1. INTRODUCTION:

Carcinoma is referred to as “an ailment characterized by an unrestrained growth of abnormal cells which if untreated and unchecked eventually kills the patient”. Cancer has become one of the ten leading causes of death in world. It is estimated that there are nearly 2 - 2.5 million cancer cases at any given point of time. Over 7lakh new cases and 3lakh deaths occur annually due to cancer.

Data from population-based registries under National Cancer Registry Programme indicate that the leading sites of cancer are skin oral cavity, lungs, esophagus and stomach amongst men and cervix, breast and oral cavity amongst women. Curcumin has an excellent safety profile having various pleiotropic actions, including anti-inflammatory, antioxidant, antitumoral, anticancer, and antimicrobial activities, with the potential for neuroprotective activity but the poor oral bioavailability limits its use as oral dosage form. Hence the objective of the present investigation was to fabricate and develop curcumin loaded ethosomes to increases its solubility thereby permeability for topical application in the treatment of skin melanomas.

Topical delivery of the curcumin can improve its bioactivity with reduction of the side effects and enhance the therapeutic efficacy. Ethosomes are novel lipid carrier developed by Touitou et al showing enhanced skin delivery of drugs. Ethosomal drug delivery system is a new state of the art technique and easier to prepare in addition to safety and efficacy. Ethosomes have become an area of research interest, because of its enhanced skin permeation, improved drug delivery, increased drug entrapment efficiency etc. Ethosomes were reported to improve in-vivo and in-vitro skin delivery of various drugs. Ethosomes can improve skin delivery of drugs both under occlusive and non‐occlusive conditions. The present work focuses on the formulation and characterization of Ethosomal systems in enhancing the trans-dermal delivery of curcumin. E. Touitou et. al (2000) [1]in their research article have described a novel carrier for enhanced skin delivery, the ethosomal system, which is composed of phospholipid, ethanol and water. Zhen Zhang et. al. (2011) characterized a novel transdermal delivery carrier, ethosomes containing 5-fluorouracil. The delivery of drugs from ethosomes in human hypertrophic scar (HS) and the mechanisms of action of ethosomes in human HS were investigated. Donatella Paolino et. al. (2012) [2] reported Paclitaxel-loaded ethosomes as topical drug delivery systems for the treatment of this pathology due to their suitable physicochemical characteristics and enhanced skin penetration ability for deep dermal delivery. Chung Kil Song et. al. (2012) describes a novel carrier, transethosome, for enhanced skin delivery of voriconazole. Transethosomes (TELs) are composed of phospholipid, ethanol, water and edge activator (surfactants) or permeation enhancer (oleic acid).

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

The main ingredient used in this study is curcumin obtained from Molychem Fine Chemicals, Mumbai. The chemicals used were cholesterol, ethanol, soya lecithin (Himedia laboratories, Mumbai).

2.2 Development of Calibration curve:

A stock solution of curcumin was prepared by dissolving 100 mg of curcumin in 10 ml ethanol to give a stock solution of concentration 10 mg/ml. From the stock solution, different aliquots were taken in series of 10ml volumetric flasks and volume made up with ethanol to get a series of working standard solutions of concentrations, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml and 5mg/ml. The l_max of the drug was determined, and standard curve was plotted between concentration and absorbance. [3].

2.3 Preparation of Ethosomes:

Thin film dispersion method was used to prepare the dispersion of ethosomes by using various concentrations of lecithin (10, 15, 20 mg) and cholesterol (10, 15, 20 mg). This method involves dissolving Curcumin, lecithin and Cholesterol in ethanol in a round bottom flask and heating the mixture at the temperature above the lipid transition temperature (55±2°C) at 60 rpm until complete evaporation. This follows formation of homogenous thin film on the flask wall by maintaining the condenser (chilled) for complete removal of organic solvent. Purging of Nitrogen was done to oxidize the lipids for at least 12h prior to hydration. Then the film was hydrated with Phosphate buffer pH 7.4 and ethanol (10ml) solution for 30 mins. The dispersion was left undistributed at room temperature for 2 h to allow complete swelling of the lipid to obtain the vesicular dispersion of Curcumin and then subjected to probe sonication (Bandelin RK 100 H, Germany) for total of 45 mins for 3 cycles of 15 mins each (15 sec on/off cycle). Formulation was then stored in refrigerator until further characterization [4]

2.4 Evaluation of Curcumin loaded Ethosomes:

2.4.1 Entrapment efficiency (EE):

Centrifugation method was used to determine the entrapment efficiency of drug. Drug loaded ethosomes were kept overnight at 4°C before centrifugation. Then they were subjected to centrifugation at 12000 rpm for 30 mins using ultracentrifuge (Remi instrument). The supernatant liquid was collected and analysed for free Curcumin by UV-Visible spectrophotometer (UV-1700 series, Shimadzu) at 429 nm. The percent entrapment efficiency was calculated as follows [5].

