Formulation and Evaluation of Sustain Release Microsphere of Tretinoin for the Treatment of Acne vulgarise

 

Pooja Shivane, Sridevi G.*, Gopkumar P. and Sujit Pillai

G.R.Y. Institute of Pharmacy, Borawan, Khargone -451228

*Corresponding Author E-mail: srinitk@gmail.com

 

Tretinoin (TRT) is a metabolite of vitamin A, which is indicated primarily in the topical treatment of comedonal and papulopustular Acne vulgaris. TRT suffers several disadvantages which can strongly limit its utility. To overcome problems associated with topical delivery of TRA in the plain form, microsphere based sustained delivery formulation were studied in present research.

 

Topical microspheres of tretinoin were prepared using different methods (double emulsification and solvent evaporazation) and compared. Microspheres of tretinoin prepared in this study were nearly spherical. SEM pictures indicated spherical particles with smooth surface and free of surface irregularities. The particle size range of microspheres was between 20-150 μm, which is suitable enough for the preparation of topical gel formulations. The drug entrapment efficiency by both method of preparation was admirable. Nearly 90% of the drug content of microspheres was released in 12 hrs. In vitro drug release from microspheres indicated Zero and Higuchi kinetics of drug release pattern. Prepared microsphere based formulation helps in molecular tretinoin movement at the skin surface l rather than the crystalline form of tretinoin, which may cause irritation and other skin disorders. Side effects of tretinoin are reduced dramatically when it is applied as microbased formulation which also adds in faster absorprion of drug via skin surface. Microsperes also helps in protecting Tretinoin against photo-degradation

 

 

INTRODUCTION:

Tretinoin (TRT) is a widely used drug in the topical treatment of acne, photo-aged skin, psoriasis and other skin disorders. It also has been used to treat mottled hyper pigmentation, roughness, and fine wrinkling of photo damaged skin and to induce remission in acute promyelocytic leukemia. In addition, topical TRT has been tried in a wide range of skin conditions such as rosacea, keratinisation disorders, pigmentation disorders, and some neoplastic disorders. The effect of TRT in the treatment of acne is to reduce the number and the size of comedones and therefore, TRT is commonly used at various dosage forms, such as lotions, hydrogels or creams.^1-3. As these formulations are easily removed by wetting, movement, and contacting, the efficacy is unpredictable.³ Its use is limited by skin irritation, erythema, scaling, burning sensation and increased susceptibility to sunlight.^2,3.

 

Despite having these potentials advantageous its photo degradability can cause skin sensitization. Furthermore, topical TRT often causes transitory stinging, erythema, peeling, edema, dryness, or itching, which can result in poor patient compliance⁴. To overcome problems associated with topical delivery of TRT, microspere based formulations for topical delivery has been considered in the present study. The particulate delivery systems may be considered for topical delivery of medications because of their potentials of providing a controlled release rate and enhanced deposition of drug into the hair follicles, where drug is most needed ⁵.The use of microspheres has provided for a higher concentration of drugs in deeper layers of the skin and a reduction in Percutaneous absorption and unwanted side-effects.^6,7. Different investigators reported an increased skin accumulation of TRT in-vitro and a reduced irritancy in-vivo after treatment with these type of carriers. It was observed that the maximum comedolytic activity of TRT is reached at a concentration of five to ten times lower when TRT is incorporated into liposomes, compared to the conventional alcoholic gels⁸. On the other hand, it was found that the microspheres Tretinoin can protect the drug against photodegradation.⁹ In the current study we incorporated TRT into microspheres. Secondly, the characterization and release study of these formulations was evaluated.

