Materials
Chitosan (low molecular weight, 13000–23000 Mw) (Sigma Aldrich), Bovine skin Gelatin type B, Glutaraldehyde (GA), Sodium Hydroxide (NaOH), Sodium Bromide (NaBr), PLGA (Poly (lactic-co-glycolic acid)) 50:50 (7000–17000 Mw), PVA (Poly (vinyl alcohol)) (13000–23000 Mw), N-hydroxysuccinimide (NHS), Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)- hydroxyl valerate content = 12 mol %, poly (L-Lactic acid) (PLLA), Trifluoroacetic acid (TFA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), Piperidine, 4-(Hydroxymethyl)phenoxyacetic acid (HMPA) Linker, Ninhydrin, N-hydroxybenzotriazole (HOBT), Diethylether, N,N-Diisopropylethylamine (DIEA),, Dichloromethane (DCM), Triisopropylsilane (TIS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethylformamide (DMF) and O-Benzotriazole-N,N,N’N’-Tertamethyl-Uronium-hexafluorophosphate (HBTU) were purchased from Sigma Aldrich, Hexafluoro-2-propanol (HFIP) from Spectrochem, Glacial Acetic Acid and Trifluoroacetic acid (TFA) from Merk, Tentagel-S-NH2 (0.29 meq/g), Fetal bovine serum (FBS) was purchased from Hyclone, Fmoc protected amino-acids from Peptide International Inc., Trichrome Staining Kit from Abcam and live/dead assay kit were from Invitrogen. The bacterial strains (Pseudomonas aeruginosa and Staphylococcus aureus) were acquired from ATCC (American Type Culture Collection) and the cell lines were obtained from NCCS (National Centre for Cell Science), Pune, India.
LLKKK18 and Tylotoin peptides: Synthesis and Characterisation
The peptides LLKKK18 (KLFKRIVKRILKFLRKLV) and Tylotoin (KCVRQNNKRVCK) were synthesized by solid-phase method using the Fmoc-Strategy [16]. TentaGel™–S–NH2 resin was linked to HMPA linker and peptide synthesis was carried out. After synthesis, the peptide was manually cleaved using a cleavage cocktail. Diethyl ether was used to precipitate the peptides followed by multiple washes. The peptides were then dissolved, lyophilized, and stored at -20°C. Peptide identities were verified using Matrix-Assisted Laser Desorption Ionization Mass Spectroscopy (MALDI-TOF MS). The high-performance liquid chromatography (HPLC) profile of Tylotoin peptide was done using a Shimadzu LC-AD HPLC system with a reverse phase C18 column. Buffer A (water containing 0.1% TFA) and Buffer B (acetonitrile: water (80:20) with 0.01% TFA) at the rate of 1 mL/min was used. The eluents from the column were monitored by UV (214 and 254 nm), and retention time was analysed.
Preparation of LLKKK18 peptide carrying PLLA-Chitosan-PHBV electrospun nanofiber
Chitosan was dissolved in TFA and PLLA, PHBV were dissolved separately in chloroform. The solutions were then combined in the ratio of 0.5:1.5:3 (PLLA: Chitosan: PHBV). The solutions were electrospun using HO-NFES- 043 Halmarc Nano Fiber Electrospinning Unit (flow rate: 12µl/min, Voltage of + 12 kV and the tip to collector distance of 10 cm).
To prepare LLKKK18 peptide-loaded electrospun mats, 1mg/ml peptide solution was activated with EDC/NHS and added to PLLA-Chitosan-PHBV electrospun nanofiber and kept in 4°C for 24 hours. The electrospun fiber was thoroughly washed, dried and stored (-20°C). The nanofiber mats were characterised using FEI- Nova NanoSEM 450 Scanning Electron Microscope (SEM), PerkinElmer Spectrum 65 Fourier transform infrared (FTIR) spectroscopy by attenuated total reflectance method, PerkinElmer 6000 Differential Scanning Calorimeter (DSC).
Synthesis of Tylotoin peptide entrapped PLGA nanoparticles
PLGA 50:50 (7000–17000 Mw) was dissolved in Dichloromethane and 2.5 ml of this solution was added with 100 µl Tylotoin solution and 10 ml of 1% PVA solution, mixed thoroughly and sonicated twice (2 minutes, 50 amps). The solution in the tube was added drop-wise to 0.3% PVA solution while it was being magnetically stirred at around 600 rpm and kept for 1 hour. PLGA nanoparticles were collected from the solution by centrifugation, washed and lyophilized. The size of the particles was analysed using Beckman Coulter–Delsa Nano C Dynamic Light Scattering (DLS) and TEM (Transmission electron microscopy) using JEOL 1011 (Japan).
