1. INTRODUCTION:

Transdermal drug delivery (TDD) is one of the trendy preferable routes of drug administration as it solves some of the main popular problems of other routes of drug administration such as the oral and parenteral routes¹. A pharmaceutical agent has to comply with certain physicochemical characteristics to be formulated as TDD such as low molecular weight, low melting point, high partition coefficient (lipophilicity) and low dose (i.e., ideally less than 25mg/day).

Unfortunately, the selective property of the skin barrier, limits the presence of many drugs in the transdermal drug market segment¹.

Vesicular systems act as successful pharmaceutical carriers due to their ability to achieve sustained release pattern. They can offer targeted drug release at specified site, and subsequently, they can solve any bioavailability issues². Vesicular drug carriers are colloidal, concentric vesicles where one or more bilayer membranes, mainly composed of natural or synthetic lipids enclose an aqueous core. The biphasic nature of vesicular drug delivery systems allows delivery of both hydrophilic and hydrophobic drugs². Vesicular drug carriers include liposomes, niosomes, sphingosomes, ethosomes, transferosomes and ufasomes³. Unfortunately, liposomes can deliver drugs in epidermal layer, this is much preferable in treatment of skin disorders, e.g: psoriasis or skin fungal infections as liposomes allow the drug to reside in the skin much longer. However, liposomes fail to deliver drugs to the blood stream through the skin, owing to the rigidity of liposomal vesicles due to presence of cholesterol within the phospholipid bilayer². Ethosomes are modified liposomes with relatively high alcohol (ethyl alcohol) content that enables drug moieties to pass into deep skin layers and reach the systemic circulation⁴. The high ethanol content gives great elasticity for vesicular membranes owing to its permeation enhancing effect. Consequently, ethosomes can squeeze themselves easily across skin pores of much smaller diameters⁵.

Carvedilol (CAR) works as a non-selective β- receptor antagonist and an α1 receptor blocker with in-vitro antioxidant properties. Carvedilol is mainly prescribed in the management of cardiovascular disorders; e.g: angina pectoris and hypertension⁶. Nevertheless, CAR suffers from substantial hepatic pre-systematic metabolism which causes massive decline in its oral bioavailability (25–30 %)(6)^.Also, CAR is weak base, which causes its extremely low solubility in the alkaline pH of small intestines⁷. This low solubility which hinders the complete absorption of CAR in the small intestine and colon⁸. Therefore, the poor bioavailability experienced by carvedilol in addition to its insufficient solubility together with its low molecular weight (406.5) and lipophilic nature make it a potential candidate for transdermal route⁹. Different strategies have been conducted for CAR transdermal delivery^6,10. Ethosomes are phospholipid-based elastic nanovesicles with relatively high ethanol concentrations (20–45%)⁴. The goal of our work was to prepare a stable ethosomal gel system for transdermal CAR administration and evaluate its potentiality in improving CAR bioavailability.

2. MATERIALS:

Phospholipon 90G (PL90G) was gifted from Lipoid GmbH, Ludwigshafen, Germany. Carvedilol (CAR) was gifted from Global Napi Pharmaceuticals, Egypt. Carvid ® oral tablets were gifted from Multi Pharma Co., Egypt. Hydroxypropyl methylcellulose (HPMC), acetonitrile and formic acid were procured as HPLC grade from Sigma-Aldrich Co., USA. Ethanol, Methanol and Tertiary butyl methyl ether (TBME) were procured as HPLC grade from Fisher Scientific Co., UK.

3. METHODS:

3.1. Preparation of CAR ethosomes:

For preparation of our formula, we used the cold method previously described by Dayan and Touitou¹¹. The ethanolic vesicular system was composed of 1% (w/v) PL 90G, 30% (v/v) ethanol, 50mg (w/v) CAR and distilled water to 100% (v/v). Briefly, phospholipid and drug were mixed with ethanol in a tightly sealed vessel¹². The ethanolic solution obtained was heated to 30°C by a thermo-regulated water bath (Memmert, SV 1422, Germany) while being stirred at 700rpm with a magnetic stirrer (Wise Stir, Korea)¹¹. Double-distilled water preheated to 30°C was poured dropwise through a syringe to the ethanolic solution. Stirring was continued for 5 min. after complete addition of water¹¹. The final milky dispersion was left for 30 min. to allow cooling down to room temperature before storing in the refrigerator overnight¹². The ethosomal formulation was probe sonicated (Model-Q125, Newtown, CT, USA) at 4°C in an ice-bath at 40 % output frequency (at 40W) for 3 min. to obtain smaller vesicles size¹².