 

Microspheres are small spherical particles, with diameters in the micrometer range (typically1 μm to 1000 μm). These sustained release microspheres may be produced by several methods utilizing emulsion system (oil-in-water, oil-in-oil, water-in-oil-in-water), as well as by spray drying¹⁰. In the present study Tretinoin microspheres were prepared by oil-in-water (o/w) emulsion system with microspheres being produced by the emulsion solvent evaporation method. This relatively simple method enables the entrapment of a wide range of hydrophobic drugs. And in the second method solvent evaporization the polymers are dissolved/dispersed in aqueous medium followed by dispersion in the non aqueous medium. Ex: oil. In the 2nd step, cross linking of the dispersed globule is carried out either by means of heat or by using chemical cross linkers. The chemical cross linking agents used –gluteraldehyde, formaldehyde, terephthalate chloride, diacidchloride etc. Cross linking by heat is effected by adding the dispersion to previously heated oil. Heat denaturation is not suitable for the thermolabile drugs while the chemical cross-linking suffers disadvantage of excessive exposure of active ingredient to chemicals if added at the time of preparation. Tretinoin microspheres prepared by both the method were examined and evaluated for their suitability to incorporate into derma gel formulations.

 

MATERIAL AND METHODS:

Tretinoin obtained as a gift sample from Shalaks Pharmaceutical Pvt. Ltd, New Delhi-110 015 (India) and Ethyl cellulose was kindly gifted from Lupin Pharmaceuticals, Indore (India). Acetone, liquid paraffin, n-Hexane was purchased from Loba Chemi Pvt. Ltd., Mumbai. All other chemical used were of analytical grade.

 

Preparation of Tretinoin Microspheres by Solvent Evaporization Method^{11, 12}:

Specific quantity of polymer (Ethyl Cellulose) was dissolved in Acetone to this weighed quantity of drug (TRT) was added and stirred well to make drug polymer dispersion [Solution no. 1]. Measured quantity (150 ml) of Light Liquid Paraffin was taken in a beaker and in this 1% of span 80 (1.5 ml.) was added and stirred for 5 min. [solution no.2]. Then mix solution 1 drop wise in solution 2 and stirred for 2 to 3 hours, 5ml of n-Hexane was added finally with continuous stirring for another 10min, then filtered and washed with 15ml. of pet ether. Collected microspheres were air dried.

 

Preparation of Tretinoin Microspheres by Double Emulsification Method:

Microspheres containing TRT were prepared using an double emulsification technique. Microsperes are prepared as per the formula stated in table number 2.  Preliminary studies were conducted to evaluate the influence of various experimental conditions such as temperature, stirring rate, state of saturation of external phase and organic to aqueous phase volume ratio on the formulation of microsphere particle size and production yield.

 

Table 1: Formulation of Tretinoin microspheres by solvent evaporization technique:

S.No	Formulation code	Drug (mg)	Polymer (Ethyl cellulose) mg	Stirring rate (rpm)
1	F1	50	100	500-1000
2	F2	50	150	500-1000
3	F3	50	200	500-1000
4	F4	50	100	500-1000
5	F5	50	150	500-1000
6	F6	50	200	500-1000

 

Weighed amounts of ethyl cellulose were dissolved in 50 ml mixture of acetone and ethyl acetate (2:3) to produce 2% or 4% w/v polymer solutions, as indicate in Table 1. TRT was dissolved in the polymer solutions so as to obtain various drug/polymer ratios of 0.15 or 0.3 (w/w). A solution of PVA in water (60 ml, 1% or 2% w/v) was prepared, saturated with ethyl acetate, and placed in an ice-bath. Drug/polymer solution was dripped into the PVA solution, while being stirred using a paddle mixture at 1000 rpm for 60 min. The emulsion obtained was removed from the ice bath and subjected to gradual addition of 340 ml 1% PVA solution while stirring the mixture at the same speed at room temperature for 1 h. The microspheres so formed were separated from the mixture by centrifugation at 10,000 rpm for 5 min. To further remove remaining organic solvent from the microspheres and harden their walls, 200 ml 0.1% PVA solution was added to the microspheres and stirring the mixture was carried out under the conditions previously described for another 2 h. The microspheres were separated by centrifugation, washed with water, dried at room temperature, and then kept in a dark vessel in a desiccator. The supernatants of all centrifugation steps were pooled and kept for TRT analysis.

