INTRODUCTION:

Microsponges have the ability to acquire the flexibility to entrap a wide range of active ingredients that are mostly used for prolonged topical administration with extended release of drug as these are sponge like structure with highly cross linked, patented, spongy, polymeric microspheres which are used¹. These are prepared in such a way so that it can deliver active pharmaceutical ingredient efficiently at the minimal amount of dose and also to enhance stability, reducing side effects and modify drug release profiles².

These microsponges have the capacity and ability to entangle a wide range of active ingredients such as emollients, fragrances, essential oils, sunscreens and anti-infective etc which are used as topical carrier system². Further, difficulties occur with the drug which is of poor solubility in topical delivering during the formulation of conventional dosage form. One of the difficulties occur with it less bioavailability and defective absorption³.

Moreover, most of the conventional topical products has an ability to act on the superficial layers of the skin, which when applied releases their active components for fast absorption by producing a highly concentrated layers of actives⁴. Hence, a novel strategy delivery system is required which may prolong the existence of active agents either on the skin surface or within the epidermis, and by reducing transdermal penetration.

Microsponge delivery system(MDS) overall executes all these requirements and thereby giving an assurance of drug localization on the surface of the skin and within epidermis without entering into the systemic circulation to a large extent.

The aim of the proposed work involves formulation and evaluation of gel loaded with Luliconazole microsponge which is prepared by using different ratios of drug:polymer (Ethyl cellulose and Eudragit RS 100) by quasi-emulsion technique.

Hence, in this present work an attempt was to develop controlled release microsponges to minimize frequent dosing, increase the pharmacological effect and thus improve patient compliance by using synthetic polymer.

Luliconazole is used as the topical antifungal agent belonging to the class of organic compounds known as dichlorobenzene. It is hydrophobic in nature, has poor solubility and high permeability and it comes under BCS class II drugs with molecular weight 354.28g/mol. It acts by suppressing an enzyme lanosterol demethylase which results in decreased amounts of ergosterol. It is a topical dosage form available in the form of cream. It is used for the topical treatment of athlete’s foot, jock itch and ringworm caused by dermatophytes such as Trichophyton rubrum, Microsporum gypseum and the Epidermophyton floccosum⁵. LCZ is selected to prepare the gel formulation using microsponges because Microsponge Drug Delivery System (MDS) is the unique versatile technique and has the ability to overcome the drawbacks of topical formulation such as-

· Lack of patient compliance is a result of applying ointments, which are often aesthetically unappealing, greasiness, stickiness etc.

· These vehicles require high concentration of active agents for effective therapy because of their low efficacy of delivery system, resulting into irritation and allergic reactions in significant users.

· Uncontrolled evaporation of active ingredients, unpleasant odour and potential incompatibility of drugs with the vehicles^4,6.

MATERIAL AND METHOD:

Material:

Luliconazole was a gift sample from Virupaksha Organics Ltd., Telangana, India. Ethyl cellulose and Eudragit RS 100 was purchased from SD. Fine Chem ltd., PEG 400 and Carbopol 934 was purchased from Quali Chem, New Delhi 11060, DCM was purchased from SD. Fine Chem Ltd. Mumbai 400025.

Method:

Preparation of Microsponges

The luliconazole microsponges were prepared by 2 step process known as quasi-emulsion solvent diffusion method. In this, Ethyl cellulose and Eudragit RS 100 polymers along with different drug : polymer ratio are used (as shown in table 1).

The two step process i.e., internal phase includes polymer which is dissolved in solvent i.e., dichloromethane and then the drug is added to the polymer solution followed by PEG 400 under ultrasonication at 35⁰C. The inner phase is then poured dropwise in the external phase which is prepared by using PVA dissolved in distilled water and then was stirred continuously for 2 hours until the solvent is evaporated and the microsponge are formed. The prepared microsponge were separated by filteration, washed off and dried in hot air oven at 40⁰C for 24 hours. Then, the microsponges are weighed to determine production yield⁸.

Characterization and Evaluation of LCZ Microsponges:

1. Production yield and loading efficiency:

The production yield of the microsponges of various batches was determined by calculating accurately the initial weight of the raw materials and the final weight of the microsponge obtained⁹.

Practical mass of microsonges

Production yield= –––––––––––––––––––––––––––––– × 100

Theoritical mass (polymer+drug)

The loading efficiency (%) of the microsponges can be calculated by using the following equation.

Actual drug content in microsponge

Loading efficiency = ––––––––––––––––––––––––––––––––– × 100

Theoritical drug content

2. Drug Content:

All the batches of microsponge formulation was weighed accurately and transfer into 100ml of volumetric flask containing PBS of pH 7.4 and mix it well. Then, the solution was filtered through whatmann filter paper. Drug content was determined by UV spectrophotometer at 295nm^9,10.

