The drug, carvedilol, was procured from Chandra Laboratories, Hyderabad India. Soy lecithin, ethanol, PG, tri-ethanolamine and Carbopol-934 were purchased from Research Lab Fine Chem Industries, Mumbai India while cholesterol was obtained from Merck Ltd., Mumbai, India. The purchase of ultrapure water was done from Cortex Laboratories, Hyderabad, India. Centrisart filters with molecular cut off at 20000 were purchased from Sartorius Research Lab Fine Chem. Industries, Mumbai, India. The remaining chemicals used were of analytical grade and solvents were of HPLC grade.
5.2 Preparation of carvedilol loaded ethosomes
The ethosomes were prepared using ethanol [(20–40% (v/v)], PG [10% (v/v)], 2–5% (v/v) soy phospholipids and 0.005% (v/v) cholesterol in ultrapure water. The soy phospholipids, cholesterol, carvedilol drug solution, and PG were added to ethanol gradually followed by vigorous stirring to solubilise properly. The mixture was heated to 30 o C and distilled water was added slowly drop wise while the mixture was being stirred magnetically at 700 rpm. After addition of water, the stirring was carried out for an additional 5 minutes. The formed ethosomal suspension was then sonicated for reduction of the vesicular size to the desired extent(30). The final step was refrigeration of the suspension at 4 ℃(31).
5.3 Solubility studies
The extent of solubility of carvedilol was tested using ethanolic solutions in water of varying concentrations – 20, 30 and 40% (v/v). Before centrifugation, in each vehicle (2 ml centrifuge tube) an excess of amount of 1.5 ml of carvedilol was taken. After vortexing, the centrifuged tubes were kept for incubation in an orbital shaker (Remi Electrotechnik Ltd, Mumbai, India) for 48 hours at an ambient temperature of 25 °C to ensure proper solubilisation (32, 33). For removal of the excess undissolved drug, the incubated samples were undergone centrifugation at 3000 rpm. The supernatant taken at regular intervals were quantified for determining the drug concentration using RP-HPLC method.
5.4 HPLC quantification of dissolved drug
The HPLC unit used for the drug quantification (Shimadzu, Japan) had the following specifications: LC-10AT solvent module with SPD-10A column, PDA detector with LC10 software. The RPCL column had the specifications of C18 (150 × 6 mm) with 5 µm packing material. The mobile phase used was acetonitrile and 15 mM of ortho-phosphoric acid in the volumetric ratio 37:63 with the addition of tri-ethylamine in the concentration of 0.25%(v/v). The pH of the mobile phase was adjusted to 2.5 using ortho-phosphoric acid. A 20 µl of each sample was injected and the eluent detection was done at a wavelength of 242 nm. The flow rate was maintained at 1 ml/min with a runtime of 12 minutes (34).The final step was refrigeration of the suspension at 4 ℃(31).
5.5 Preparation of carvedilol loaded ethosomal hydrogel
Using various concentrations of the polymer Carbopol 934, the hydrogel was formulated, the concentrations of Carbopol being 1% (w/w), 1.5% (w/w) and 2% (w/w). Accurately weighed quantities of the polymer were dissolved in specific quantities of the prepared ethosomal suspension using a magnetic stirrer at 1000 rpm. The process was continued until smooth lump-free homogenous gels were attained. Appropriate quantity of tri-ethanol amine was added to adjust the pH of the prepared gel. The pH was adjusted to 5.5. The final semi-solid gel was stored overnight at room temperature.
5.6 Characterization of Ethosomosal Formulation
5.6.1 Assay of the encapsulated drug in ethosomes
The diluent used for dissolving the prepared ethosomal formulations was chloroform and methanol in 1:1 (v/v) ratio and diluted with the mobile phase. The HPLC parameters used and assay determination was done the same way as for quantification of the dissolved drug earlier(35).
5.6.2 Drug entrapment efficiency
Firstly, the prepared ethosomal formulation was undergone centrifugation at 8000 rpm for 30 minutes. The centrifuge tubes used were Centrisart tubes. The free unencapsulated drug concentration present in the supernatant was determined by HPLC and entrapment efficiency was calculated using Eq. 1
5.6.3Particle sizing and distribution
The average vesicle size, PDI and zeta potential of the ethosomes were determined by using the DLS method. To avoid the error due to multi-scattering action, a 2 ml quantity of each sample was undergone dilution with distilled water by proper mixing. The diluted sample was then injected into a clean disposable zeta cell and measurements were recorded using a zetasizer (Malvern Nano-ZS90).
