Materials

Resveratrol (RSV) (high purity > 98%) was purchased from Guangzhou Phytochem Sciences Inc. (Guangzhou, China). Thymol was purchased from Oxford Lab Fine Chem I.I.P (India). Dialysis bags (molecular weight cut off 12,000–14,000 Da) (Serva, Heidelberg, Germany). Coumarin-6 dye was obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was supplied by SERVA Electrophoresis GmbH (Germany). TRIzol® Reagent was provided by Invitrogen, Thermo Fisher Scientific (Waltham, MA, USA). The primers were acquired from Sigma-Aldrich (UK) and a one-step RT qPCR kit (SYBR Green with low ROX) was purchased from Intron Biotechnology, Korea. B-cell Lymphoma/Leukemia 2 ELISA kit was purchased from Novus Biologicals, UK (NBP2-69946), and the Nuclear Factor Kappa B ELISA kit was obtained from Wuhan Fine Biotech Co., Ltd (Wuhan, Hubei, China). Acetonitrile and DMSO (HPLC grade) were purchased from Fischer Scientific (Loughborough, UK). Methanol, chloroform, ethanol, and formalin were provided by Al-Gomhureya Co. (Cairo, Egypt). All other reagents and chemicals were of analytical grade.

Preparation and optimization of RSV-loaded invasomesPreparation of invasomes

Invasomes were prepared by thin film hydration method with some modifications [7]. Five milligrams (5 mg) of RSV, 100 mg soybean phosphatidylcholine (PC), and 100 mg thymol (terpene) were dissolved in a 10 mL mixture of chloroform and methanol (2:1) in a dry clean round bottom flask to achieve the final RSV concentration of 0.5 mg/mL. The organic solvent was then evaporated using a rotary evaporator (Buchi AG, Switzerland) under vacuum using an oil-free vacuum pump (ROCKER®, Model Rocker 801, Taiwan) for around 25 min at 60 °C (above transition temp. of lipid) (Tc) until a thin uniform film was formed. Ten milliliters of hydration solution (0.5: 9.5 v/v ethanolic PBS, pH 7.3) was added to the film and left on the rotary evaporator at room temperature for 30 min. Unloaded invasomes were prepared using the same method but without adding RSV.

Optimization of the prepared invasome formulation

To achieve an optimum formulation with reduced particle size and uniform vesicular structure, different methods of particle size reduction were assessed such as homogenization, probe sonication, and extrusion. During sample homogenization, a high shear homogenizer (Ultra Turrax; IKA Labortechnik, Staufen, Germany) was used at different speeds; 5000, 6000, 7000, and 10,000 rpm. Homogenization was carried out in an ice bath for 3 cycles, 1.5 min or 3 min each with a constant rest time of 5 min between each cycle. As for probe sonication, samples were subject to sonication (60% amplitude for 5 min with a pulse 2 s on, 2 s off). As for the extrusion method, the samples were subject to 5, 9, or 11 cycles. Regardless of the method used for particle size reduction, all samples were left overnight at 4 °C in the fridge to stabilize the vesicles until further characterization.

Physicochemical characterization of invasomesParticle size, polydispersity index, and zeta potential determination

Samples were first diluted with deionized water 1:50 dilution and sonicated (ELMA, Germany) for 5 min before being measured. Samples were measured using Malvern Zetasizer Nano-ZS, Malvern instrument (Malvern, UK) at 25 °C and 173° angle to assess particle size (PS), polydispersity index (PDI), and zeta potential (ZP). Measurements were done in triplicate and all results were expressed as mean value ± (SD) [26].

Transmission electron microscopy (TEM)

The vesicular morphology of RSV-loaded invasomes and unloaded invasomes was investigated using a transmission electron microscope (TEM) (JEM-1400 Plus 120 kV, Joel, Peabody, MA, USA) [26]. Both samples were first diluted with deionized water (1:50) and bath sonicated for 5 min at room temperature. Samples were then added to a carbon-coated grid and stained with 1% uranyl acetate for 30 s. Samples were left to air-dry and were then examined at 20 K × magnification power [27].

