Chemicals and biochemical
IL-6, IL-10, TNF-⍺, TGF-β, VEGF, ELISA kits were purchased from eBiosciense (San Diego, CA, USA). Bradford and N-acetyl-beta-D-glucosaminidase (NAG) were purchased from Thermo Scientific (Rockford, IL, USA). Hydrated chloride was purchased from Vetbrands (Paulinia, SP, Brazil). Other's reagents used were of analytical grade and were obtained from various commercial sources.
Preparation of the stem cells extract from Coffea canephora
Leaves of C. canephora were harvested from 10 individuals, located at the Espírito Santos Institute for Research, Technical Assistance and Rural Extension (INCAPER), Marilândia - ES - Brazil (latitude 19o23’56’’S, longitude 40o32’07’’O). The leaves were washed with distilled water followed by immersion in 70% ethanol and 3.5% NaOCl solution for surface disinfestation. Subsequently, leaves were washed three times in sterile distilled water into a laminar flow cabinet. Leaf explants were inoculated in Petri dishes containing an induction culture medium for callus and incubated at 24°C in the dark for 30 days . 0.5 g of the friable callus were transferred to Erlenmeyer flasks containing 30 mL liquid culture medium for establishment and multiplication of cell aggregate suspensions . The culture was kept under stirring, at 110 rpm, in the dark at 24°C, and subcultivated every 14 days for two months to increase biomass. In the end, cell aggregates in suspension and culture medium were processed using an ultrasonic probe followed by lyophilization. For extraction, the lyophilizate was resuspended in ultrapure water (10% w/v), homogenized, and centrifuged at 10,000 rpm at 4°C. The supernatant obtained (extract) was named stem cell extract from C. canephora (SCECC).
Phosphatidylcholine, cholesterol, polyethylene glycol (PEG 400), and soy lecithin were dissolved in a solvent mixture of chloroform and methanol (2:1 v/v) in a 500 mL round bottom flask. Organic solvents were evaporated at 50°C. This mixture was added in the proportion of 20% (v/v) of the SCECC and stirred at room temperature (25°C) at 15000 rpm for 15 min . The liposome of stem cell extract from C. canephora (LSCECC) was stored in a refrigerator at 4°C until final preparation and application. A similar void liposome (without the sample) was also prepared and stored for further experiments. The schematic LSCECC is represented in Fig. 1.
Formulation stability testing
To carry out the in vivo experiments, two emulsions in a non-ionic base were prepared. The positive control with 10% by weight of LSCECC and the negative control with 10% by weight of the void liposome. To determine the stability of emulsions and the shelf life, the accelerated stability method according to the National Health Surveillance Agency of Brazil was used . Both emulsions were stored in a tightly closed glass bottle, protect from light, and submitted to the heating and cooling cycles. For 28 days, the emulsions were heated to 45°C ± 2°C for 24 h and cooled to 4°C ± 2°C for another 24 h. After this period, the organoleptic characteristics of the emulsions were evaluated: appearance, odor, color, viscosity, and pH.
Particle size analysis for dynamic light scattering
The synthesized emulsions were taken to the Microtrac device to determine the diameter of the particles, and thus characterize the formed liposome through the technique of the dynamic light scattering (DLS). The colloid was inserted into the reading vat, through which the light-emitting laser passes. The result was given in a table and a particle size histogram [6, 7]. The equipment used was an NPA152 Zetatrac, Microtrac, USA.
Atomic force microscopy
The atomic force microscopy (AFM) was performed according to Ruozi et al., (2011). The sample preparation occurred through the deposition of a drop of the emulsion on a cleaved mica slide. Subsequently, a second blade was used to spread the liquid. The sample was decanted for 30 min and was then taken to the AFM. The intermittent contact mode was used, where the tip vibrates at a resonant frequency, practically all the time interacting with the sample surface.
Transmission electron microscopy
The transmission electron microscopy (TEM) was performed according to Ruozi et al., (2011). The equipment used was a Jeol JEM1400 microscope, with tungsten filament and acceleration up to 120kV. The surface on which samples are deposited on a copper device is called a Grid. Grids are covered with a carbon film called Formvar. One drop of the diluted emulsion is added and stored overnight in a Petri dish to dry. The contrast used was phosphotungstic acid.
In vivo experiments were conducted by the National Council for the Control of Animal Experimentation and were approved by the Ethical Committee, Bioethics and Animal Welfare of the Universidade Vila Velha (UVV) (ID n.575–2020). A total of 40 adult male Wistar rats (Rattus norvegicus) weighing 240–310 g and aged 8–9 weeks were used. All animals were kept under standardized conditions and monitored at a controlled temperature of 24°C ± 2°C with 12 h light-dark period in the bioterium of Universidade Vila Velha. Access to water and food was free during the entire experimental period.