5.6.4 FT-IR studies
FT-IR studies were conducted for the pure drug, the excipients used, the ethosomal suspension and the ethosomal hydrogel to determine the occurrence of any physio-chemical interactions and compatibility between the drug and the excipients used. The K-Br pellet technique was used. The scanning range and the resolution were kept at 400–4000 cm− 1 and 4 cm− 1 respectively(36). The FT-IR instrument used was of making Bruker Optics Germany Model-200.
5.6.5 Particle morphology
SEM was used to examine the surface morphology of the prepared ethosomes. After adhering the ethosomal suspension onto the carbon-coated stubs, they were sputtered with platinum using a coating machine (Auto Fine Coater, JFC-1600, JEOL, Japan) and then observed under the SEM in high vacuum atmosphere(37, 38). The SEM used was of the make JSM-6501LA, JEOL, Japan.
5.6.6 Particle appearance
The shape determination and overall appearance of the prepared ethosomes were observed using TEM. The sample preparation was done by placing a drop of the diluted ethosomal suspension on a carbon coated grid and followed by addition of a drop of aqueous 2% phosphotungstic acid solution. After the removal of excess liquid, the suspension was air-dried and TEM imaging was done at an acceleration voltage of 100 kV(39).
5.6.7 Physical Examination and pH measurement of ethosomal gel
The physical characteristics of the prepared ethosomal gels were determined by visual examination. The gel samples were visually examined to determine the homogeneity, consistency, phase separation and appearance of any aggregate formations. The pH was measured by using a digital pH meter (Remi, Hyderabad, India). For proper measurement, it was ensured that the glass electrode of the pH meter was completely dipped into the gel system (40).
5.6.8 Viscosity measurement of gels
The viscosity was measured using a viscometer (Brookfield Viscometer, CAP 2000L) under high torque and low-temperature mode. The cone used was of No. 1 type. About 500 mg of each sample was taken for analysis. 5 minutes of prior settling time was ensured before viscosity determination (41).
5.6.9 Spreadability of the gels
The extent and degree of the gel Spreadability were measured using the glass slide apparatus with the help of a modified wooden block. Using a glass side, a quantity of gel of known weight was placed on the movable pan using a glass slide and then placed on the fixed glass slide to make sure the gel was properly sandwiched between the glass slides for 5 minute duration. The excess gel exiting from the sides was continuously removed. The Spreadability was determined using Eq. 2.
5.6.10 Skin irritation test of the ethosomal gel
All the animal studies were conducted on Wister rats after obtaining permission from CPCSEA with the wide permission being documented as No.51/01/C/CPCSEA/2013/13. Using a clipper, the hair from the dorsal portion of nine rats was removed and the ethosomal gel was applied on the blank skin portion. Before the application, the rats were divided 3 groups with each group consisting of 3 members. Each group had a characteristic based on the application of the gel as follows
Group 1 - No application of gel on the rats
Group 2 - The prepared ethosomal gel was applied on the rats
Group 3 – Blank (without drug) ethosomal gel was applied on the rats
Each time the amount taken for the application was 500 mg with uniform spreading over the blank skin portion of area 4 cm2. Any sign of erythema or redness of skin was observed after every 24 hours up to 72 hours. The time is measured from the point of gel application.
5.6.11 In-vitro drug release studies
The dialysis bag method was used to carry out the in-vitro release studies of both the ethosomes and ethosomal gel. Before the test, it was made sure that the membrane of the dialysis was properly hydrated with complete wetting of the membrane(24). The hydration medium used was PBS of pH 6.8 and the hydration was carried out for 2 hours. The samples of both ethosomal suspension and ethosomal gel each containing the drug were transferred to the dialysis bags with both ends sealed. The bags were then suspended in bottles containing 200 ml of the buffer solution and rotated at 100 rpm in a thermostatically temperature-controlled water bath shaker. The temperature was maintained at 37 ± 0.5 °C throughout the process. For each sample, 1 ml of the aliquot was taken at pre-determined time intervals. For the first 6 hours, the aliquot was taken on an hourly basis and then after the sample was taken after 8, 16, 24, 48 and 72 hours. The drug concentration after each time interval, was determined at 242 nm spectrophotometrically.