Entrapment

efficiency (%)

Total amount of drug loaded

Free drug in supernatant

Total amount of drug used in formulation

2.4.2 Vesicles size and zeta potential analysis:

Vesicle size (nm), polydispersity index (PDI) and zeta potential of ethosomal formulations were analyzed by Malvern Zetasizer Nano ZS90 (Malvern Instruments) that works on the Mie theory to determine at given temperature. Then dilution of formulation was done by deionized water and readings were recorded using a scattering angle of 900 at 25^oC using disposable polystyrene cells and disposable plain folded capillary zeta cells, respectively, after appropriate dilution [6].

2.4.3 Polydispersity index:

Polydispersity was determined according to the equation,

Polydispersity = D (0.9)-D (0.1)/ D (0.5)

Where,

D (0.9) corresponds to particle size immediately above 90% of the sample.

D (0.5) corresponds to particle size immediately above 50% of the sample.

D (0.1) corresponds to particle size immediately above 10% of the sample. (Muller R.H et. al., 2008)

2.5 Preparation of Ethosomal gel:

1g of Carbopol 940 was dispersed in 100ml of water

(1% w/v) with constant stirring by using magnetic stirrer (RQ122, Remi instruments, India). Dispersion was kept overnight for soaking. Ethosomes containing drug equivalent to 1% curcumin (100mg) was added into 10ml of the 1% Carbopol 940 and mixed until uniformity was attained. The mixture was neutralized with triethanolamine to form the gel [3].

2.6 Evaluation of Ethosomal gel:

2.6.1 Physical examination:

Physical parameters like color, oiliness/greasiness, softness/grittiness, phase separation and ease of application have been examined as they play vital role in patient’s compliance [4].

2.6.2 pH determination:

The pH of formulation was determined by readings was recorded using digital pH meter (pH 510, EUTECH instruments) [3].

2.6.3 Estimation of drug content:

This was done by dissolving 0.5 g of each formulation in 50 mL of ethanol followed by filtration through the whatman filter paper (No.41) to remove the residues. Placebo sample also filtered through whatman filter paper as of formulation and is used as a blank. Drug content was estimated at 429 nm by using UV spectroscopy [3].

2.6.4 Measurement of viscosity:

Viscosity was determined using Brookfield viscometer with the spindle no.7, with a speed of 10 rpm at 25⁰C (DV II plus, Brookfield) [7].

2.6.5 In vitro permeation studies:

Curcumin release from ethosomal gel was measured by passing through the semipermeable cellophane membrane which is previously immersed in phosphate buffer pH 5.5 for 24 h. The membrane was placed in a franz diffusion cell which have a diffusion area of 1.813 cm² and receiver compartment was filled with 10 ml of pH 5.5 phosphate buffer saline which was constantly stirred with a magnetic stirrer at 300 rpm. The water bath was upheld at a temperature of 37±0.5^◦C to mimic the skin environment. 100 g of ethosomal gel was placed on the diffusion barrier in the donor compartment.1 ml of the solution from the receiver compartment was withdrawn at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10 and 12 h and replaced with a same quantity of buffer. Samples were analyzed by using UV spectrophotometry [8].

2.6.6 Ex vivo permeation studies:

Permeation studies of optimized formulation were performed using Franz diffusion cells with a diffusion area of 1.813 cm². The receptor chamber was filled with 10 mL of Phosphate buffer pH 5.5 solution containing 0.3% of tween 80 a hydrophilic surfactant which acts as a solubilizer and constantly stirred with a magnetic stirrer at 300 rpm. The water bath was maintained at temperature of 37±0.5^◦C. The known amount (100 mg) of formulations was applied on the rat skin surface, facing stratum corneum to donor compartment. Three franz diffusion cells were run simultaneously at pH 5.5. Two mL of sample was withdrawn from each cell during the predetermined time intervals of 0.25, 0.5, 0.75,1,2,4, 6,8,10 and 12 h respectively. The amount of curcumin content in the collected samples was determined by U.V at 429 nm against solvent blank [9].

