Enhancement and Extended Release of the Anti-hypertensive Drug Carvedilol using Optimized Ethosomal Gel via Transdermal Route

Background Carvedilol, a popular anti-hypertensive drug, when orally administered has very poor bioavailability on the account of undergoing hepatic metabolism and therefore it becomes primal to explore an alternative drug delivery route for carvedilol. For a drug to be delivered by undergoing the least number of stages of metabolism and achieve high target specicity, transdermal delivery is the most preferred route. Hence, a study was conducted to test the potential of ethosomes as a candidate for transdermal delivery of carvedilol. A statistical study by using Central Composite Design (CCD) was also conducted for optimizing the quantity of the primary constituents present in the ethosomes. The optimized ethosomal formulation was then incorporated into a hydrogel to prepare the ethosomal gel. Results The optimized formulated ethosomal suspension and the ethosomal gel were undergone physicochemical, compatibility and in-vitro drug release studies along with characterization studies. The incorporation of the ethosomes into the hydrogel proved to be effective for skin application thereby ensuring better transdermal delivery. The optimized ethosomal gel has showed credible physical appearance, spreadability, viscosity and in-vitro drug release. The pharmacodynamic studies conducted on Wister rats revealed that the anti-hypertensive action was gradual and sustained lasting up to a period of 24 hours. The stability studies conducted also showed that prepared formulations maintained its consistency within the range for the measured parameters of physical appearance, rheological properties and entrapment eciency for a period of 3 months.


Abstract Background
Carvedilol, a popular anti-hypertensive drug, when orally administered has very poor bioavailability on the account of undergoing hepatic metabolism and therefore it becomes primal to explore an alternative drug delivery route for carvedilol. For a drug to be delivered by undergoing the least number of stages of metabolism and achieve high target speci city, transdermal delivery is the most preferred route. Hence, a study was conducted to test the potential of ethosomes as a candidate for transdermal delivery of carvedilol. A statistical study by using Central Composite Design (CCD) was also conducted for optimizing the quantity of the primary constituents present in the ethosomes. The optimized ethosomal formulation was then incorporated into a hydrogel to prepare the ethosomal gel.

Results
The optimized formulated ethosomal suspension and the ethosomal gel were undergone physicochemical, compatibility and in-vitro drug release studies along with characterization studies. The incorporation of the ethosomes into the hydrogel proved to be effective for skin application thereby ensuring better transdermal delivery. The optimized ethosomal gel has showed credible physical appearance, spreadability, viscosity and in-vitro drug release. The pharmacodynamic studies conducted on Wister rats revealed that the anti-hypertensive action was gradual and sustained lasting up to a period of 24 hours. The stability studies conducted also showed that prepared formulations maintained its consistency within the range for the measured parameters of physical appearance, rheological properties and entrapment e ciency for a period of 3 months.

Conclusions
The incorporation of the drug loaded into hydrogel and its effect on regulating systolic blood pressure in a sustained way lasting 24 hours proved to be better than the present available marketed formulation which has a rapid action with the anti-hypertensive effect lasting only for 10 hours. The chosen route for delivering the drug transdermally hence proved to be effective with better enhancement and permeation capability and shows the high potential of ethosomes to be considered for novel delivery of other anti-hypertensive drugs.