5.6.12 Ex-vivo skin permeation studies
The ex-vivo skin permeation studies were carried for both ethosomal suspension and ethosomal gel. For both the suspension and gel, the batch which showed the most promising results in terms of physical studies, entrapment efficiency and in-vitro drug release studies was selected. After sacrificing the rats, the skin from the abdominal portion was chosen for conducting the studies. The hair from the skin was removed thoroughly using a razor blade and the skin was separated from the connective tissue diligently using a scalpel to prevent perforations or incisions. After removal, the skin was then washed thoroughly with double distilled water and stored at − 18 °C to retain its metabolic efficiency. The skin was then hydrated overnight at 25oC in PBS (pH 6.8 and containing 0.02% sodium azide as a preservative). The overnight hydration was done to ensure the removal of extraneous debris and leachable enzymes(42, 43).
A skin sample of appropriate size was fixed at the ends of a diffusion cell ensuring a permeation area of about 5.3 cm2 was available. The SC portion of the skin layer faced the donor compartment while the dermis side of the skin met the receptor compartment. A 200 ml of a solution of transcutol, ethanol and PBS at pH 6.8 in the ratio 10:40:50 (v/v) was used as the hydration medium. Such a hydration medium was chosen to ensure proper sinking during the permeation studies (20). The diffusion cells were maintained in a thermostatically controlled water bath shaker at 37 ± 1oC at 100 rpm. At pre-determined time intervals (0, 1, 2, 3, 4, 5, 6, 8 and 24 hours), a 5 ml sample of the receptor medium was withdrawn, and then filtered using a nylon syringe filter of 0.22 µm size. Every time a sample was taken, an equivalent amount of fresh receptor medium was added to maintain the volume constant. The assay of the drug in the sample taken was determined at 242 nm using spectrophotometry. For the selected sample batches, the cumulative drug permeation through the skin was plotted against time to see the release pattern. The steady state flux (Jss) was also determined. The measurements were taken in a triplicate manner and compared with those of a control batch.
5.6.13 Pharmacodynamic study
The pharmacodynamic study was conducted for both ethosomal suspension and ethosomal gel. A comparative study was done with control as well as the marketed formulation. One group of rats were not administered with any drug whatsoever. In another groups of rats, in which the pure drug or its various formulations were administered, the hypertensive effect was induced using sodium chloride solution and MP separately. The sacrificed rats weighed in the range of 220–250 g and were fed ad libitum as per the standard procedure. After two weeks from inducing hypertensive effect, the rats in which the mean systolic BP was 150–160 mm Hg were selected and the drug and its various formulations were administered. The marketed formulation was administered orally (10 mg/kg of body weight) while the rest of the formulations were administered transdermally (10 mg/kg of body weight). Before the blood pressure measurement was done, the rats were properly trained to stay calm and non-aggressive in the cages. The systolic BP was measured by the tail-cuff method (Bio-pack system Inc., Santa Barbara, USA) at pre-determined time intervals after the drug administration (1, 2, 4, 6, 10, 12, and 24 hours) for all the groups(44, 45).
5.6.14 Stability studies
The stability studies were conducted for both ethosomal suspension and ethosomal gel. Two batches were used for each of the formulations, one was stored at 4 °C and the other at room temperature at 23–30 °C. The parameters determined for stability studies were mean vesicle size, PDI, zeta potential, entrapment efficiency (EE%) and assay using HPLC. The stability studies were conducted at 0, 1, 2, 3 and 6 months(46, 47).
5.6.15 Statistical studies for optimizing the formulation of ethosomes
The CCD modelwas implemented to optimize the correct formulation of the carvedilol-loaded ethosomes. The software used for the study was DoE (Version 11, Stat-Ease Inc., Minneapolis, USA). For CCD modelling, the variables are chosen to be either dependable or independent. The independent variables were the different constituents of ethosomes namely amount of phospholipids (X1), amount of ethanol (X2) and amount of PG (X3). While the dependent variables (responses) were the vesicle size (Y1), % EE (Y2), and % CDR (Y3). Based on the experimental setup and number of factors involved in the formulation, a quadratic relation between the factors was chosen governed given by Eq. 3. The ANOVA method was used to know the significant effect of the factors and their interactions.