2.6.7 Release kinetics:

In vitro dissolution has been recognized as an important element in drug development. Under certain conditions it can be used as a surrogate for the assessment of bioequivalence. Several theories/kinetic models describe drug dissolution from immediate and modified release dosage forms. There are several models to represent the drug dissolution profiles where f_tis the function of t (time) related to the amount of drug dissolved from the pharmaceutical dosage system. To compare dissolution profiles between two drug products models dependent (curve fitting), statistical analysis and model independent methods can be used (Costa P., et al., 2001).

To elucidate mode and mechanism of drug release, the in vitro data was transformed and interpreted at graphical interface constructed using various kinetic models. The zero-order release Eq. (1) describes the drug dissolution of several types of modified release pharmaceutical dosage forms, as in the case of transdermal systems, matrix tablets with low soluble drugs, coated forms, osmotic systems etc., where the drug release is independent of concentration.

Q_t= Q_o+ K_ot(1)

where, Q_tis the amount of drug released in time t, Q_ois the initial amount of the drug in the solution and K_ois the zero-order release constant

The first order Eq. (2) describes the release from the system where release is concentration dependent e.g. pharmaceutical dosage forms containing water soluble drugs in porous matrices.

log Q_t= log Q_o+ K₁ t /2.303(2)

where Q_tis the amount of drug released in time t, Q is the initial amount of drug in the solution and K is the first s order release constant.

Higuchi described the release of drug from insoluble matrix as a square root of time

Q_t= K_H√t (3)

where, Q_tis the amount of drug released in time t, K_His Higuchi’s dissolution constant. (Suvakanta dash et al; 2010) (Paulo Costa et al; 2000).

3. RESULTS AND DISCUSSION:

3.1 Development of calibration curve:

Calibration curve of the drug was developed to found out the linearity between concentration of drug in the solution and its optical density. It was concluded that the perfect linearity between the concentration and absorbance was observed when the concentration range was from 1 to 5mg/ml. Table 1 and fig 1 represent the calibration of curcumin using ethanol.

Table 1: Calibration curve of curcumin in ethanol (λ_max= 429nm)

Concentration(mg/ml)	Absorbance
0	0
1	0.140
2	0.272
3	0.412
4	0.526
5	0.643

Fig. 1: Calibration curve of curcumin in ethanol

3.2 Preparation of curcumin loaded ethosomes:

Eight batches of curcumin loaded ethosomes were prepared by hot method and evaluated for vesicle size, polydispersity index, percent entrapment efficiency and zeta potential.

3.3 Evaluation of curcumin loaded ethosomes:

3.3.1 Vesicle size and zeta potential analysis:

Ethosomes loaded with curcumin were prepared using hot method with a slight modification by employing probe sonication. Different ratios of soya lecithin, ethanol and cholesterol were used for various batches to obtain desired size and entrapment efficiency. During the preparation, an increase in size of ethosomes was observed. Employing probe sonication has helped in achieving the lesser particle size. The results of vesicular size propose that the formulation F4 prepared with 10 % of soya lecithin, 4.5 % of ethanol and 10% of cholesterol showed smallest vesicle size with 197.7 nm with a zeta potential of -11.9 mV shown in fig.2 and 3. The results of all the formulations are tabulated in table 2.

3.3.2 Polydispersity:

The Polydispersity index (PDI) is the measure of size distribution of the nanoparticle’s formulation. PDI was measured using Malvern zeta sizer. PDI values range from 0.000 to 1.000 i.e. monodisperse to very broad particle size distribution. PDI values of all the formulations indicate that particle size distribution was unimodal. The optimized F4 batch is a having a PDI of 0.34 are shown in fig 2. The results of all the formulations are tabulated in table 2. Based on the obtained results the F4 batch shows a least vesicle size, with a good zeta potential and PDI indicating the uniform distribution of the vesicles throughout formulation leading to maximum stability.

3.3.3 Scanning electron microscopy (SEM):

The External morphological studies (SEM)revealed that maximum nanoparticles were nearly spherical and smooth surface (fig 4). The nanoparticle size observed by SEM correlated well with the particle size measured by zeta sizer (Malvern instrument).