 

 

Table 2: Table representing Tretinoin microsphere formulation by of Double emulsification method:

S.No	Formulation code	Drug (mg)	Acetone and ethyl cellulose  50 (ml)	PVA solution % (W/V)	Stirring rate(rpm)
1	F1	50	2:3	1% (340)m l0.1%(200)m	500-10,000
2	F2	50	2:3	1% (340)m l0.1%(200)m	700-10,000
3	F3	50	2:3	1% (340)m l0.1%(200)m	1000-10,000
4	F4	50	2:3	1% (340)m l0.1%(200)m	500-10,000
5	F5	50	2:3	1% (340)m l0.1%(200)m	700-10,000
6	F6	50	2:3	1% (340)m l0.1%(200)m	1000-10,000

Evaluation of Microspheres¹²:

Determination of drug content:

The 3 different batches of the microspheres were subjected for drug content analysis. Accurately weighed 100mg of microsphere samples were mechanically powdered. The powdered microspheres were dissolved in 100ml quantity of phosphate buffer PH 7.4 and Ethanol (8:2) ratio and kept for six hour for mixing then filtered using 0.45µm membrane. The UV absorbance of the filtrate was measured using a UV spectrophotometer at 344 nm. The result of analysis was reported in table number 3.

 

Drug loading and encapsulation efficiency:

Drug loading and encapsulation efficiency was determined for all batches using the following formulas. Values are expressed as percentage.

 

%Drug content = (amount of TRA loaded microsphere / weight of microsphere)x100   %EE = (amount of TRA loaded in the microsphere / initial amount of TRA) x 100

 

Particle Size Distribution studies:

The size distributions of prepared microspheres were determined by the optical microscopy method using a calibrated stage micrometer (µm). And the size distribution ranges of prepared microspheres were measured and number particles in each size distribution range were noted from the sample size of around 300 microspheres from each formulation. Graph of size distribution range against number of microspheres was plotted (figure 5).

 

Surface Morphology characterization using SEM:

The most widely used procedures to visualize micro particles are conventional light microscopy (LM) and scanning electron microscopy (SEM). Both can be used to determine the shape and outer structure of micro particles. LM provides a control over coating parameters in case of double walled microspheres. The microspheres structures can be visualized before and after coating and the change can be measured microscopically. SEM provides higher resolution in contrast to the LM. SEM allows investigations of the microspheres surfaces and after particles are cross-sectioned, it can also be used for the investigation of double walled systems. Conflocal fluorescence microscopy1 is used for the structure characterization of multiple walled microspheres. Laser light scattering and multi size coulter counter other than instrumental methods, which can be used for the characterization of size, shape and morphology of the microspheres. Shape and surface characteristics of the microsphere were investigated and photographed using scanning electron microscope.

 

In –Vitro Drug release from microsphere:

The release rate of Microsphere was determined using USP Dissolution Testing Apparatus 2 (paddle method). The dissolution test was performed using 900 ml of phosphate buffer of pH 7.4 and ethanol in  (8:2) ratio at 37 ± 0.5°C and 50 rpm. A 5 ml solution was withdrawn from the dissolution media at hourly interval and the samples were replaced with 5ml of fresh dissolution medium. The samples were filtered through a 0.45μ membrane filter and diluted to suitable concentration with dissolution medium.  Absorbance of these solutions was measured by UV/Visible spectrophotometer at 344nm.

 

In -vitro drug release Kinetics:

In order to study the exact mechanism of drug release profile from the prepared microspheres different kinetic studies were carried out. Kinetics of release was analyzed using different kinetic models like Zero-order kinetic model, First-order kinetic model, Higuchi’s model, korsmeyer equation. The regression analysis from the plots of Cumulative percent drug released versus time, Log Cumulative percent drug remaining versus time, Cumulative percent drug released versus square root of time, Log cumulative percent drug released versus log time  indicated zero order and Higuchi’s model as best fit kinetic release models.