Percentage entrapment efficiency and drug loading measurement (EE% and DL%)

To measure the amount of RSV entrapped in the prepared invasomes, samples were centrifuged at 14,000 rpm for 1 h at 4 °C (Model3K-30; Sigma Laborzentrifugen GmbH, Osterode, Germany). The supernatant was then separated and used to quantitatively measure the amount of free unentrapped RSV using an ultraviolet and visible spectrophotometer at λmax = 305 nm (Cary 60 UV-Vis Spectrophotometer, Agilent, Santa Clara, CA, USA). Measurements were done in triplicate and represented as mean value ± SD. EE% was calculated using the following Eq. (1) [27]:

$$\%\;EE\;=\;\frac\;\times\;100$$

(1)

Drug loading was calculated using the following Eq. (2):

$$\%\;DL\;=\;\frac\;\times\;100$$

(2)

Formulation of invasome-loaded gel

To increase contact time on the skin, the optimum prepared invasome formulation was loaded in a gel as developed by Teaima et al. [28] with slight modifications. The gel was prepared by dispersing Carbopol (Carbomer 941) polymer (3% w/v) in the invasomal dispersion (10 mL) and left on a magnetic stirrer (IKA Labortechnik, Staufen, Germany) at 25 °C and 800 rpm till complete dispersion. The dispersion was then neutralized by adding triethanolamine (TEA) dropwise until a soft viscous gel was obtained [28].

Characterization of invasomal gelDrug content in the gel

To calculate drug loading of RSV in the gel, 1 gm of RSV-loaded invasomal gel was dissolved in 20 g ethanol by heating on a magnetic stirrer (IKA Labortechnik, Staufen, Germany) at 70 °C and 1200 rpm for 30 min [29]. To break down the vesicles, the sample was subjected to probe sonication (Bandelin, Berlin, Germany) (60% amplitude continuously for 15 min) and then centrifuged at 14,000 rpm at 21 °C for 10 min to get the supernatant where drug content was analyzed by UV-Vis spectrophotometry at λmax = 305 nm.

Spreadability and rheological measurement

The spreadability of loaded and unloaded gels was measured by spreading 0.5 g gel on a 2 cm diameter circle pre-marked on a flat glass plate after which a second glass plate was employed. A weight of 500 gm was allowed to rest on the upper glass plate for 5 min after which the diameter of the circle formed after spreading of the gel was measured [30].

As for rheological measurement, the shear thinning and thixotropic ability of both loaded and unloaded gels was performed by measuring the viscosity with a Viscometer (Brookfield RV head multipoint viscometer with spindle CP-52). A sufficient amount of gel was added to the receiving chamber at room temperature where the gel was rotated at 0.1, 0.5, 1, 5, 1, 0.5, and 0.1 rotations per minute.

In vitro release of RSV

In vitro drug release was carried out using the dialysis bag method [27]. Constant sample volumes of 1 mL RSV suspension in PBS (D), 1 mL optimized RSV-loaded invasome liquid formulations (F2), and 1 g RSV-loaded invasomal gels (G) were added into dialysis bags (molecular weight cut off 12,000–14,000 Da) (Serva, Heidelberg, Germany) with an amount equivalent to 0.5 mg/mL drug. The bags were tightly sealed from both sides and immersed in 30 mL phosphate buffer saline (pH 7.3) as a release medium to ensure sink conditions (solubility of RSV in PBS pH 7.3 was 100 µg/mL). All samples were placed in a shaking water bath maintained at 37 ± 0.5 °C and 100 rpm. One milliliter of samples was withdrawn at different sampling time intervals (0.5, 1, 2, 4, 6, and 24 h) and replaced by equal volumes (1 mL) of fresh PBS to maintain sink conditions. Withdrawn samples were then analyzed spectrophotometrically at λmax = 305 nm. Percent cumulative drug release (mean ± SD) was plotted against time after correction where % cumulative drug released was calculated using the following Eqs. (3) and (4) [31]:

$$Amount\;of\;drug\;release\;\left(mg/mL\right)\;=\;\frac$$

(3)

$$\%\;Cumulative\;drug\;release\;=\;\frac\;\times\;P\;\left(t\;-\;1\right)\;+\;Pt$$

(4)

In this equation: P(t-1) is the percentage of the amount released before time (t) and Pt is the percentage of the amount released at time (t).