3.3.4 Drug loading efficiency:

The loading of curcumin into ethosomes was determined by the analysis of the supernatant for free drug using a UV-Visible spectrophotometer after centrifugation of the curcumin loaded ethosomes. Table 2 represents the drug loading efficiency for all formulated batches. From the results we observed that the concentration of ethanol and lecithin is the determinant factors in maintaining the stability of the ethosomes. Ethanol possess the net negative charge which maintains the zeta potential of the system whereas, lecithin retains the morphology and rigidity of ethosomes. As observed F4 batch prepared with 10 % of soya lecithin, 4.5 % of ethanol and 10% of cholesterol showed maximum entrapment of 71.2±3.12.

Fig.4: SEM image of formulation F4

Table 2: Vesicle size, zeta potential, PDI and Drug loading efficiency of various batches

Formula-tions	Vesicle size (nm)	Zeta potential (-mV)	Polydispersity Index (PDI)	Drug loading efficiency (%)
F1	786.1	-12.3	0.21	70.3±2.58
F2	515.1	-9.2	0.23	76.9±3.20
F3	370.3	-9.9	0.13	82.8±2.96
F4	197.7	-11.9	0.34	71.2±3.12
F5	228.4	-10.3	0.39	75.8±2.0
F6	372.1	-12.9	0.11	79.5±2.12
F7	350.9	-12.1	0.27	73.3±2.49
F8	402.1	-16.2	0.31	68.8±3.14

3.4 Evaluation of the ethosomal gel:

Curcumin loaded ethosomes (F4) was incorporated in carbopol gel. Physical examination was performed for colour, smoothness/grittiness, oiliness/greasiness and ease of application. The results of physical examination are shown that the gel was yellowish in colour with a smooth feel, non-greasy and free from grittiness. The pH of the formulation was found to be in the range of 5.4 with a viscosity range of 5164 cps.

3.4.1 In vitro drug release studies:

The release profile was obtained by placing 100 mg of the gel on the cellophane membrane in the Franz diffusion cell. The results for release studies i.e. % drug release (receptor chamber), are shown in the table 3 and fig 5. It was observed that drug concentration steadily increased in the receptor chamber with increase in time, where the permeation profile generally followed Fick’s diffusion law. The % cumulative amount of drug release from the ethosomal gel at the end of 12^th hr after application was found to be 92.10±2.36, whereas inhouse paclitaxel gel showed on only 86.26±2.73.

Table 3: In vitro drug release studies for ethosomal gel and inhouse paclitaxel gel through cellophane membrane

S. No	Time (h)	Cumulative drug permeated (%) at pH 5.5
S. No	Time (h)	Ethosomal gel	Inhouse Paclitaxel gel
1	0	0	0
2	0.25	2.25±0.12	1.25±0.10
4	0.50	12.0±0.66	3.0166±1.01
5	0.75	18.0±0.96	6.64±1.10
6	1	21.0±1.02	8.37±0.98
7	2	26.0±1.95	12.52±1.45
8	4	34±1.89	18.0±1.64
9	6	46.21±2.10	20.0±1.99
10	8	61.99±2.03	21.07±2.63
11	10	75.96±1.99	29.49±2.52
12	12	82.0±2.36	40.26±2.73

n=3*

3.4.2 Release kinetics for ethosomal and inhouse paclitaxel gel through cellophanemembrane at pH 5.5

The in vitro drug release data obtained from release studies were fitted into various kinetic models like zero order, first order, Higuchi, Hixson Crowell, Korsmeyer-Peppas to study the mechanism of drug release from the ethosomal and inhouse paclitaxel gel containing curcumin and paclitaxel. The values are presented in table 4, fig 6 and 7. The ethosomal gel was found to have higher r² value of 0.9896 for zero order kinetics which is close to 1.0 indicates the drug is released at a constant rate and independent of time which is the goal of all controlled-release drug-delivery mechanism. Whereas inhouse paclitaxel gel showed an r² value of 0.9882 for peppas which is close to 1.0 indicates the mechanism of drug release i.e, release of drug from the formulation is by diffusion, erosion, swelling and may by the combination of diffusion and swelling.