Peptide release from PLGA nanoparticles and PLLA-Chitosan-PHBV nanofibers
The bioactive peptides release study from PLGA nanoparticle (carrying Tylotoin) and PLLA-Chitosan-PHBV nanofibers (carrying LLKKK18) was conducted separately using Thermo Fisher Micro Bicinchoninic acid Protein Assay Kit. 5 mg of lyophilized Tylotoin entrapped PLGA nanoparticles and LLKKK18 conjugated PLLA-Chitosan-PHBV nanofibers were taken in separate 1.5 tubes and added with 1000 µl of Phosphate buffered saline (PBS, pH 7.4). The Tylotoin entrapped PLGA nanoparticles containing tubes were centrifuged at 10000 rpm. The samples were kept at 37°C, after which 200 µl of the supernatant was collected at different time intervals and fresh PBS was used to replenish the solvent volume. The standard curve of both the peptides were plotted and the peptides release from PLGA nanoparticles and PLLA-Chitosan-PHBV nanofibers at the different time intervals was quantified by taking OD at 562 nm. The loading efficiency of Tylotoin in the PLGA nano-particles was analysed by calculating the concentration of Tylotoin in supernatant collected during nanoparticle preparation.
Preparation of 3D Bi-layered hydrogel and nanofiber sponge synthesis
Chitosan was dissolved in 1% glacial acetic acid. Gelatin was dissolved in distilled water at 60°C. Tylotoin peptide entrapped nanoparticles (500µg/ml) were added to the gelatin solution while it was being stirred after which the two solutions were mixed to form a 1:1 chitosan-gelatin solution. The chitosan-gelatin (CG) solution was kept at room temperature and 0.25% of Glutaraldehyde (GA) solution was slowly added to it while it was being magnetically stirred for 1 hour. The solution was poured into small plastic molds. The peptide conjugated PLLA-Chitosan-PHBV electrospun nanofiber mat was added to the mold containing CG solution and was kept undisturbed at room temperature for GA crosslinking. After which, the material was frozen and then lyophilized. The dried hydrogels were soaked in 1% NaOH for 2 hours to neutralize the acetic acid and then washed. The hydrogels were then soaked in 5% NaBr solution for 1 hour to neutralize the cytotoxic effect of GA. After which, the material was washed numerous times, kept for freezing and then lyophilized.
The 3D scaffold was characterised by SEM (FEI- Nova NanoSEM 450), ATR-FTIR spectroscopy (PerkinElmer Spectrum 65) and DSC (PerkinElmer 6000).
Porosity measurement
The material porosity was measured by the liquid displacement method using ethanol for easier permeability into the developed 3D bi-layered sponge dressings without causing shrinking or swelling [27, 28]. The material was completely immersed in a tube carrying ethanol with volume V0. The combined volume of the 3D bi-layered sponge and ethanol (V1) was measured. So, the volume of the ethanol-occupied sample was calculated as V1-V0. The sample containing ethanol was then removed, leaving the remaining ethanol in the tube with volume V2. The volume of ethanol inside the sample was calculated as V0- V2. Therefore, the volume (VS) of the sample is the total of the volume of 3D sponge pores and volume carrying ethanol and the, calculated as VS = (V1–V0) + (V0 –V2) = V1– V2. The porosity of the 3D layered Sponge was calculated using the formula: P = (V0 – V2)/ VS.
Fluid absorption property
The liquid absorption ability of the 3D bi-layered hydrogel and nanofiber sponge were investigated. The material was completely immersed in PBS (pH 7.4) for 1 hour and then retrieved. Surplus liquid was removed using a blotting paper. The fluid absorption ability of the developed 3D layered Sponge was calculated as:
Absorption ratio (%) = (A1- A0)/A1 × 100, where A0 is the weight of the dry 3D sponge and A1 is the weight of the wet 3D sponge [29].
In-vitro degradation of 3D bi-layered hydrogel and nanofiber sponge
The degradation rate of the 3D bi-layered hydrogel and nanofiber sponge was studied by using phosphate buffer, pH 7.4. 3D sponge with pre-determined weight was incubated in 0.1M phosphate buffer at 37 0C for 22 days. The material was then removed from the buffer at different time intervals, washed, freeze-dried, and weighed. The in-vitro degradation ratio of the sample (DRS) was determined as:
DRS = (W0- Wx)/W0× 100 (W0 is the initial weight of 3D sponge and Wx is the weight of the 3D sponge at different time intervals) [30].
Antibacterial Studies
The antibacterial action of developed 3D bi-layered sponge dressing was tested by Kirby-Bauer disc diffusion technique [31]. Bacterial strains Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) were taken as test organisms. 100 µl of the bacterial suspensions (106 Colony forming unit (CFU)/ml) was spread on Mueller-Hinton Agar (MHA) plates and sterile 3D sponge discs were placed on the surface of the plates, followed by incubation for 24 hours at 370C. Ciprofloxacin (5mg/ml) was kept as positive control. PBS (pH 7.4) and peptides free 3D sponge disc were kept as negative controls. The inhibition zone of bacterial growth was measured using a ruler.
To perform the release ablation study, the bacterial suspensions in the logarithmic phase were resuspended in Luria–Bertani (LB) broth for achieving a cell density of 1 × 106 CFU/ml. Sterile 3D bi-layered sponge carrying LLKKK18 peptide (8mm disc) and free peptide (LLKKK8, 100µg) were added to 1 ml bacterial suspensions in 24 well plates, then incubated for 6 h at 370C while continuously shaking at 100 rpm. Ciprofloxacin was kept as positive control and peptides free 3D sponge discs, PBS (pH 7.4) were kept as negative controls. 10 µl of the bacterial suspension were collected after incubation time from each well and plated on Mueller-Hinton agar plates and evaluated the release ablation abilities by counting the CFU/ml.