Background
Transdermal drug delivery poses a better advantage over other established routes of delivery because the concerned drug does not undergo pre-systemic metabolism resulting in better bioavailability and better patient compliance. The primary challenging part for transdermal drug delivery is crossing the barrier made by the stratum corneum (SC). One of the primary reasons is that SC is tightly connected which limits the drug permeation (1). To improve transdermal drug penetration, various physical and chemical methods have been tried and tested which include iontophoresis, microneedles and nanocarriers like liposomes and nanolipid particles (2). Microneedles have been used for successful transdermal delivery of cisplatin for synergistic chemo-therapy of breast cancer (3). Even though lipid nanoparticles have garnered attention in recent times, their mechanism of penetration and transdermal properties have not been fully understood (4). Moreover, conventional liposomes with their poor penetrating capability are not ideal candidates for transdermal delivery (5).
In the recent years, specially designed vesicular drug carriers, other than liposomes, have been developed and characterized for effective delivery of the drug involved by crossing the barriers of the skin layers (6,7).One among such vesicular carriers is ethosomes. Ethosomes are lipid based vesicular drug carriers in which the concentration of ethanol is high. The spherical shape of binary ethosomes is well de ned and has an enclosing of a lipid bilayer. They can penetrate through SC with ease and therefore has been established as an effective candidate for transdermal drug delivery of a variety of drugs (2,8). The size of the synthesized ethosomes is usually smaller in size and exhibit better stability than liposomes. The predominant presence of ethanol in high concentration in ethosomes impart them the ability to modify the highly dense alignment of the lipid bilayers in the SC thereby ensuring deeper penetration and moreover the low-toxicity and less irritability on the skin also promote the use of ethosomes for transdermal delivery (9).
Carvedilol, a popular anti-hypertensive drug, when administered orally, has low bioavailability mainly because of its lipophilicity and hence undergoes hepatic metabolism. It also has low dissolution capability belonging to the class II category in biopharmaceutical classi cation system (8,10). However, its high lipophilicity and low molecular weight make the probability of transdermal drug delivery better. One of the feasible ways to increase the bioavailability of carvedilol is its incorporation into lipid based carriers. Some of the lipid based carriers include microspheres and solid lipid nanoparticles which have been administered via routes which escape hepatic metabolism. As mentioned earlier, ethosomes with its various favourable characteristics, can be considered to be a better choice among the available lipid based carriers for transdermal delivery.
Through this study, as shown in Fig. 1 successful entrapment of carvedilol loaded ethosomes in hydrogel for transdermal delivery was possible and the anti-hypertensive effect of the drug was tested on Wister Rats by tail-cuff method. Liposomes in the form of gel have been used to deliver drugs transdermally for psoriasis treatment (11). Hence, in this study hydrogels were tried and tested to ensure better stability and deposition of the intended drug on the skin (12). The use of hydrogels also made sure the involved drug was of proper consistency in its nal stages when administered.
Beyond a certain limit, the content of ethanol in the formulated ethosomes has the tendency of making the vesicular membrane leaky which can result in low entrapment e ciency and stability (13,14). Hence, the optimal formulation of ethosomes was the solution to tackle this problem. For proper optimization of ethosomal formulations statistical design studies have to be conducted under a given set of conditions. Based on the designs available, the central composite design (CCD), a robust form of response surface methodology (RSM), is the most preferred for determining the best possible formulation. The CCD model is very much e cient for the estimation of the extent of effectiveness of many individual variables (15,16). Therefore, in this study besides the in-vitro and in-vivo studies, the focus was also given on determining statistically the various factors involved in ethosomal gel formulation of carvedilol (17,18).

Results
The e cacy of transdermal drug delivery is highly dependent on the extent of drug solubility. In this study, the solubility of the drug carvedilol in ethanol was found to be very effective, the solubility increased as the concentration of ethanol in the buffer medium was increased. This trend can be seen from Table 1. However, when the concentration of ethanol was increased to 45% (v/v) during ethosomal preparation, the drug-loaded ethosomes could not be obtained. When the ethosomes were observed under SEM (at 12 kV and 30.0 kV), the ethosomes were found to be nearly spherical and properly dispersed with minimal aggregation as shown in Fig. 2. TEM was used to examine the periphery of the ethosomes formed it was seen that ethosomes formed were multilamellar and smooth surfaced as in Fig. 3.
As mentioned before, to nd a statistical relationship among the various components involved in ethosomal formulation, CCD was used. The determined independent variables (X i ) were the amount of lipid (X 1 ), ethanol (X 2 ) and propylene glycol (X 3 ) added. The levels or extremities decided for each independent variable can be seen from Table 2.
The dependant variables (Y i ) chosen were vesicle or particle size (Y 1 ), entrapment e ciency (%EE) (Y 2 ), cumulative drug release (CDR) (Y 3 ). The desired response for each dependent variable can be seen from Table 3.  Zeta Potential (-mV) Maximize The software DoE was used and from the regression equations, it was observed that a quadratic relation between the dependent and independent variables is more suitable with a better co-relation among the variables. This can be seen in   A PDI value of less than 0.5 is an indicator of homogenous size distribution of vesicles (19). From Table 6 it can be seen that for every formulation, the PDI value was less than 0.45 which ensured the homogenous distribution of vesicles. The value of zeta potential, an indicator of the stability of the suspension formed, should always have negative value to show that the suspension has good stability. All the formulations had a negative value for zeta potential.
For the sake of clarity and conformation, the FT-IR spectra ( As far as the in-vitro drug release pattern from the various prepared ethosomal formulations (EF1-EF20) was concerned, from Fig. 6 we can see that almost all the formulations, had a linear pro le release up to 10 hours and then the curve plateaued without much release. From the graph, it can be deduced that the formulation EF1 had a better sustained release which lasted more than 72 hours. Moreover, the other characteristics associated with EF1 namely vesicle size, zeta potential and entrapment e ciency were also credible. Therefore this formulation was chosen to be incorporated into various hydrogel formulations (20).
Similarly, an in-vitro drug release studies from different formulations (G1-G6) of the ethosomal incorporated hydrogels were also done and release pro le for each batch can be seen in Fig. 7. The hydrogel formulation G7 was loaded with the pure drug was chosen as a control. For G7 (the control), an almost 100% release of the drug was observed within the rst ten hours with a linear pro le while for the batches G1-G6 a linear release pro le was observed with 60-80% of the drug released. While for batch G2 only 50% was released but also showed a better sustained release. For G6, the release stopped after 25 hours with 80% release, while for G4 and G5 90% release was observed which stopped after 50 hours. For batches G1 and G3 90% release could be seen lasting up to 75 hours but G2 batch showed better sustained release. Hence, the batch G2 was selected for further in-vitro and ex-vivo permeation studies. The composition of each gel formulation can be seen from Table 6. The drug release from the hydrogel formulations depends on the presence of three-dimensional polymeric cross-links of hydrogels which in turn is governed by a converse relationship with the viscosity of the hydrogel(21). For a better understanding of the pharmacodynamic study, a comparative model was adopted and the study was done for both ethosomes and ethosomal gel along with the marketed formulation. The case studies were done for hypertensive effect induced by both SC solution and MP. The SC solution and MP were orally administered to the rats.
From Fig. 8 and Fig. 9, it can be observed that in the group of rats, in which no drug of any kind was administered, the induced systolic BP remained constant. The marketed formulation of carvedilol exhibited a rapid decrease in the systolic BP and normalizing it within 10 hours. Meanwhile, in case of both ethosomes and the ethosomal gel, a gradual reduction in systolic BP was observed which was brought down to the normal in 24 hours. This exhibited the sustained and extended action or release of carvedilol in case of both ethosomes and ethosomal gel.
When it came to the physicochemical properties of the drug loaded ethosomal gel formulations, as seen in Table 7 the pH value was well within the expected range as that for skin (5.5-6.8) and hence would not cause skin irritation. The spreadability of the gels were found to be low thereby ensuring better localization of the loaded drug and better skin penetration when applied. In all the gel formulations, the assay was found to be above 90%.