3.2. Physicochemical characterization of the prepared ethosomal formulation:

3.2.1. Vesicle size and polydispersity index (PDI) measurement:

The vesicle size (VS) of ethosomal formulation was measured at 25^°C with a Zeta-Sizer (Malvern, Nano Series ZS90, Malvern Instruments, Ltd., UK) by dynamic light scattering method (DLS)¹³. Before size measurement, the formulation was appropriately diluted with ethanol-water (30% v/v). PDI was also measured to ensure acceptable particle size homogeneity. Measurements were taken from three independent samples and the results were displayed as mean±SD.

3.2.2. Measurement of entrapment efficiency percentage (EE %):

The entrapped amount of CAR was calculated indirectly using the ultracentrifugation technique¹⁴. The ethosomal formulation was spun for 1 hour at 4°C and 20,000 revolutions per minute (Stratos centrifuge, Germany)^14,15. The supernatant was carefully collected after centrifugation and filtered through a 0.2µm Millipore membrane filter, followed by proper dilution with ethanol-water (30% v/v). A UV spectrophotometer (Shimadzu, UV-2401 PC, Japan) was used to measure the amount of unentrapped medication in the supernatant at 240nm using a 1: 1 water-ethanol solution as a blank. The experiment was repeated three times and the mean value and standard deviation (SD) were calculated.

Finally, the EE% was calculated as follows¹⁵:

EE % = (Total drug amount - (unentrapped) drug in the supernatant )/(Total drug amount)×100

3.2.3. Stability study:

The formulation was packed in well-sealed amber glass bottles at two different storage temperatures: refrigeration (4±2°C) and room temperature (25±2°C) with a relative humidity (RH) of 60±5% for 3 months period. EE% and VS were measured periodically at one, two and three months¹².

Also, size change rate (SCR) was calculated from the following equation¹⁶:

SCR=(S3-S0)/S0*100 (2)

3.2.4. Zeta potential and Transmission electron microscopy (TEM):

Zeta analysis was performed on the ethosomal formulation by DLS using a ZS90 Zeta-Sizer (Malvern Instruments, Ltd., UK). The sample was diluted with double distilled water at a ratio of 1:100 (v/v) and measured at 25°C¹⁸. Transmission electron microscopy was also used to examine the morphology of the samples (JEOL Co., JEM-2100, Japan). Briefly, the formulation was diluted in double distilled water at a ratio of 1:100 (v/v), then stained with a sufficient amount of 2 % (w/v) phosphotungstic acid and gently mixed. A drop of the mixture was placed on the carbon-coated grid. The grid was allowed to dry before TEM was used to examine the vesicles¹⁶.

3.3. Preparation of CAR-loaded ethosomal gel system:

The ethosomal formulation was incorporated into 3% (w/w) HPMC gel system¹⁹. An accurately weighed amount of HPMC (3% (w/w)) was added to hot distilled water under stirring at 1000 rpm to yield a drug-free gel base. The gel base was left for 24 hours in refrigerator to ensure complete polymer swelling and homogeneity²⁰. The ethosomal gel was made by continuously stirring the appropriate volume of the ethosomal formulation (0.5 mg Carvedilol/ mL) with the pre-prepared gel base at a ratio of (1:1.5 v/w). At the same ratio, CAR aqueous suspension containing 0.5 mg/mL was added into HPMC gel base for comparison purpose.

3.4. Characterization of ethosomal gel system:

3.4.1. Visual inspection (Appearance and colour):

The prepared gel formulation was examined for appearance, homogeneity, colour, and the presence of any lumps or aggregates²¹.

3.4.2. pH measurement :

pH of 10 % (w/w) aqueous solution was measured using a digital pH meter (3510 Jenway pH meter, UK) to test the compatibility of the gel with skin. Using a magnetic stirrer, 1 g of the ethosomal gel was dissolved in 9 g of distilled water to make the solution²². The test was repeated three times, and the mean value and standard deviation (SD) were calculated.

3.4.3. Spreadability test and rheology measurement:

About 0.5g of the prepared gel formulation was squeezed between two glass slides and left for about 5 minutes, or until no more spreading was visible. The diameter of the generated circle after gel spreading was measured to determine spreadability²². Brook-field Viscometer (DV-III, programmable rheometer, spindle 52, USA) was used to assess the rheology of the produced ethosomal gel at 25°C²².

3.4.4. Drug content:

An accurately weighed quantity of the ethosomal gel (about 2g) was mixed with 25mL methanol in a stoppered flask. To allow complete drug extraction, the mixture was sonicated for 15min with the aid of a bath sonicator (SH 150 – 4L, California, USA).The obtained solution was filtered, suitably diluted with methanol and assayed at 242nm against methanol as a blank²¹.