Ex vivo drug deposition

The ex vivo drug permeation/deposition study on animal skin was approved by the Ethics Committee of Medical Research, Faculty of Pharmacy, Alexandria University, according to the requirements of the Institutional Animal Care and Use Committee (IACUC), Alexandria University, Egypt (AU-06-2022-1191133). This study was applied on rat skin using Franz diffusion cells with some modifications [7]. The full-thickness dorsal skin was isolated from Wistar rats (average 200 g weight), dorsal hair was shaved, the subcutaneous tissue was removed, and the skin was washed in saline. The skin was mounted on Franz cells between donor and receiver cells with the epidermal layer facing the donor cell and the dermis inner layer facing the receptor cell. The receiver compartment was filled with 20 mL ethanolic PBS (3:7) to ensure sink conditions. The donor cells were filled with 1 mL drug suspension in PBS (D), 1 mL optimized RSV-loaded invasomes dispersion (F2), or 1 g RSV-loaded invasomal gels (G) where the final drug concentration in each sample was equivalent to 0.5 mg/mL over a skin diffusion area of 1 cm2. Samples were placed in a shaking water bath at 32 ± 0.5 °C and 80 rpm. Samples (0.5 mL) were then withdrawn from receptor cells at different time intervals (0.5, 1, 2, 4, and 6 h) and replaced with 0.5 mL fresh ethanolic PBS (3:7) to maintain constant volume and sink condition. The amount of RSV was analyzed by reversed phase isocratic HPLC (Agilent Technologies-1260 Infinity, Germany) with a UV detector (G1314F, C18 column (Agilent HC-C18 [4.6 × 250 mm], and 5 μm particle size)) and Agilent ChemStation® software (32-bit version) (revision B.02.01 SR1). The mobile phase used was acetonitrile-deionized water mixture (40:60 v/v) at a 1.0 mL/min flow rate at room temperature. An aliquot of 100 μL sample was injected and detected at (λmax) 305 nm [32].

At the end of the study, the amount of RSV deposited in the skin was measured where skin samples were collected at the end of the experiment, gently rinsed with the release medium, and immersed in a methanol/PBS mixture 1:1 then thoroughly homogenized (Ultra Turrax; IKA Labortechnik, Germany) for complete drug extraction. Digested samples were then centrifuged, and the supernatants were filtered via a 0.22 um syringe filter before HPLC quantification.

The amount of drug permeated through the skin and deposited in the skin layers was carried out in triplicate and the results were presented as means ± SD.

Ex vivo cell cultureCell culture

All cell culture experiments were carried out at the Centre of Excellence for Research in Regenerative Medicine and its Applications (CERRMA), Faculty of Medicine, Alexandria University, Alexandria, Egypt. Squamous skin carcinoma cells (SCCs) of passage number 25 were grown in DMEM high glucose cell culture media supplemented with 100 U/mL penicillin-streptomycin and 10% (v/v) FBS in a humidified environment of 5% CO2.

Cytotoxicity test

Cytotoxicity of RSV-loaded invasome, unloaded invasome, and RSV solution in ethanol was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay where IC50 of RSV was calculated in each of the prepared formulae [27]. Briefly, SCCs were cultured in 96-well plates (Greiner Bio-One, Germany) at a seeding density of 5 × 103 cells/well and incubated at 37 °C for 24 h to ensure cell adherence to the plates. The cells received serial dilutions of RSV-loaded, RSV-unloaded invasomes or RSV solution in ethanol (concentration range 0–50 ug/mL) and left to incubate for 48 h. The medium was then removed and replaced with MTT solution in PBS (5 mg/mL). The cells were further incubated for 4 h in the dark at 37 °C and 5% CO2. MTT solution was then replaced with 100 µL of DMSO and left for 10 min with gentle shaking on an orbital shaker (Heidolph Instruments, Schwabach, Germany) to dissolve the formed purple formazan crystals. Absorbance was measured using an automated microplate reader (BioTekR Instruments, VT, USA) at 570 nm to calculate the percentage of cell viability with respect to control untreated cells, in triplicate using Eq. (5) [27]. Half of the maximum inhibitory concentration (IC50) of RSV was calculated using a dose-response curve with non-linear regression analysis using GraphPad Prism (version 9).