Discussion
The effect of ethanol on increasing the solubility of carvedilol can help to avoid drug precipitation and hence we can prepare better stable ethosomes which will exhibit enhanced drug entrapment e ciency (22). This can be attributed to the fact that disruption of lipid molecules involved which then will affect the structural integrity of the ethosomes and hence lead to drug precipitation. Moreover, there is an increase in the vesicular size of the ethosomes formed when the concentration of ethanol is increased thereby making the transdermal delivery of the drug less effective (22,23).
The multi-lamellar nature of the formulated ethosomes can be reasoned to the fact that the presence of ethanol contributed to the enhancement in the exibility as well as the uidity of the phospholipids bilayers (24). Out of the 20 batches of ethosomes prepared, the formulation which consisted of 2-5% phospholipid, 20-40% ethanol along with the appropriate amount of water had multi-lamellar structure. This again shows the in uential nature of ethanol presence in con rming the type of vesicular structure formed during the ethosomal preparation. Another point to be noted is that, ethosomal formation is con rmed when the concentration of the hydrophilic phase is increased, that is when the phospholipid molecules reorganize leading to an increase in turbidity of the preparation medium.
From the statistical data obtained it can be deduced that the ethosomal formation as well as their size are dependent on the amount of phospholipid, ethanol and PG used in the formulation which thereby in uence the % EE and the % CDR. To ensure proper transdermal drug delivery, the preferred approximate ethosomal size is 300 nm. It can be seen that for the different formulations from EF1 to EF 20 the vesicular size increased as the addition of phospholipid increased. The increase in phospholipid content was intended to enhance the structural rigidity of the vesicles. At the same time, changes in ethanol concentration were also done to test the stability of the vesicles. The effect was that ethanol addition favoured a reduction in the vesicle size which is due to the steric stabilization of the net charge of the system and edge activation mechanisms (25). The increase in PG concentration in the range of 10% (v/v) also resulted in the decrease of vesicle size, which shows the ability of PG to interpenetrate the phospholipid layer which provided better exibility to the ethosomes.
The change in zeta potential was noted whenever a change in the concentration of the additives was made. An increase in the zeta potential value was observed with the increase in concentration of ethanol and PG which indicate that the polar nature of ethanol and propylene glycol boosted the net surface charge hence resulting in strong electrostatic repulsion among the ethosomes. This phenomena prevented vesicle aggregation and therefore better stability and uniformity for the suspension (26).
Similarly, the % EE was also affected by the change in concentrations of the additives during ethosomal formulations.
The amount of phospholipid had a directly proportional relation for %EE, suggesting the increase in phospholipid bilayer formed around the vesicle and hence better holding capacity of the lipophilic drug. However, the effect of ethanol showed a different trend. Up to a concentration of 40%(v/v), the %EE increased which can be attributed to the increased uidity of the ethosomal membrane and also due to increased solubility of the lipophilic drug in the inner polar ethosomal core. But when ethanol concentration was above 40%(v/v), the %EE decreased due to increased solubilisation of the drug in ethanol causing disruptions in the vesicular membrane (27,28), This was a contradicting the effect from the normal, which can be reasoned to increase in the uidity of the membranes thereby the entrapped drug leaching out (29).
The decrease in the in-vitro release of carvediliol when the amount of phospholipids was increased can be because of the increased rigidity in the assembly of phospholipid bilayer as concentration increased. While the increased drug For skin retention studies, both the ethosomal suspension and ethosomal gel showed better retention capability than the pure drug loaded hydrogel. This occurred due to the combined effect of ethanol in strengthening the penetration effect of carvedilol by its high solubility in ethanol along with improving the elasticity of the prepared vesicles which allowed them to pass through the skin pores even though the skin pores have much smaller diameter than that of vesicles.