In vivo skin regeneration experiment
All animals were anesthetized, shaved, and had their back cleaned with 70% ethanol. Then, four full-thickness excisional wounds were created on the back region of each rat with a sterile 15-mm punch biopsy. The rats were separated into 2 distinct groups (n = 20). Rats were daily treated with the developed liposomal formulation containing 10% LSCECC (positive control) or with the emulsion containing 10% void liposome. The wounds were covered with gauze to keep them protected. Five animals were euthanized on days 2, 7, 14, and 21 according to a standardized protocol. The biopsies were collected and stored for future histological and biochemical investigations [18, 22].
Evaluation of wound healing index
The morphometric analysis of the wounds was performed using images of the wounds at 0, 2, 5, 7, 10, 14, and 21-days post-wounding. The wound areas were calculated using ImageJ software (Wayne Rasband do Research Services Branch, National Institutes of Health – NIH, Bethesda, Maryland, EUA). The determination of wound area size reduction was calculated by comparing the current size and the initial size in percentage using the following formula: (wound area day 0 – wound area days 2, 5, 7, 10, 14, and 21)/wound area day 0) x 100. Values were expressed as the percentage of healed wounds .
From each animal on each corresponding day, two wounds were collected, conditioned for 24 h in 3.7% formaldehyde phosphate buffer, followed by processing and paraffin inclusion. Serial histological sections with approximately 3–6 \(\mu\)m thicknesses were mounted on glass slides and stained with hematoxylin and eosin (H&E) for evaluation and quantification of the inflammatory infiltrate and with a Picrosirius Red for quantification of collagen .
Evaluation of inflammatory infiltrate
The semi-quantitative evaluation of the inflammatory infiltrate was performed as previously described in the literature [18, 22], and the results were reported as the average of the total number of inflammatory cells per group.
Evaluation of collagenesis by imaging
The semi-quantitative study of collagenesis was performed as previously described , using the histomorphometric image analysis of the filled areas corresponding to stained collagen fibers, using picrosirius staining. The image analyses were determined by digital densitometry recognition using computer-aided ImageJ software. The results were reported as the average distribution of collagen per treatment.
Cytokines and growth factors measurements
The biopsies collected from each animal on days 0, 2, 7, 14, and 21 post-wounding were homogenized on ice using Lysing Matrix A tubes and a Fast Prep-24 homogenizer (MP Biomedicals, Santa Ana, CA) and centrifuged (1500g) for 10 min. The homogenate obtained from the ulcers was used to measure pro and anti-inflammatory cytokines IL-6, IL-10, TNF-α, and the growth factors TGF-β and VEGF using the immunoenzymatic assay (ELISA - "Enzyme Linked Immuno Sorbent Assay") following the manufacturers' specifications for each assay (eBioscience®). Optical densities were measured at 450 nm in a microplate reader device. The cytokine and growth factors quantitation were expressed in pg; sensitivities were > 10 pg/mL.
Total protein quantification
The total protein concentration for each tissue biopsy (homogenate) was determined by colorimetric method using a commercial Pierce® kit, as per the manufacturer's specifications (Labtest Diagnóstica - Lagoa Santa, MG, Brazil). Experiments were performed in 96-well plates, and protein concentrations were calculated by regression analysis using a standard curve of a solution of bovine serum albumin by colorimetric measurements at 595 nm in an ELISA plate reader.
Measurement of neutrophilic infiltrate
The neutrophilic infiltrate into the wound site was determined through the colorimetric myeloperoxidase (MPO) assay, as previously described [18, 22]. Quantitation of neutrophils was determined against a standard neutrophil curve. The results were described as the total number of neutrophils per mg of tissue.
Measurement of macrophage tissue accumulation
The presence of macrophages in the wound site was determined by N-acetyl-β-D-glycosaminidase (NAG) assay as previously described . Thus, 100 µL of the supernatant from the biopsy samples was added to a 96-well plate. To the samples, 100 µL of the substrate (p-nitrophenyl-N-acetidyl-β-D-glucosamine) 2.23 mM diluted in citrate-phosphate buffer pH 4.5 was added. It was incubated at 37°C for 60 min. To stop the reaction, 100 µL of glycine buffer pH 10.6 was added to each well. Absorbance was measured by spectrophotometry in a microplate reader at 405 nm. Results were expressed as NAG activity in optical density (OD/mg tissue).
Statistical analyses were performed using GraphPad software (San Diego, CA, 176 USA). Results are presented as the mean ± standard deviation (SD). Comparisons between two groups were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s post-test or two-way ANOVA when appropriate. The level of significance was p < 0.05.