Fig 5: Invitro drug permeation profile of ethosomal gel and inhouse paclitaxel gel through cellophane membrane at pH 5.5

Fig 6: Zero order kinetics of ethosomal gel at pH 5.5 through cellophane membrane

Fig 7: Higuchi kinetics of ethosomal gel at pH 5.5 through cellophane membrane

Fig 8: Peppas kinetics of ethosomal gel at pH 5.5 through cellophane membrane

Fig 9: First order kinetics of ethosomal gel at pH 5.5 through cellophane membrane

Fig 10: Hixson kinetics of ethosomal gel at pH 5.5 through cellophane membrane

Fig 11: Zero order kinetics of inhouse paclitaxel gel at pH 5.5 through cellophane membrane

Fig 12: Higuchi kinetics of inhouse paclitaxel gel at pH 5.5 through cellophane membrane

Fig 13: Peppas kinetics of inhouse paclitaxel gel at pH 5.5 through cellophane membrane

Fig 14: First order kinetics of inhouse paclitaxel gel at pH 5.5 through cellophane membrane

Fig 15: Hixson kinetics of inhouse paclitaxel gel at pH 5.5 through cellophane membrane

Table 5: Ex vivo drug permeation studies of ethosomal and inhouse paclitaxel gel using rat skin

S. no	Time (h)	Cumulative drug permeated (%) at pH 5.5
S. no	Time (h)	Ethosomal gel	Inhouse Paclitaxel gel
1	0	0	0
2	0.25	0.39±0.01	0.10±0.1
4	0.50	0.59±0.01	0.36±0.16
5	0.75	1.55±0.03	1.52±0.5
6	1	6.42±0.01	4.63±0.4
7	2	10.25±0.02	9.12±1.1
8	4	21.02±0.02	12.32±1.45
9	6	28±0.02	16.0±1.98
10	8	38.5±2.63	19.24±2.1
11	10	46.1±1.02	20.10±1.56
12	12	64.24±1.73	23.21+1.89

3.4.3 Ex vivo Permeation studies:

The permeation profile was obtained by placing 100 mg of the ethosomal gel and inhouse paclitaxel gel on the Rat dorsal ear skin in the Franz diffusion cell. The results for permeation studies i.e. % drug permeated (receptor chamber), were listed in the table 5 and fig 16. It was observed that drug concentration steadily increased in the receptor chamber with increase in time, where the permeation profile generally followed Fick’s diffusion law. The % cumulative amount of drug permeated from ethosomal and inhouse paclitaxel gel at the end of 9^th hr after application was found to be 46.1±1.02 and 20.10±1.56. The comparatively higher permeability of ethosomal gel could be due to increased diffusion coefficient of drug. Moreover, smaller vesicle size of ethosomes provides larger area for permeation of drug in to skin and high drug concentration on the affected area results in a larger concentration gradient, which is a necessity for efficient dermal drug delivery.

3.4.4 Release kinetics for ethosomal and inhouse paclitaxel gel through rat skin at pH 5.5

The ex vivo drug release data obtained from release studies were fitted into various kinetic models like zero order, first order, Higuchi, Hixson Crowell, Korsmeyer-Peppas to study the mechanism of drug release from the ethosomal and inhouse paclitaxel gel containing curcumin and paclitaxel. The values are presented in table 6, fig 17 and 18. The ethosomal gel was found to have higher r² value of 0.9766 for zero order kinetics which is close to 1.0 indicates the drug is released at a constant rate and independent of time which is the goal of all controlled-release drug-delivery mechanism. Whereas inhouse paclitaxel gel showed an r² value of 0.9812 for peppas which is close to 1.0 indicates the mechanism of drug release i.e, release of drug from the formulation is by diffusion, erosion, swelling and may by the combination of diffusion and swelling.

Fig 16: Ex vivo drug permeation profile of ethosomal gel and inhouse paclitaxel gel through rat skin at pH 5.5

Fig 17: Zero order kinetics of ethosomal gel at pH 5.5 through rat skin

Fig 18: Higuchi kinetics of ethosomal gel at pH 5.5 through rat skin

Fig 19: Peppas kinetics of ethosomal gel at pH 5.5 through rat skin

Fig 20: First order kinetics of ethosomal gel at pH 5.5 through rat skin

Fig 21: Hixson kinetics of ethosomal gel at pH 5.5 through rat skin

Fig 22: Zero order kinetics of inhouse paclitaxel gel at pH 5.5 through rat skin

Fig 23: Higuchi kinetics of inhouse paclitaxel gel at pH 5.5 through rat skin

Fig 24: Peppas kinetics of inhouse paclitaxel gel at pH 5.5 through rat skin

Fig 25: First order kinetics of inhouse paclitaxel gel at pH 5.5 through rat skin

Fig 26: Hixson kinetics of inhouse paclitaxel gel at pH 5.5 through rat skin

4. CONCLUSION:

· Study on various formulation excipents revealed that all the excipients shown some variation which affected the vesicle size, zeta potential, PDI and entrapment efficiency. Batch prepared with 10 % of soya lecithin, 4.5 % of ethanol and 10% of cholesterol showed maximum entrapment efficiency of 81.2±3.12.