Conclusions
From the studies conducted, it can be brought to light that nano-sized ethosomal suspension loaded into hyrdogelcan be considered for transdermal delivery of the anti-hypertensive drug, carvedilol. A statistical study using CCD helped to know better the in uence of the factors that directly affect the synthesis as well as the entrapment e ciency of ethosomes. The resulting size and morphology of the ethosomes were well within the acceptable range to be considered for transdermal delivery. The ethosomes and ethosomal gel exhibited the ability to penetrate the skin layers to a greater extent. It was also shown that successful incorporation of the drug-loaded ethosomes into hydrogel was possible without compromising the molecular integrity of the drug involved. The in-vitro and ex-vivo release of the drug showed a sustained release pattern and the amount of encapsulated drug released was in the expected limit. The pharmacodynamic studies revealed that the ethosomes and ethosomal gel had gradually decreased the systolic BP and the anti-hypertensive action lasted for 24 hours while for the present marketed formulation, the action was rapid and lasted only for 10 hours. The skin irritation studies conducted showed that the ethosomal gels were safe to use. Moreover, the stability of the ethosomes and ethosomal gel was credible lasting up to3 months. Overall, the use of ethosomes as a drug delivery vehicle for an anti-hypertensive drug was found to very effective. 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 nal step was refrigeration of the suspension at 4 ℃(31).

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 quanti ed for determining the drug concentration using RP-HPLC method.

HPLC quanti cation of dissolved drug
The HPLC unit used for the drug quanti cation (Shimadzu, Japan) had the following speci cations: LC-10AT solvent module with SPD-10A column, PDA detector with LC10 software. The RPCL column had the speci cations of C18 (150

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 speci c 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 nal semi-solid gel was stored overnight at room temperature.

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 quanti cation of the dissolved drug earlier(35).

Drug entrapment e ciency
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 e ciency 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).

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.

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.

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).

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).

Viscosity measurement of gels
The viscosity was measured using a viscometer (Brook eld Viscometer, CAP 2000L) under high torque and lowtemperature 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).

Spreadability of the gels
The extent and degree of the gel Spreadability were measured using the glass slide apparatus with the help of a modi ed 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 xed 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.

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 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 rst 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.

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 e ciency and in-vitro drug release studies was selected. After sacri cing 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 e ciency.
The skin was then hydrated overnight at 25 o C 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).  (20). The diffusion cells were maintained in a thermostatically controlled water bath shaker at 37 ± 1 o C 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 ltered using a nylon syringe lter 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 ux (J ss ) was also determined. The measurements were taken in a triplicate manner and compared with those of a control batch.

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 sacri ced 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).

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 e ciency (EE%) and assay using HPLC. The stability studies were conducted at 0, 1, 2, 3 and 6 months (46,47 where A total = total amount of carvedilol in ethosomal suspension; A unentrapped = unentrapped carvedilol in ethosomal Schematic showing the preparation of optimized ethosomes and its subsequent loading on to hydrogel followed by pharmacodynamic study for examining the anti-hypertensive effect of the drug carvedilol Figure 1 Schematic showing the preparation of optimized ethosomes and its subsequent loading on to hydrogel followed by pharmacodynamic study for examining the anti-hypertensive effect of the drug carvedilol In-vitro drug release studies of formulation (EF1-EF20) (EF-Ethosomal formulation) Figure 6 Page 31/33 In-vitro drug release studies of formulation (EF1-EF20) (EF-Ethosomal formulation) Figure 7 In-vitro drug release studies of gel formulation (G1-G7) (G-Ethosomal Hydrogel) Figure 7 In-vitro drug release studies of gel formulation (G1-G7) (G-Ethosomal Hydrogel)