced this value to 10.17 ± 0.21 mg/cm 2 /hr which both values are significantly lower than the control group (the open container) (p < 0.05).
As shown in Table 1, the water uptake ratio of CA/Gel was 12.78 ± 0.32%, and the incorporation of berberine increased the ratio to 14.37 ± 0.42%. This enhancement in the water uptake ratio can be related to the hydrophilic  www.nature.com/scientificreports www.nature.com/scientificreports/ nature of berberine. These results demonstrate that the fabricated CA/Gel and CA/Gel/Beri can properly absorb the wound exudates and subsequently improve the wound healing process.
Weight loss assay findings. The degradation rate of the prepared CA/Gel and CA/Gel/Beri was measured in PBS at days 7 and 14 ( Table 1). The results indicated that the fabricated dressings undergo significant weight loss despite remaining structurally stable during 14 days, and the highest weight loss, around 80%, was observed in CA/Gel/Beri group on days 14.
As shown in Table 1, the incorporation of berberine accelerated the weight loss of CA/Gel nanofibers at both time intervals, which was statistically significant at 7 days (p < 0.05). The observed increased weight loss can be related to the hydrophilic nature of berberine, which enhanced the interactions between CA/Gel nanofibers and water molecules. Moreover, berberine may reduce the physical interactions between the polymer chains, which facilitates the degradation rate. However, in the wound dressing applications, there is no need for biodegradability.

Microbial evaluations findings. Microbial penetration.
A proper wound dressing must withstand against microbial invasion through the dressing and show acceptable microbial barrier property. In this experiment, the negative control was the tube closed with a cotton ball to test the sterilization procedure, while the positive control was open tube and tested to ensure the growth possibility of bacteria in the nutrient broth.
As shown in Fig. 2, the fabricated dressings prevent bacterial transition, and negligible colonies were grown in the culture media, which was statistically significant compared with the positive control (P < 0.005). Moreover, the cloudiness of the nutrient broth was further evaluated by Spectrophotometer at 600 nm, and the results are shown in Fig. 3.
The turbidimetry assay results confirmed the colony counting assay findings, which indicated that the fabricated dressings exhibited excellent microbial barrier property. The results demonstrated that the microbial  www.nature.com/scientificreports www.nature.com/scientificreports/ penetration of CA/Gel/Beri dressing was lower than CA/Gel (p < 0.23), which can be related to the antibacterial property of berberine. The antibacterial activity of berberine was shown by Peng et al. 55 . Moreover, it is shown that even 64 layers of gauze were not able to stand against bacterial penetration into the wound 56 . Hence, these results imply that the fabricated dressings are suitable for wound care applications.
Antibacterial assay findings. An effective wound dressing should possess the antibacterial activity along with bacterial penetration barrier. The antibacterial activities of the fabricated dressings were assessed by time-kill assay against gram-positive and gram-negative bacterium Staphylococcus aureus and Pseudomonas aeruginosa, respectively ( Table 2).
The results exhibited that the antibacterial activity of the fabricated CA/Gel/Beri dressing was significantly higher than the positive control and the CA/Gel dressing (p < 0.005). Previous studies confirmed the potential antibacterial activity of berberine against various bacteria 55,57,58 . Kang et al. 57 reported that berberine exhibited time and concentration dependence antibacterial activity against Actinobacillus pleuropneumoniae with the MIC of 0.3125 mg/mL. Wojtyczka et al. 59 reported that berberine has antibacterial activity against coagulase-negative staphylococcus strains with the MIC ranged from 16 to 512 μg/mL. The higher bactericidal effect of CA/Gel/Beri dressing observed in our study is related to the incorporation of berberine in the matrix of CA/Gel nanofibers, while this value is effective for wound dressing applications. These results imply that the fabricated dressings are not only a barrier against bacterial penetration but also an antibacterial dressing.
The hemocompatibility results. Since wound dressings are designed to contact with bloody wounds, it is essential to assess the hemocompatibility of the fabricated dressings. The hemolysis induced by the fabricated dressing was measured as an indication of hemocompatibility, and the obtained results are presented in Fig. 4a. The obtained results from the hemocompatibility test showed that the hemolysis induced by the fabricated dressings were significantly lower than the positive control (distilled water-lysed RBC) (p < 0.05). Moreover, it is shown that the berberine incorporated dressing exhibited lower hemolysis than pure CA/Gel dressing. Vuddanda et al. 60 synthesized berberine chloride nanoparticles and reported less than 10% hemolysis under incubation with human blood samples. They proposed that the electrostatic interaction between positively charged nanoparticle and negatively charged RBCs may have induced the observed hemolysis.