 

RESULTS AND DISCUSSION:

The Tretinoin loaded microsphere were prepared by selected methods, Solvent evaporization and double emulsification method using Ethyl cellulose as a rate controlling polymer. The microspheres obtained under these methods were mostly spherical and without aggregation. Drug entrapment efficiency (DEE) of all formulation was found to be around 90%. The mean geometric particle size of microspheres prepared by (W/O) Emulsification solvent evaporization method and Double emulsification method was found in a range of 20-150µm 30-130 µm respectively as represented in graph(Fig 3).      

 

The surface morphology of the microspheres were studied using SEM indicated that prepared microspheres were almost spherical in shape with smooth rounded surface without any surface irregularities. Scanning electron microphotographs of microsphere prepared by solvent evaporization and double emulsification methods represented in (Fig 4).

 

In vitro drug release from Tretinoin loaded Microspheres was represented in (Fig 1) and (Fig 2). All the formulations found to release Tretinoin in a controlled manner over the time interval of 12 hours. To describe the kinetic of drug release from microspheres, release data was analysed according to different kinetics equations describe in (table 4).   

 

Drug Content:

Table No 3: Table indicating %Drug content and % of drug entrapment from different microsphere formulation:

S No.	Formulation Code%	% of Drug Content	% of Drug entrapment
1	F1	81±0.334	84±0.324
2	F2	89±0.351	91±0.385
3	F3	87±0.389	90±0.395
4	F4	85±0.412	88±0.435
5	F5	91±0.435	93±0.495
6	F6	84±0.456	87±0.498

 

In –Vitro Drug release from microsphere:

Cumulative drug release (Tretinoin) from the microsphere prepared by solvent evaporization method (F1, F2, F3, Formulation).

 

Figure1. Cumulative drug release from Tretinoin microsphere by (solvent evaporization) method.

 

Figure 2. Cumulative drug release of Tretinoin drug microsphere by Double emulsification method.

 

Particle size and shape:

Particle Size Analysis by Microscopic method:

The high shearing rate required for emulsification caused the breakdown of various drug polymer solution into fine globules resulting in small microsphere. The mean particle size of the microsphere significantly increased with increasing ethyl cellulose concentration and was in the range of 10-100 µm.

 

Surface Morphology studies by SEM:

The viscosity of the medium increases at a higher polymer concentration resulting in enhanced interfacial tension. Shearing efficiency is also diminished at higher viscosity. There resulting in the formation of large particles. Scanning Electron Microscopy showed that formulation produced spherical microspheres as shown in figure 5.

 

Figure 3: Particle size of microsphere:

 

Figure 4: Morphology of Tretinoin microsphere by solvent evaporization method and Double emulsification method

 

Table No 4: In vitro drug release kinetics of drug release:

S.No.	Formulation code	Zero order Kinetic model	First order Kinetic model	Higuchi’s model	Korsmeyer equation
1	F1	0.9884	0.867	0.9884	0.7535
2	F2	0.8388	0.877	0.9706	0.7116
3	F3	0.9813	0.792	0.9813	0.7643
4	F4	0.9563	0.834	0.9563	0.7855
5	F5	0.9613	0.892	0.9613	0.7169
6	F6	0.9706	0.795	0.9706	0.7116

 

DISCUSSIONS:

Drug entrapment efficiency (DEE) was found to be more than 80% of all formulations. Microspheres prepared using formulation F5 and formulation F2 were found to have maximum yield of more than 90%. The particle size of all the formulation was found to be satisfactory. The surface morphology of the microsphere as indicated by SEM pictures was having narrow size range. This narrow range of particles size can be attributed to the effect of stirring time, stirring speed and rate of solvent vaporization during preparation of microsphere. All batch of Tretinoin microsphere show sustain drug release drug delivery system for 12 hours but formulation F2 and F5 were showed more controlled effect on release over the extended period of time in comparison to all other formulation. Zero order and higuchi kinetics were more suitable release pattern found from the pattern of drug release.

 

ACKNOWLEDGMENTS:

The authors are thankful to Principal GRY institute of Pharmacy, Borawan for providing all facilities and his kind support to successful completion of Research project.

 

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Accepted on 06.07.2013         © RJPT All right reserved