3.4.5. Skin permeation study:

3.4.5.1. Preparation of rat skin

Adult male Wister albino rats weighing 170 to 190g were used in the study. A total of six rats were used during the permeation study. Three rats were randomly selected for each formulation i.e, drug loaded-ethosomal gel and CAR plain hydrogel²¹^.

The rats were sacrificed by cervical dislocation. The hair on the dorsal skin of the rats was shaved with utmost attention using an electrical clipper to avoid skin damage¹⁸. The animals' hairless skin was excised, and the subcutaneous fat tissue was carefully removed. Following that, the removed skin was thoroughly rinsed with phosphate buffer solution PBS, pH=7.4, and visually checked for any cracking or furrows. The excised skin was then stored at -20^oC until further use¹⁸.

3.4.5.2. Skin permeation test:

Skin permeation test was conducted using locally fabricated assembled diffusion cell to study the permeation of CAR from ethosomal gel and CAR plain hydrogel using animal skin model. The dimensions of the used receptor compartment were 100mL volume and 3.4cm²diffusion area. The donor compartment was filled with 20% v/v methanolic PBS pH 7.4 to maintain sink conditions¹⁸. The dermis of the excised dorsal rat skin was fixed on the receptor compartment with the stratum corneum layer facing the donor compartment. Appropriate amount of the ethosomal gel, equivalent to 1 mg carvedilol was applied on the effective area of stratum corneum layer (3.4cm²). The methanolic PBS buffer was continuously stirred at 100 rpm and maintained at 37±1^oC using a thermostatically-controlled magnetic stirrer¹⁸. At suitable time intervals, 2mL samples were collected from the sampling port up to 24 hr and filtered using 0.2µm membrane filter²³. Drug concentrations in the filtered samples were obtained after appropriate dilution at 242nm using a UV-spectrophotometer²⁴. The receptor cell was immediately replaced with 2mL of fresh diffusion medium throughout the experiment. The same procedure was conducted on CAR plain hydrogel containing 1mg carvedilol for comparison. The experiment was performed three times and mean values ± SD were calculated. Also, steady-state flux (Jss, µg/hr.cm²) and permeability coefficient (Kp, cm/hr) were calculated using the formulae below²¹^:

J_ss = (Amount of drug permeated)/(Time*area of exposed skin) (3)

3.5. Pharmacokinetic study:

3.5.1. Animal handling:

The pharmacokinetic study was conducted on twelve adult male healthy Sprague-Dawley rats weighing between 200g±10%²⁵. Rats were allocated randomly in two groups each having six rats.

Group I: received ethosomal gel containing 0.5mg CAR /1mL ethosomal dispersion incorporated in 3% HPMC i.e: equivalent to 2.25mg/kg.

Group II: received marketed oral tablet, namely (Carvid ® 6.25mg tablet) in a suspension form (0.5mg/1mL) prepared with distilled water equivalent to 2.25mg/kg²⁵.

3.5.2. Study design:

The study design was performed in a parallel manner. Concerning rats in group (I), approximately 9cm² of dorsal skin was shaved 24 hr before the time of the experiment and the rats were kept under observation to observe any undesirable effects of shaving. The ethosomal gel was applied on the shaved area of animals skin. After application, the rats were restrained with their hands for 5min. before being put in separate cages²⁶.Group (II) rats received oral tablet suspension (Carvid® 6.25mg) after its crushing and reconstitution in sufficient volume of distilled water. Each rat in group (II) was administered one dose of CAR aqueous suspension i.e, CAR equivalent to 2.25mg/kg using an oral gavage needle²⁶. Ether was used to anesthetize rats before the collection of blood samples²⁵. Blood samples (0.75mL) were collected from the rats' retro-orbital veins using heparinized tubes at the following time intervals: 0 (pre-dose), 1, 2, 4, 6, 8, and 24 hr post dosing. Collected blood samples were centrifuged at 3500rpm for 5 minutes (Biofuge, primo Heraeus Germany, Centrifuge)²⁷. Plasma was separated and stored at -20^oC for further LC/MS analysis.

3.5.3. Chromatographic system and conditions:

Plasma samples were analysed using reversed-phase HPLC-MS/MS (AB Sciex Instruments, Canada)²⁸. The system comprised an API 4000 triple Quadruple LC/ mass spectrophotometer detector equipped with a Shimadzu LC-20AD pump (Shimadzu, Japan) and a Shimadzu SIL-20A/HT auto sampler injector (Shimadzu, Japan). The isocratic mobile phase was composed of acetonitrile and 0.1% formic acid (80:20% v/v) mixture²⁹. The run time and column flow rate were 1 min. and 0.80mL/min respectively and the injection volume used was 6µL.