Cell proliferation assay results. The proliferation of the L929 murine fibroblastic cell line was measured by MTT assay at 24 and 72 h defter cell seeding, and the results are present in Fig. 4b. It is shown that the maximum cell growth was obtained with the CA/Gel/Beri dressing, which was statistically significant compared with the control group (tissue culture plate) (p < 0.005). It is also apparent that the incorporation of berberine enhanced the proliferation of the L929 murine fibroblastic cells. Liu et al. 61 reported that berberine is able to promote the   www.nature.com/scientificreports www.nature.com/scientificreports/ proliferation of periodontal ligament stem cells (hPDLSC) through the activation of the ERK-FOS signaling pathway via binding to the EGFR on the cell membrane.
The morphology of the cells on the nanofibers. The morphology of the seeded L929 murine fibroblastic cell on the CA/Gel/Beri was observed using SEM after fixation and dehydration. As shown in Fig. 5, the cultured cells well spread onto the nanofibers after 24 h cell seeding. This image clearly depicts that the cells are attached and spread onto the fabricated nanofibers.
Animal study findings. Histopathology and Histomorphometry analysis results. The histopathology examinations evaluated the wound healing efficacy of the fabricated CA/Gel and CA/Gel/Beri dressings, and the results are presented in Fig. 6. The obtained tissues were stained with H&E and MT stainings. Figure 6a shows the positive control (healthy skin) with intact dermal and epidermal, whereas the negative control group (untreated wound) exhibited polymorphonuclear inflammatory cells (PMNs) infiltration and granulation tissue formation; however, the epidermal layer has not been formed yet (Fig. 6b).
The CA/Gel dressing group showed the completed epithelialization and less infiltrated PMNs in comparison with the negative control group (Fig. 6c). The anti-inflammatory activity of berberine has been shown in other studies. Xiao et al. 62 reported that berberine has an anti-inflammatory effect through the adjusting PCSK9-LDLR pathway and reducing the plasma concentration of IFNγ, TNFα, IL-1α, and 8-isoprostane. Yi et al. 63 demonstrated that berberine acts as an antiatherosclerotic agent through the inhibition of COX-2 expression via the ERK1/2 signaling pathway. In another study, Kim et al. 64 conducted a study to assess the anti-inflammatory effect of berberine on normal human keratinocytes. They reported that berberine treatment reduced IL-6 expression, activity, and expression of matrix metalloproteinase-9 (MMP-9). Moreover, they observed that berberine suppressed AP-1 DNA binding activity and ERK activation.
The epidermal proliferation and the epidermal layer enlargement were evident in CA/Gel/Beri group (Fig. 6d), while the inflammatory response and the granulation tissue decreased in this group. Rejuvenation of skin appendix was seen and developed in CA/Gel/Beri group. Moreover, this group exhibited more resemblance to healthy skin with a thin epidermis, the presence of normal rete ridges, and a standard thickness of skin layers. The histopathological observations showed that the fabricated CA/Gel/Beri dressing resulted in the best outcome compared to negative control and CA/Gel groups.
The histomorphometric analysis was done to further evaluate the healing process of the wounded skin ( Table 3). The minimum re-epithelialization was observed in the negative control group, while the CA/Gel/Beri exhibited the highest re-epithelialization after 16 days (P < 0.05). The wound site in the negative control group was filled with immature granulation tissue, which indicated the slow and ineffective wound healing process.
The wound healing process is critically dependent on the angiogenesis, as well as the collagen synthesis in the wound site. In this regard, the angiogenesis and collagen synthesis were measured to further assess the wound healing process under the influence of the fabricated dressings. The highest collagen density, 88.8 ± 6.7%, was observed in CA/Gel/Beri, which was statistically significant compared with the CA/Gel, 71.7 ± 5.2%, and the negative control group, 29.5 ± 2.3%, (P < 0.01 and P < 0.001). Moreover, CA/Gel/Beri exhibited the highest angiogenesis, 19.8 ± 3.8, while the lowest angiogenesis, 9.3 ± 1.2, was observed in the negative control group (P < 0.001). Previous studies demonstrated the wound healing potential of berberine treatment. Pashaee et al. 44 evaluated the wound healing activity of berberine on STZ-induced diabetic rats and conducted that the treatment improved the wound healing process. They proposed that the observed healing process is related to the anti-diabetic, anti-inflammatory, as well as antimicrobial effects of berberine. Our findings indicated that the incorporation of berberine into the dressing significantly improved the healing process. www.nature.com/scientificreports www.nature.com/scientificreports/ conclusion DFU is one of the main disabling complications of Mellitus diabetes, which requires excessive consideration and wound care management. The healing process in DFU is delayed and takes a long time to heal, so accelerating agents should be applied to enhance the healing process. Moreover, the wound site must be isolated from pathogenic microorganisms via a well-designed dressing. Electrospun nanofibrous dressings are ideal wound dressing due to their nanometric scale, high surface to volume ratio, adjustable porosity, and ability to load various drugs and bioactive molecules. In the present study, CA/Gel nanofibrous dressing containing berberine was fabricated using the electrospinning technique and after characterization used as a DFU dressing material. The characterization results demonstrated that the fabricated dressing was suitable as the wound dressing material and did not induced any adverse effect on the cultured cells and also exhibited antibacterial activity against gram-positive and gram-negative bacterium. The animal studies on the STZ-induced diabetic rats demonstrated that the CA/Gel/ Beri dressing enhanced the wound healing process. The evidence from this study suggests that the fabricated CA/  www.nature.com/scientificreports www.nature.com/scientificreports/ Gel/Beri dressing is suitable dressing material to enhance the healing process of DFU and also isolate the wound site from the pathogenic microorganisms invasion.