· In in vitro drug permeation studies using dialysis bag, the samples withdrawn in equal time intervals shown more drug release from the dialysis bag in comparison with that of inhouse paclitaxel gel which conform that the curcumin loaded ethosomal gel is a better formulation for the further studies.

· In ex vivo, permeation study using pork ear skin, curcumin loaded ethosomal gel showed the greater drug deposition on the skin > 60 % in 12 hrs for the better curability for the melanoma. whereas inhouse paclitaxel gel shown < 60 % of drug deposition in 12 h.

· The comparison of both ethosomal gel and inhouse paclitaxel gel reveals that ethosomal gel shown maximum release within 12 hrs in both in vitro and ex vivo studies, which can be taken for the further in vitro cell line and in vivo study.

To conclude, ethosomal vesicular drug delivery system was suitable for treatment of melanoma. Lipophilic drug curcumin was loaded into the vesicles and then incorporated in the ethosomal gel provided a control release of the curcumin. The ethosomal gel shown good deposition on the which allowing the curcumin to retain in the deeper layers of the skin for a longer time that helps in eradicating the melanoma cells completely without causing any pain on the skin.

However, future studies are required to prove the efficacy of the prepared ethosoamal gel both preclinical and clinical studies.

5. REFERENCES:

1. E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, Ethosomes — novel vesicular carriers for enhanced delivery: characterization and skin penetration properties Journal of Controlled Release 65 (2000) 403–418

2. Donatella Paolino, Giuseppe Lucania, Domenico Mardente, Franco Alhaique, Massimo Fresta, Ethosomes for skin delivery of ammonium glycyrrhizinate: In vitro percutaneous permeation through human skin and in vivo anti-inflammatory activity on human volunteers Journal of Controlled Release 106 (2005) 99–110

3. Cavalli, R., Marengo, E., Rodriguez, L., and Gasco, M. R. (1996). Effects of some experimental factors on the production process of solid lipid nanoparticles. European journal of pharmaceutics and biopharmaceutics, 42(2), 110-115.

4. Chen, Y. J., Jinetal. (2006). [Preparation of solid lipid nanoparticles loaded with Xionggui powder-supercritical carbon dioxide fluid extraction and their evaluation in vitro release]. Zhongguo Zhong yao za zhi= Zhongguozhongyaozazhi= China journal of Chinese Materia Medica, 31(5), 376-379.

5. Elldem, T., Speiser, P., and Hineal, A. (1991). Optimization of spray-dried and congealed lipid microparticles and cha-racterization of their surface morphology by scanning electron microscopy. Pharm Res, 8, 47-54.

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		RELEASE KINEITCS
		ZERO	HIGUCHI	PEPPAS	FIRST	Hixson Crowell
		1	2	3	4	5
		R(CvT)	R(CvRoot(T))	Log T vs Log C	TIME vs LOG % REMAINING	TIME Vs (Q1/3-Qt1/3)
Ethosomal gel	Slope	11.642	32.361	0.952	-0.009	0.279
	Correlation	0.9948	0.9656	0.9462	-0.9740	0.9860
	R 2	0.9896	0.9324	0.8953	0.9486	0.9722
Inhouse Paclitaxel Gel	Slope	5.094	14.102	1.010	-0.005	0.091
	Correlation	0.9829	0.9502	0.9941	-0.9731	0.9772
	R 2	0.9662	0.9028	0.9882	0.9469	0.9549

		RELEASE KINEITCS
		ZERO	HIGUCHI	PEPPAS	FIRST	Hixson Crowell
		1	2	3	4	5
		R(CvT)	R(CvRoot(T))	Log T vs Log C	TIME vs LOG % REMAINING	TIME Vs (Q1/3-Qt1/3)
Ethosomal gel	Slope	8.910	23.753	1.701	-0.006	0.176
	Correlation	0.9882	0.9200	0.9803	-0.9593	0.9718
	R 2	0.9766	0.8463	0.9609	0.9203	0.9443
Inhouse Paclitaxel gel	Slope	3.604	10.113	1.938	-0.005	0.060
	Correlation	0.9851	0.9653	0.9906	-0.9889	0.9877
	R 2	0.9705	0.9318	0.9812	0.9779	0.9756