3.5.4. Sample preparation:

About 0.5mL of the withdrawn plasma was mixed with 100µL of Toremifene (internal standard) in plastic tubes. About 5mL of TBME were added to each tube. Samples were vortexed for 1 min and left for 15 min. at room temperature to facilitate plasma proteins precipitation. After 15 min., samples were centrifugated (3500 × g for 5 min.) at 4^oC to separate the precipitated plasma proteins. The organic layer was withdrawn with a micropipette and evaporated to dryness in a vacuum concentrator. The collected residue was mixed with a 250µL mobile phase^20,29.

3.6. Pharmacokinetic parameters:

The trapezoidal method was used to calculate the area under the curve [AUC]_0–24. C_max, peak plasma concentration of drug and t_max, time needed by the drug to reach C_max were attained from the plot of plasma concentration against time²⁰.

F_rel, percentage relative bioavailability was determined from the equation below²⁶:

Frel=(Dpo*AUC_TS)/(D_TS*AUCpo) * 100 (5)

Where, D po= dose of oral formula, D_TS= dose of transdermal formula, AUC_TS= AUC of the transdermal formula curve and AUC po= AUC of the oral formula curve.

4. RESULTS AND DISCUSSION:

4.1. Physicochemical characterization of CAR ethosomal formulation (VS, PDI and EE %):

The ethosomal formulation showed small vesicle size (46.75±9.41nm), low PDI (0.25±0.09) and high EE% (97.10±2.30 %). The low PL content and high ethanol content (1g PL90G and 30% ethanol) may be the factors responsible for the small vesicle size¹⁵. The high EE% can by ascribed to the sufficient intermediate amount of carvedilol added which was 50mg/100mL. Low PDI value (< 0.3) is an indication for the homogenous vesicle size distribution²⁰.

4.2. Stability study:

Ethosomes stored at cold temperature (4±2^oC) and room temperature (25±2°C) showed significant difference in EE % after three months at p˂0.05 (Table 1)³⁰. The increase in vesicle size was acceptable at refrigeration temperature where the SCR was found to be 7.37% i.e. ˂10%. However, the ethosomal formulation showed remarkable particle size agglomeration with SCR of 347.06% after being stored at room temperature as shown in Table 1. This huge particle size enlargement may be due to fusion of phospholipid bilayers between the adjacent vesicles. Consequently, storage of lipid-based nanovesicles at lower temperatures is recommended for minimization of vesicle size enlargement and potential drug loss²⁰.

4.3. Zeta potential and TEM:

Generally, high positive or negative ZP values (> |30| mV) result in stable colloidal dispersions as a result of the electric repulsion between particles having identical electrical charge³¹. The ethosomal formulation showed satisfactory zeta potential value of + 38.45mV. The positive sign of zeta potential may be due to the high pka value of weakly basic carvedilol (pKa = 7.8-8.7)³². The photomicrograph of the ethosomal formulation showed spherical unilamellar vesicles with nanometric size (Figure 1)³³.

Fig.1 photomicrograph of ethosomes using TEM

4.4. Characterization of the ethosomal gel system:

The prepared gel was homogeneous and had a smooth texture without coarse particles. pH value of the ethosomal gel was 6.0±1.31 which indicates the suitability of the gel formulation for skin application due to the high tolerability and absence of skin irritation signs upon application of a gel having a pH close to the pH of the skin (4-7units)²².The ethosomal gel showed mean spreadability circle diameter of 6.40±0.12cm which indicates sufficient spreadability¹⁹. The viscosity of the ethosomal gel was 30979.5 mPa.s (mPa.s =1 cP). The percentage of incorporated drug in the prepared gel was 95.21±1.2% which assures uniform distribution of CAR within the ethosomal gel system.

4.5. Ex vivo permeation study:

The percentage of CAR permeated across rat skin from the ethosomal gel after 24 hr was significantly higher than that permeated from CAR plain hydrogel (p< 0.05)¹³.As shown in figure 2, the percentage of drug permeated from both CAR ethosomal and non ethosomal gels was found to have linear behaviour up to 6 hr, then, steady state behaviour was observed till the end of the 24 hr¹³. The flux values for the ethosomal gel and CAR plain gel were 11.33±0.09µg/hr.cm² and 6.26±0.14µg/hr.cm² respectively. The Kp values for the ethosomal gel and CAR plain gel were 1.10±0 and 0.60 ±0 respectively. The flux and Kp of the ethosomal gel were relatively higher in comparison to CAR plain hydrogel at p˂0.05. The efficient permeation enhancing mechanism of ethanol may be the cause for such good permeation.