Fabrication of nanofibrous wound dressing. A proper amount of gelatin and CA were separately dissolved in HFP to obtain the final concentration of 6% (w/v). CA (6% (w/v)) and Gel (6% (w/v)) were combined at the weight ratio of 25:75 and stirred for 24 h. In the next step, the berberine was added to the resulted solution in the amounts of 1% (w/w) 65 of the total polymer content and stirred for 24 h. The commercial electrospinning apparatus (NanoAzma, Tehran, Iran) was used to fabricate the electrospun wound dressing. Briefly, the CA/Gel/ Beri solution was loaded into a 10 mL disposable syringe ending to a 25-gauge stainless steel blunted needle. The needle was connected to the power supply and the polymer solution was pumped onto the tip of the needle via a syringe pump at the constant rate. The applied voltage, the feeding rate, and the nozzle to the collector distance were 15 kV, 0.2 mL/h, and 15 cm, respectively. The cross-linking was conducted to prevent the dissolution of gelatin in the biological situation. The fabricated mats were incubated with the vapor of 10% (w/v) glutaraldehyde (GA) for 16 h to induce cross-linking between the nanofibers 66 . The cross-linked nanofibers then washed thoroughly by distilled water to remove unreacted GA followed by drying at room temperature.

Characterization of the wound dressing.
Scanning electron microscopy analysis. The produced wound dressings were sputter-coated with a thin layer of gold using a sputter coater (KYKY Technology Development, Beijing, China) and imaged using a scanning electron microscope (SEM; KYKY Technology Development, Beijing, China) at an accelerating voltage of 26 kV.
Mechanical strength measurement. The tensile strength of the fabricated wound dressings was measured by a uniaxial tensile testing device (Santam, Karaj, Iran) with an extension rate of 1 mm/min, according to ISO 5270:1999 standard test methods.
Contact angle measurement. The water contact angle was measured as the indication of hydrophilicity/hydrophobicity nature of the dressings via a static contact angle measuring device (KRUSS, Hamburg, Germany). The water droplet was poured onto the three different points of each sample, and the resulted angle between the water droplet and the surface of each specimen was averaged and reported.
Water vapor permeability (WVP) test. The flexible bottle permeation test (Systech, UK) was used to measure WVP of the fabricated dressings. The dressings-capped bottles were incubated at 33 °C for 12 hours, the evaporated water through the dressing was measured, and Eq. 1 was used to calculate WVP at steady-state.
Where W, A, and T stand for the mass of water lost, area (1.18 cm 2 ) of dressing, and exposure time, respectively.
Water uptake ratio evaluation. The water uptake ability of the mats was measured based on our previous studies 47,67 . The dry dressing was weighed (W 0 ), immersed in distilled water at room temperature for 24 h, and then the wet samples were weighed again (W 1 ). Equation 2 was used to calculate the water-uptake capacity of the dressings. Three measurements were conducted for each specimen and the averages were reported.
Porosity assessment. The porosity of the electrospun dressings was measured based on the liquid displacement technique using Eq. 3 68 . Briefly, the mat of weight W was immersed in a graduated cylinder containing a known volume (V1) of ethanol, and the resulted volume then was recorded as V2. After 10 min, the samples were removed, and the residual ethanol volume recorded as V3.
Weight loss measurement. Weight loss of the dressings was measured as the function of degradation based on our previous study 67  Microbial penetration test. The microbial penetration test assessed the resistance of the fabricated dressings against microbial penetration. Briefly, 10 ml vials (test area: 0.8 cm 2 ) containing 5 ml of BHI broth culture medium was capped with the prepared mates, kept at ambient conditions, and growth of the bacteria into the culture medium was measured after 3 and 7 days. Bottles covered with the cotton ball and open vials served as negative and positive controls, respectively. The turbidity as the indication of microbial contamination was measured by spectroscopy approach at 600 nm using a microplate spectrophotometer (n = 3).