$$\%\;Cell\;viability\;=\;\frac\;\times\;100$$

(5)

Cellular uptake

Cellular uptake was assessed using invasomes loaded with coumarin-6 fluorescent instead of RSV prepared using thin film hydration method (0.5 mg/mL) and free coumarin-6 solution (C6) in ethanol (0.5 mg/mL). SCCs were cultured at 5 × 105 cells/well in a 6-well plate and then incubated for 24 h. After incubation, coumarin-6-loaded invasome and free coumarin-6 solution were applied to the cells to reach a final concentration of 100 ng/mL and incubated for a further 4 h. Cells were washed 3 times with PBS then fixed with 4% v/v paraformaldehyde in PBS solution at room temperature and left for 15 min. Cellular uptake was assessed by measuring the fluorescence intensity of C6 fluorescent dye using a confocal laser scanning microscope (CLSM) at 355 nm (LeicaR Microsystems Inc. Model DMi8, Metzler, Germany). All experiments took place away from direct light to prevent the detrimental effects of ambient light. The fluorescence intensity in images produced by CLSM was quantified using ImageJ 1.52a software acquired by the National Institutes of Health, USA to determine fluorescence intensity [27].

In vivo evaluation of anticancer efficacy of the prepared formulaeAnimals

Forty male Swiss Albino CD1 mice weighing 18–25 gm each were purchased, housed, and maintained at the animal house of Medical Research Institute, Alexandria University, Egypt. Mice were kept under ambient conditions at 25 ± 1 °C and 50% relative humidity with dark/night cycle for 12 h. They had free access to food and water throughout the study. This in vivo study on animals was approved by the Ethics Committee of Medical Research, Faculty of Pharmacy, Alexandria University, according to the requirements of the Institutional Animal Care and Use Committee (IACUC), Alexandria University, Egypt (approval number) (AU-06-2022-1191133). An Ehrlich ascites cancerous (EAC) cells mouse was obtained from the National Institute of Cancer, Egypt. The mice’s back skin was shaved before tumor induction and treatment.

Tumor induction

Ehrlich ascites–induced skin cancer mice model was used to induce skin cancer in Swiss Albino male CD1 mice. The Ehrlich ascites cancerous cells (EAC) were obtained from the ascitic fluid–bearing tumor of a mouse (Swiss albino) which was cultured inside the mouse for 8–10 days. Skin cancer was induced by intradermal injection of 100 µL Ehrlich ascites cancerous cells (EAC) (containing approximately 1*106 of tumor cells) in the dorsal side [33] with some modifications. Forty mice were divided into 5 groups, 8 mice in each group (n = 8), as follows: group 1 (Gp1) for healthy negative control mice, group 2 (Gp2) for positive control (untreated cancerous) mice, group 3 (Gp3) for blank gel-treated mice, group 4 (Gp4) for unloaded invasomal gel-treated mice, and group 5 (Gp5) for drug-loaded invasomal gel-treated mice.

Treatment started 10 days after tumor induction where 0.5 g gel was applied topically on mice skin daily (equivalent to 1 mg/kg RSV) over a 1 cm diameter area on the dorsal side of each mouse and covered with bandage and plaster to decrease leakage of gel. The treatment duration post-induction was 21 days. During the treatment period, mice weights and tumor sizes were measured every other day using a balance and a caliber respectively.