Actual drug content in microsponges

Drug content (%) = ––––––––––––––––––––––––––––––––– × 100

Weighed quantity of microsponges

3. In-vitro Drug Release from Microsponges:

Accurately weighed loaded microsponges (50 mg) were placed in 100ml of methanol in 100ml glass bottles. The later were horizontally shaken at 37°C at predetermined time intervals¹¹. Aliquot samples were withdrawn (replaced with fresh medium) and analysed UV spectrophotometrically at 295nm for Luliconazole. The contents of drugs were calculated at different time intervals up to 6 hrs.

Preparation of Microsponge entrapped Luliconazole gel:

Batch F1 (Microsponge using Ethyl cellulose) and F2 (Microsponge using Eudragit RS 100) was found better in its controlled drug release and is selected for the further topical gel formulation. Accurately weighed amount of carbopol 934 (0.5%w/w) in water was taken and soaked overnight. In another beaker, microsponge containing Luliconazole was dissolved in ethanol and then, added to the carbopol solution by stirring continuously followed by addition of PEG 400. Triethanolamine (2%v/v) was added slowly in a dropwise manner with constant stirring until the transparent gel was obtained. Further formed gel was stored in an air tight tubes or container for its proposed study¹². Calculated amounts of LCZ micropsonge formula was incorporated into the base, so that the final concentration of LCZ is 1% w/w in the final gel formulation.

Evaluation of Microsponge Loaded Gel:

1. Drug content:

The drug content was determined by dissolving the gel formulation in the 100ml of volumetric flask containing PBS of pH 7.4 then, sonicated and filtered¹⁰. The solution is analysed in UV Spectrophotometer at 295nm of LCZ.

2. Spreadability Test:

Spreadability of gel formulation was determined by using horizontal plate method by placing 1gm of gel sample between two glass slides then compressed to uniform thickness by applying 125gm of weight on it for specific time (5min). And, the time noted for the gel as it Slide off from from one plate to other¹³

The spreadability was then calculated using the following formula:

S= M×L / T

(where, S = spreadability, M= weight placed, L= length moved by glassslide and T= time)

3. Physical appearance and pH determination:

The prepared microsponge gel was determined by using digital pH meter by immersing the electrodes in it and then its colour, homogeneity, consistency and pH was recorded^13,15.

4. Viscosity measurement:

The viscosity of gel formulation was analysed by using Brookfield Viscometer spindle (52) at 1.5 rpm at temperature 37±0.5°C. It reveals that with increase in polymer concentration the viscosity also increases¹⁵.

5. In-Vitro drug release by Franz diffusion cell:

The in-vitro study of gel was carried out by Franz diffusion cell, using an egg shell membrane which was extracted by using concentrated Hcl. The receptor compartment were filled with phosphate buffer pH 7.4 and 1gm of gel were smoothly spread over the egg membrane on donor compartment which was placed on a thermostatic magnetic stirrer and the temperature was maintained at 37˚C throughout the study. Selected batches FG1(using EC) and FG2(using ERS 100) were used for release study are carried out over a period of 12 hrs at regular interval shown in Table 9. Aliquots, each of 1ml volume were withdrawn at specific time interval and replaced with equal volume of fresh phosphate buffer. The samples withdrawn are assayed spectrophotometrically at wavelength of 295nm.

RESULT AND DISCUSSION:

Characterization of Pure Drug:

Physical Characterization of drug:

Luliconazole (LCZ) was found to be solid, off white to pale yellow crystalline powder with good flow behaviour which complies with the description given in the literature.

Melting Point Determination:

The melting point of Luliconazole (LCZ) was in the range of 150^oC (literature studied 149-154 ^oC). As the procured drug was supposed to be good and pure.

Ultraviolet Visible Spectroscopy:

Caliberation curve of LCZ was plotted using Phosphate Buffer Solution (PBS) of pH 7.4 by keeping concentration range of 2-20 μg/ml (shown in Table 3). The drug was analysed spectrophotometrically in the range of 400-200 nm. The λ max of the LCZ was found 295nm which indicates the purity and confirmation of drug sample.