Fig. 2 Permeation profile of CAR from ethosomal and non ethosomal gel through excised rat skin (n = 3)

4.6. In vivo Pharmacokinetics study:

Several pharmacokinetic parameters including C_max, t_max, K_{el ,}t1/2, AUC(s) and MRT were calculated for both test and oral formulae. Mean plasma concentration against time after transdermal application of ethosomal gel system, was compared to the commercially available oral tablet (Carvid) as illustrated in Figure 3. The mean peak plasma concentration values were 33.60±2.1ng/ mL and 24.14±1.28ng/mL for oral tablet and ethosomal gel respectively, (n=6). T_max was1 hour and 6 hours for oral tablet and carvedilol ethosomal gel respectively (Figure 3)²⁸. At p<0.05, the ethosomal gel had significantly lower C_max as well as delayed t_max,compared to the commercial oral tablet. Mean exposure [AUC]_0-24 for the transdermal ethosomal/24hr was significantly higher than the [AUC]_0-24 of the same dose i.e; 2.25mg/kg. AUC values for test and oral formulae were 0.301±0.01µg.hr/mL versus 0.106±0.01µg.hr/mL respectively, at p<0.05). The high C_max and small t_maxvalues after oral administration may be claimed to rapid absorption of CAR from the gastro intestinal tract as well as extensive metabolism by liver enzymes known as first pass effect. As for the transdermal ethosomal gel, the lower C_maxand prolonged t_maxvalues may be due to barrier feature of upper skin layer that leads to slow release of drug into skin dermis followed by sustained spontaneous delivery in the blood circulation and hence the drug bypasses the portal circulation. As a matter of fact, drug delivery systems having low C_max and prolonged t_max – result in slower absorption and hence reduced amplitude between the successive peaks and troughs of plasma drug concentrations – while maintaining high AUC and therapeutic drug concentration level. This subsequently reduces the dose-related side effects³⁴. Mean residence time (MRT) data showed higher value for the transdermal formula in comparison with the alternative oral route i.e; 7.2 ± 0.4 hr and 3.3 ± 0.1 hr. This significant difference in C_max, AUC and MRT (where p˂0.05) was emphasized and assured by the high value of relative bioavailability (F_rel) of the ethosomal gel which was 281.92% compared to the oral tablet (Carvid).

Fig. 3 Plasma drug concentration against time profile following transdermal and oral administration of CAR (2.25 mg kg⁻¹) for six rats (mean±SD)

5. CONCLUSION:

The ethosomal formulation was more stable when stored at 4°C for 90 days. Ethosomal formula showed good zeta potential and was observed under TEM. The ethosomes appeared as spherical unilamellar vesicles. Ethosomal gel showed good pH, spreadability, homogeneity and suitable drug content. Furthermore, a significantly higher flux and Kp were obtained from the ethosomal gel than the plain CAR hydrogel formulation after a 24-hr permeation study. The ethosomal gel of carvedilol depicted a satisfactory prolonged t _max, higher AUC and longer MRT for CAR which nominates it as novel sustained drug delivery carrier in addition to other transdermally delivered antihypertensive agents. In summary, ethosomal gel system is a promising transdermal delivery model for carvedilol since it showed significantly improved bioavailability.

6. ETHICAL APPROVAL:

Ethical Compliance: the ethical standards were complied. The experiment protocol for ex vivo and in vivo studies was reviewed and approved by the Institutional Animal Ethics Committee of the National Research Center, Egypt (Registration no.15188) and Al-Azhar University, Faculty of Pharmacy for girls (Registration no.⁴¹).

The authors have no competing interests to declare that are relevant to the content of this article.

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Received on 16.04.2022 Modified on 02.06.2022

Research J. Pharm. and Tech 2022; 15(9):4017-4023.

DOI: 10.52711/0974-360X.2022.00673

Formulation	Time (days)	Temperature (^oC)
		4 ± 2^oC		25 ± 2^oC
		*Size (nm)	*EE (%)	*Size (nm)	*EE (%)
Ethosomal formulation	0	46.75 ± 9.41	97.10 ± 2.34	46.75 ± 9.41	97.10 ± 2.34
	30	48.10 ± 2.19	95.71 ± 1.39	84.0 ± 3.10	93.21 ± 1.04
	60	49.90 ± 2.86	95.38 ± 0.37	124.70 ± 10.41	91.07 ± 1.01
	90	50.20 ± 4.85	94.20 ± 0.53	209.0 ± 10.41	87.67 ± 1.20