Skin tumor volume measurement

Skin tumor size was measured for all mice groups except gp 1 (healthy) every other day using a caliper tool where length and width were determined to evaluate the antitumor effect of the treatment formulae compared to the untreated positive group (gp 2). Length and width measurements of tumors were presented as mean ± SD. Mean tumor volume was calculated as shown in the following Eq. (6):

where X and Y are the width and length diameters, respectively. Percent change in tumor volume is represented in the following Eq. (7) [34]:

$$\%\;change\;in\;tumor\;volume\;=\;\frac\;\times\;100$$

(7)

Termination

At the end of the treatment, mice were terminated by exsanguination after exposure to a high dose of inhaled isoflurane. Blood was collected from the orbital sinus of the eye and the collected blood samples were centrifuged at 3000 rpm at 4 °C for 10 min to separate plasma. Plasma was then stored at −80 °C till further biochemical analysis. The tumor was collected where parts were snap-frozen, while others were put in 10% v/v formalin for histological examination. The frozen parts were maintained at −80 °C for further RT-PCR and ELISA examination. Other organs such as the kidney, liver, and lungs were isolated and kept in 10%v/v formalin for toxicity assessment.

Quantitative analysis of cancer biomarkers on gene level using PCR and protein level using ELISA

PCR experiment was carried out in the Pharmaceutics Molecular Laboratory, Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University.

Expression levels of BAX and Caspase-3 genes were quantified using polymerase chain reaction (PCR) to evaluate the effect of different formulations (blank gel, unloaded invasomal gel, and RSV-loaded invasomal gel) on the treatment of skin cancer in comparison to negative and positive control groups [35]. First, snap-frozen samples were homogenized with TRIZOL reagent (Invitrogen, USA). An easy spin RNA extraction kit (Intron Biotechnology, India) was used in the purification and extraction of RNA [27]. A NanoDrop ND-1000 (NanoDrop DS-11 FX; DeNovix, Delaware, USA) was used to measure the quality and concentrations of RNA at 260 nm and 280 nm absorbance. Primers used for BAX [35], Caspase-3 [27], and GAPDH are represented in Table 1. GAPDH was used as a housekeeping gene to normalize relative transcript levels. According to the guidelines of Applied Biosystems/Life Technologies, the method applied in PCR quantification for BAX and Caspase-3 expressions was the comparative threshold cycle (2−ΔΔCt) method [27]. Results were normalized to GAPDH expression and expressed as arbitrary units.

Table 1 Forward and reverse primers of BAX and Caspase-3 genes for RT-PCR test for in vivo experiment

Protein levels of B-cell lymphoma/leukemia 2 (Bcl2) and nuclear factor kappa B (NF-kB) were measured in isolated tumors using enzyme-linked immunosorbent assay (ELISA) using B-cell Lymphoma/Leukemia 2 ELISA kit and Nuclear Factor Kappa B ELISA kit, respectively. Frozen samples were homogenized in PBS to reach a homogenate concentration of 10% w/v. ELISA kits were used for measurements according to the manufacturer’s protocol.

Histological examination

Tumors preserved in formalin (10% v/v) were integrated into paraffin wax blocks. Hematoxylin and eosin (H&E) stain was used to stain the samples to be examined histologically under the light microscope (Carl Zeiss, Köln, Germany) accompanied by a Canon digital camera. Histological samples were done to evaluate the antitumor effect of the treated formulations compared to the untreated group.

Blood biochemical assay to assess toxicity

Separated plasma was used to quantify levels of urea, creatinine, alanine aminotransferase (ALT or SGPT), and aspartate aminotransferase (AST or SGOT) to assess any signs of toxicity. According to the applied manufacturer’s instructions, all biochemical markers were evaluated using identified kits: urea (BioSystems S.A. Costa Brava 30, Barcelona, Spain), creatinine (Biolabo, Cat#80107, France), ALAT (BioSystems S.A. Costa Brava 30, Barcelona, Spain), ASAT (BioSystems S.A. Costa Brava 30, Barcelona, Spain).

Statistical analysis

Data was presented in triplicate as mean ± SD. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to evaluate the difference between the mean values of the studied treatments. The analysis was done for three measurements using GraphPad Prism (Version 7.04, San Diego, CA, USA). Statistical values of p ≤ 0.05 were considered significant.