In vitro cytocompatibility and wound closure ability
The mouse fibroblast (L929) cell line and keratinocyte (HaCaT) cell line from human skin were grown in DMEM supplemented with 10% FBS and 1% antibiotics.
In-vitro cell viability and proliferation analysis of the developed 3D Layered Sponge was conducted by MTT and Live/Dead staining assays in L929 and HaCaT cell lines. For MTT assay, L929 and HaCaT were seeded and grown in 96 well plates (2×104 cells/well) for 24 hours. Media added with Tylotoin carrying PLGA nanoparticles (Tylotoin-NP 500µg/ml), LLKKK18 conjugated PLLA-Chitosan-PHBV nanofibers (LLKKK18-NF), Chitosan-Gelatin hydrogel (CG-Hydrogel) and 3D bi-layered hydrogel and nanofiber sponge (3D bi-layered sponge) and untreated media (Control) were added to the well plates and incubated for 1,2 and 3 days. The cells were then added with 10% v/v MTT mixed in PBS (pH 7.4) after desired time periods. After 3 hours, the media was removed and the plates were added with Isopropyl alcohol (IPA). BioradiMark™ Microplate Reader was used to measure absorbance at 570 nm. The skin cells viability percentage was calculated as: Treatments (average OD) / Control (average OD) × 100.
The skin cell survival ability and morphology of keratinocyte and fibroblast upon treatment with the test samples were analysed by Live/Dead Assay. Approximately 1×104 cells/well on 12-well plates were seeded and grown under serum-free media carrying treatments for 2 days. The live skin cell population were distinguished by staining with calcein AM, dead cells by ethidium homodimer-1 (EthD-1) and visualized by laser Confocal Microscope (TCS SP2; Leica Microsystems).
For the in-vitro wound closure assay, skin cells were grown till confluency in 12-well plates and wounded with a 2.5 µL pipette tip forming an even cell free zone. It was washed multiple times with PBS to get rid of debris and were added with media treated with Tylotoin carrying PLGA nanoparticles (Tylotoin-NP) 500µg/ml, LLKKK18 conjugated PLLA-Chitosan-PHBV nanofibers (LLKKK18-NF), Chitosan-Gelatin hydrogel (CG-Hydrogel) and 3D bi-layered sponge as treatments and untreated media as Control. The cells were incubated for 48 hours (for HaCaT cells) and 24 hours (for L929 cells) and imaged at different time intervals. The wound closure between the injured boundaries was calculated with Olympus CellSens computer assisted analysis system. The wound closure percentage was calculated as: Pre-migration distance – Migration distance / Pre-migration distance ×100 [18].
Diabetic wound healing animal studies
All animal study procedures were approved by IAEC (Institutional Animal Ethics committee), following appropriate guidelines (RGCB/IAEC/836/GSV/2021). Sixty 4–6 week old (weight 25–35 g) Swiss albino mice (males) were injected with streptozotocin (STZ) single dose: 180mg/ kg as per standard procedure [32, 33]. Accu-Chek glucose monitoring system was used to determine animal whole-blood glucose after 4–5 days from STZ injection. Animals with blood glucose levels higher than 300 mg/dL were marked as diabetic and used for further experiments. Before surgery, the animals were anesthetized; the dorsal skin surface was shaved to remove hair and disinfected with 70% ethanol. Double full thickness wounds of 8 mm diameter were created on the animal skin using biopsy punch [34]. The diabetic mice were allocated into groups after surgery and were applied with treatments: Control (PBS, pH 7.4), Tylotoin carrying PLGA nanoparticles (Tylotoin-NP), LLKKK18 conjugated PLLA-Chitosan-PHBV nanofibers (LLKKK18-NF), Chitosan-Gelatin hydrogel (CG-Hydrogel) and 3D bi-layered hydrogel and nanofiber sponge covering the wound area. To avoid contraction of mice skin, the wound boundary was sutured with silicone ring. The animals were then caged individually with access to water and a regular diet. The skin wound healing was monitored from day 0 to 21 days. Chronic wound closure rate (%) was calculated as Wound Area (0)-Wound Area (t)/ Wound Area (0) ×100% [Wound Area (0) represents day 0 of wound formation and Wound Area (t) denotes days 7, 14 and 21 after treatments to the wound].
Histological staining
For histomorphology analysis of the wound tissues upon treatments, the biopsy samples were excised on days 7, 14, and 21 after treatments. Before paraffin embedding, biopsy sample fixing was done with 10% Neutral Buffered Formalin, dehydrated using graded IPA series and Xylene. The wound tissue samples were then sectioned (5-µm) using Leica ultra-microtome, stained by Hematoxylin and Eosin (H&E) and Masson's trichrome stain was done using Trichrome staining kit, Abcam. Quantitative analysis of wound morphological changes using images was performed using the Image J software.