Calibration curve of Luliconazole:

Table 3: Calibration curve data for Luliconazole

Sr. No.	Concentration (μg/mL)	Absorbance at 295 nm*
1.	2	0.173
2.	4	0.208
3.	6	0.246
4.	8	0.282
5.	10	0.320
6.	12	0.345
7.	14	0.382
8.	16	0.405
9.	18	0.452
10.	20	0.498

Figure 1: Calibration curve of Luliconazole

Determination of Production yield, Encapsulation Efficiency and Drug content:

Production yield of Luliconazole microsponges using ethyl cellulose were found to be between 71.11% to 94.79% and Luliconazole microsponge using Eudragit RS 100 were between 69.82% to 91.45% (Table 4). From the production yield of LCZ microsponges it was found that F5 (using Ethyl cellulose) and F4 (using Eudragit RS 100) indicates higher production yield. As well as, the loading efficiency of microsponges using Eudragit RS 100 was found to be 41.28% to 73.06% and were found to be high in Ethyl cellulose microsponges i.e., 53.55% to 79.14%. It was found that with increase in drug:polymer ratio, drug loading efficiency also increases whereas, no significant effect of concentration of Eudragit RS 100 was found in drug loading efficiency and production yield as shown in Table 4.

Due to the presence of high polymer amount, the internal viscosity may also get increased, and the time required for the diffusion of dichloromethane takes long time for the droplet formation that which improves the production yield. Drug content of microsponges using Ethyl cellulose was found to be increase with increase in drug: polymer ratio with no such effect of Eudragit RS 100 on drug content i.e., firstly decrease and then increase. It may be due to solubilisation of drug in solvent or evaporation of actives during microsponge formulation.

In-vitro Diffusion Studies

The in-vitro release data for all microsponge formulation was conducted in Phosphate buffer Solution (PBS) of pH 7.4 is shown in table 5.

It was found that in Ethyl cellulose microsponges the drug release decreases within a range of 71.48% to 65.44% w.r.t increase in drug:polymer ratio (1:1 to 9:1) and in microsponges using Eudragit RS 100 first increase then decreased within the range of 77.11% to 66.81% (Table 5).

Table 4: Production yield, Encapsulation effiecieny (%) and drug content of Luliconazole microsponge using Ethyl cellulose and Eudragit RS 100

Formulation Code	Production Yield (%)		Encapsulation Efficiency(%)		Drug content(%)
Formulation Code	Ethyl Cellulose	Eudragit RS100	Ethyl Cellulose	Eudragit RS100	Ethyl Cellulose	Eudragit RS100
F1	71.11	81.17	53.55	57.12	51.7	55.1
F2	83.21	69.82	66.78	41.28	57.2	40.7
F3	86.48	72.45	71.23	48.44	61.5	45.2
F4	90.82	91.45	74.69	73.06	69.6	53.8
F5	94.79	86.51	79.14	69.76	73.3	64.4

Table 5: In-vitro release study of Luliconazole microsponges using Ethyl cellulose and Eudragit RS 100

Cumulative % drug release
Ethyl cellulose						Eudragit RS 100
Time (Min)	F1	F2	F3	F4	F5	F1	F2	F3	F4	F5
0	0	0	0	0	0	0	0	0	0	0
30	15.92	14.48	13.11	15.48	15.23	12.78	15.63	14.76	17.56	13.58
60	21.01	27.48	16.85	21.55	22.18	19.67	21.67	25.93	22.81	24.36
120	29.67	35.42	28.45	26.77	28.51	27.45	27.45	29.34	30.56	32.84
180	37.32	43.22	37.22	34.22	37.87	36.12	43.12	37.52	39.51	43.45
240	49.81	54.36	46.76	45.76	43.21	48.62	54.62	51.36	51.23	51.13
300	64.52	60.54	57.28	55.36	54.77	62.21	67.65	64.12	67.58	59.38
360	71.48	70.91	69.49	67.41	65.44	74.86	77.11	72.26	75.46	66.81

This could be due to thickening of the polymer matrix wall with high polymer amount which results in extended diffusion and ultimately lead to less drug release. Therefore, F1 Micropsonge (using Ethyl cellulose) and F2 Microsponge (using Eudragit RS 100) shows higher drug release of 71.48% and 77.11% respectively in 6hr and follows zero order kinetics. Both batches are further formulated as gel formulation as FG1 and FG2.

Evaluation of Microsponge Loaded Gel:

Visual Inspection And pH Determination:

The luliconazole microsponge gel using Ethyl cellulose (EC) and Eudragit RS 100 (ERS 100) i.e., FG1 and FG2 respectively, shows white, viscous preparation with smooth texture.

The pH value was found to be 6.8 for FG1 and 6.7 for FG2 formulation (as shown in table 6) which avoid risk of irritation upon application to the skin.

Spreadability and Viscosity Studies:

The value of spreadability of microsponges FG1 and FG2 was found to be 7.4 and and 5.0 g.cm/sec respectively, indicating the acceptable spreadability of gel (Table 6).

Microsponge formulation FG1 was found to be more viscous than the gel loaded with microsponge using Eudragit RS 100 i.e., as shown in Table 6.