Antibacterial growth assay. The antibacterial activities of the prepared nanofibers were conducted based on the time-kill assay against Staphylococcus aureus and Pseudomonas aeruginosa 69 . Briefly, the prepared CA/Gel nanofibers were added to bacterial suspensions (previously adjusted to 1 × 10 7 CFU/ml) at concentrations of 1/2-fold of the Minimum Inhibition Concentrations (MIC). 0.5 mL of each suspension was incubated at 37 °C with gentle agitation in a shaking water bath for 24 h. After the incubation period, the suspension (10 μL) was serially-diluted and inoculated on agar plates and incubated for 1, 2, 4, and 24 h in aerobic incubation condition at 37 °C. Then, the number of viable bacteria colonies was counted.
Blood compatibility or hemolysis assay. The hemolysis assay was conducted on human whole blood anticoagulated and diluted with normal saline. The samples were incubated with 200 µl of blood samples at 37 °C for 60 min, followed by centrifugation at 1500 rpm for 10 min. Then the absorbance of the resulted supernatant was read at 545 nm using a Multi-Mode Microplate Reader (BioTek Synergy 2). The negative control and positive control were the whole blood diluted in normal saline and the whole blood diluted in deionized water, respectively. Equation 5 was used to calculate the hemolysis percent 70 .
Where Dt is the absorbance of the sample, Dnc is the absorbance of the negative control, and Dpc is the absorbance of the positive control.
Cell culture studies. L929 murine fibroblastic cell line was obtained from Pasteur Institute, Tehran, Iran, and used to evaluate the biocompatibility of the prepared dressing. DMEM/F12 enriched with 10% (v/v) FBS, 100 unit/mL of penicillin, and 100 µg/mL of streptomycin. The dressings were cut spherically and put into the bottom of each well of a 96-well plate followed by UV light exposure for one h for sterilization. Then, the nanofibrous mat was washed with PBS twice and once with DMEM/F12 and then seeded with 1 × 10 4 cells. MTT assay kit was used to evaluate the proliferation of the seeded cells based on the previously described method 71 . The positive control was the wells without dressings, the experiment was done triplicated, and the average data reported. The morphology of cultured cells in the fabricated nanofibers was observed using SEM imaging. Briefly, after 24 h, the seeded cells were fixed in 4% paraformaldehyde for one h at room temperature, dehydrated in graded ethanol, sputter-coated with a thin layer of gold, and observed at an accelerating voltage of 26 kV.
Animal studies. Induction and assessment of diabetes. The animal studies were conducted on adult Wistar rats purchased from Pasteur Institute, Tehran, Iran. Diabetes was induced by intraperitoneal injection of a single dose of 55 mg/kg STZ in citrate buffer (pH 4.4, 0.1 M). The equal volume of citrate buffer was injected intraperitoneally to the age-matched control rats. The retro-orbital plexus technique was used to collect the blood samples, and GOD-POD (glucose oxidase-peroxidase) diagnostic kit was used to measure serum glucose levels, and the levels of more than 250 mg/dL confirmed the diabetes induction 72 .
In vivo wound healing study. The animal study was conducted on 24 male adult Wistar rats (Weight 200-230 g) based on the instruction of the ethics committee of Kermanshah University of Medical Sciences. General anesthesia was induced by intraperitoneal injection of Ketamine 5%/Xylazine 2% (70 mg ketamine and 6 mg Xylazine/1 kg body weight). The full-thickness excisional wound model was created on foot of each rat as a rectangular pattern marked on the dorsal surface of the foot using a flexible transparent plastic template, and then a layer of skin in full-thickness with a standard area of 2 mm × 5 m was removed. The animals were divided into 4 groups (6 rats per group), and the wounds were treated with the sterilized CA/Gel, CA/Gel/Beri nanofibers, and the sterile gauze as the negative control. An elastic adhesive bandage was applied to fix and secure the dressings on the wounded area and the dressings changed daily.
After 16 days post-surgery, the animals were sacrificed by ketamine overdose injection and the wound tissue was harvested and fixed in 10% buffered formalin. The harvested tissues were processed, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E) and Masson's trichrome (MT). An independent pathologist observed and interpreted the prepared slides under a light microscope (Carl Zeiss, Thornwood, USA) with a digital camera (Olympus, Tokyo, Japan) and photographed at ×100, and ×200 magnification. Epithelialization, inflammatory cell infiltration, fibroplasia, and granulation tissue formation assessed in different groups, comparatively.