Table 6: Determination of Drug Content, Viscosity, Spreadability and pH

Gel Formulation	Drug content	Viscosity (cps)	Spreadability (g.cm/sec)	pH
Gel containing Ethyl cellulose (FG1)	92.5	1380	7.4	6.8
Gel containing Eudragit RS 100 (FG2)	93.2	1296	5.0	6.7

In-vitro diffusion study for microsponge gel:

The in vitro diffusion was carried out for the formulation FG1 and FG2 using PBS (pH 7.4) over the period of 12 hr. It was observed that the formulation FG1 showed higher amount of drug diffused at the end of 12 hr as compared to FG2 i.e., 89.40% and 92.18% respectively (Table 7). Hence, the microsponge loaded gel formulation FG1 was optimized formulation which shows the drug release in a controlled form with all the acceptable properties.

Table 7: Cumulative % drug released from gels loaded with Ethyl cellulose(FG1) and Eudragit RS 100(FG2) in LCZ micrsponges

Time (min)	Cumulative % drug release
Time (min)	FG1	FG2
0	0	0
30	5.62	9.42
60	9.86	13.67
120	14.77	19.22
180	23.38	26.82
240	30.21	34.14
300	37.91	44.71
360	45.74	51.18
420	54.22	58.53
480	62.12	65.11
540	71.56	72.34
600	77.82	78.43
660	81.15	84.65
720	89.40	92.18

Figure 2: Drug release Vs Time plot of gels containing Ethyl cellulose(FG1)and Eudragit RS 100(FG2) entrapped in LCZ microsponge

Table 8: Kinetic study

Sr. No.	Kinetic model	FG1 (r²)	FG2 (r²)
1.	Zero order	0.9976	0.9958
2.	First order	0.9322	0.9186
3.	Higuchi matrix	0.9319	0.9255
4.	Peppas	0.9931	0.9859
5.	Hixon crowell	0.9725	0.9716
Best fit model		Zero order

CONCLUSION:

In the present work, formulation and evaluation of LCZ microsponges was prepared successfully for locally acting anti-fungal agent. Luliconazole is formulated as microsponges by Quasi-emulsion solvent diffusion method using five different drug : polymer ratios (1:1, 3:1, 5:1, 7:1, and 9:1) for Ethyl cellulose and Eudragit based MDS.

By increasing drug:polymer ratio, production yield and drug loading efficiency increases by using Ethyl cellulose as a polymer and no significant effect of concentration Eudragit RS 100 has been showed. So, it was found that Ethyl cellulose was selected to produce good microsponges. The in-vitro release was carried out for all the microsponge formulation prepared with different drug: polymer ratios. The formulation F1 containing 1:1 drug:polymer (Ethyl cellulose) release 71.48% and formulation F2 containing 1:3 drug:polymer (Eudragit RS 100) release 77.11% of drug at the end of 6 hours.

From the results, it can be concluded that LCZ microsponges using Ethyl cellulose could control the drug release more easily as compared to LCZ micropsonge using Eudragit RS 100 over a period of 6 hrs. Therefore, F1 and F2 were selected as optimized formulation and incorporated into gel formulation as FGI and FGII, respectively. Finally, the in-vitro release of gel containing LCZ microsponges using Ethyl cellulose (FG1) was found to be in controlled release than that of gel containing LCZ microsponges using Eudragit RS 100 (FG2). The data obtained by in-vitro release was best fitted in zero order kinetics.

Therefore, the present study suggests the potentiality of microsponges to enhance the therapeutic effect, maintain stability and reduces the side effects.

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Received on 29.07.2020 Modified on 19.11.2020

Research J. Pharm. and Tech 2021; 14(11):5775-5780.

DOI: 10.52711/0974-360X.2021.01004

Sr. No.	Characters	Specification	Result
1.	Description	Off white to pale yellow crystalline powder	Off white to pale yellow crystalline powder
2.	Melting point	149-154^oC	150°C
3.	Solubility	Freely soluble in ethanol (95%), DMSO, and DMF; slightly soluble in chloroform, ethyl acetate and poorly soluble in water	Freely soluble in ethanol (95%), DMSO, and DMF; slightly soluble in chloroform, ethyl acetate and poorly soluble in water

Formulation Code	Drug:excipient Ratio	Ethyl Cellulose and Eudragit RS 100(g)	Dichloromethane (ml)	Polyvinyl alcohol (g)	Water (ml)
F 1	1:1	0.50	10	0.8	50
F 2	3:1	0.16	10	0.8	50
F 3	5:1	0.10	10	0.8	50
F 4	7:1	0.07	10	0.8	50
F 5	9:1	0.05	10	0.8	50