INTRODUCTION:

The goal of any drug delivery system is to provide a therapeutic amount of drug to the target site in the body to promptly achieve and then maintain the desired drug concentration. For the reduction of cost, to increase the stability of product, to mask unpleasant tastes, and to improve the release properties of drug various drug delivery and drug targeting systems are currently under development¹.

A well designed drug delivery system can overcome some of the problems of conventional therapy and enhance the therapeutic efficacy of a given drug. An ideal drug delivery system (DDS) delivers drug at rate decided by the need of the body throughout the period of treatment and it provides the active entity solely to the site of action. There are various approaches in delivering a therapeutic substance to the target site, one such approach is using microspheres. Microsphers have been widely accepted to achieve controlled release². Microcapsules may also serve the role of protecting the encapsulated contents to prevent the degradation of the product³. They can be used as carriers for drugs^4,5.

Microspheres are defined as spherical microscopic particles that range from 1- 1000μm in diameter⁶. Microsphere is characteristically free flowing powders consisting of protein or synthetic polymer, which are biodegradable in nature. This is the important approach in delivering therapeutic substances to the target site in sustained and controlled release⁷. A wide range of core materials have been encapsulated in microspheres, including adhesives, agrochemicals, live cells, active enzymes, flavors, fragrances, pharmaceuticals, and ink. Microsphere consist of two components namely core material and coat or shell material. Core material defined as the specific material to be coated, can be liquid or solid in nature. The composition of the core material can be varied as the liquid core can include dispersed and/or dissolved material. The solids core can be mixture of active constituents, stabilizers, diluents, excipients and release rate retardants or accelerators⁸. Microencapsulation is the process of surrounding or enveloping one substance within another substance, yielding capsules ranging from less than one micron to several thousand microns in size⁹. Microencapsulation can be done to protect the sensitive substances from the external environment and to obtain controlled release of the drug substances¹⁰ There are various techniques available for microencapsulation of drugs including the emulsion-solvent evaporation/extraction methods¹¹ (single emulsion and double emulsion methods including solid-oil-water, water-oil-oil and solid-oil-oil methods), spray drying and spray freeze drying, ultrasonic atomization, electrospraying, microfluidic methods, pore closing method, thermoreversible gel method, microfabrication method and in situ polymerization. Each method has its own advantages and disadvantages. The choice of particular technique depends on attributes of the polymer and the drug, the site of drug action, and the duration of therapy. Bioadhesion is the state in which two materials, at least one biological in nature, are held together for an extended period of time by interfacial forces. The aim of this study was to prepare Ornidazole Microsphere in a Bioadhesive gel for Local Therapy of Vaginal Candidiasis. Vaginal Candidiasisis a fungal infection in the vulva or vagina. It can be caused by the presence of Candida in the vagina. The term ‘gels’ is broad, encompassing semisolids of a wide range of characteristics from fairly rigid gelatin slabs, to suspensions of colloidal clays, to certain greases. Gels can be looked on as being composed of two interpenetrating phases. The United States Pharmacopoeia defines gels as semisolids, being either suspensions of small inorganic particles or large organic molecules interpenetrated with liquid. The various methods of preparation of gel are Chemical reaction, Temperature effect, Flocculation with salts and non-solvents.

MATERIALS AND METHODS:

Materials:

All the chemicals and the materials used in the present work were of analytical grade. The active ingredient was bought from Endoc life care Pvt Ltd, Gujarat. The Eudragit RS 100 and Eudragit RL100 was brought from Evonik deggusa Pvt Ltd, Mumbai. Liquid paraffin was brought from Nice Chemicals Pvt Ltd, Kerala. The magnesium stearate, n – hexane, acetone was brought from Loba Chemie Pvt Ltd, Mumbai.

Methods:

Preformulation study:

Preformulation studies are the first step in the rational development of dosage forms of a drug. It can be defined as an investigation of physical and chemical properties of drug substance, alone and when combined with excipients. preformulation studies on the obtained sample of drug include physical test and compatibility studies.

Solubility analysis:

Preformulation solubility analysis was done to select a suitable solvent system to dissolve the drug and also to test its solubility in the dissolution medium which was to be used. The solubility of ODZ in 10mg/ml of different solvent was carried out.

Determination of λmax:

A solution of ODZ containing concentration of 10μg/ml was prepared in methanol and UV spectrum was taken using Shimadzu (UV-1800) double beam. Spectrophotometer and scanned between 200 to 400nm. The maxima obtained in the graph were considered as λmax for the drug ODZ.

Sample preparation and analysis by FTIR¹²:

IR Spectra of drug and excipients were taken using FTIR Spectrophotometer (Jasco 4100 type A). The drug and polymer were mixed physically in 1:1 ratio and the mixtures were stored in an oven at 400C and 75% relative humidity in closed containers for one month. FTIR Spectrum of samples was taken by using KBr pellet method. Pellets of sample and KBr (1:100) were prepared using hydraulic press and scanned over the wave number range 4000-600cm-1 at the ambient temperature.

Sample preparation and analysis by DSC¹²:

The samples were prepared by physical mixture of drug and excipients (1:1) using a clean dried glass mortar and pestle. Samples (5-10mg) were accurately weighed and hermetically sealed in aluminum pans. Thermograms were obtained using Shimadzu (DSC-60) instrument, heating at a constant rate of 10ºC/min, over a temperature range of 40 – 600ºC. To maintain on inert atmosphere nitrogen gas was purged at a rate of 10 ml/min.

Preparation of microsphere by Solvent Evaporation Method:

Ornidazole microspheres were prepared by solvent evaporation method using Eudragit RS-100 and Eudragit RL-100 polymers with different drug/polymer ratios. Weighed quantity of polymer was dissolved in 30ml of acetone with stirring. 1g of drug and 100mg of magnesium stearate as a dispersion agent were then dispersed in the polymer solution. The resultant milky white dispersion was poured into a vessel containing a mixture of 270ml of liquid paraffin and 30ml of n-hexane and stirred for 5h using a homogenizer fitted with a four blade “butterfly” propeller with a diameter of 50mm, stirring was continued for 3h at 1000rpm or until the acetone was completely evaporated. Following removal of the acetone, the resultant microspheres were harvested by vacuum filtration after which they were washed four times with 25ml of n-hexane and dried at room temperature for 24h.

Evaluation of microspheres:

Appearance:

The general appearance and elegance of microsphere was identified visually, which include size, shape, color, presence or absence of an odor, taste, surface texture.

Particle size:

Particle size analysis carried out by optical microscopic method. A minute quantity of microspheres were dispersed in glycerin and then spread on clean glass slide and average size of 100 microspheres was determined in each batch.

Percentage Yield:

Practical yield was calculated as the weight of ODZ microspheres recovered from each batch in relation to the sum of starting material.

The percentage yield of prepared ODZ microsphere was determined by using the formula

Actual weight of product

% yield = --------------------------------------------------- × 100

Total weight of excipients and drug

Determination of percentage drug entrapment efficiency (PDE):

Practical drug content

% PDE = ----------------------------------------- 100

Theoretical drug content

Scanning Electron Microscopy (SEM):

Scanning electron microscopy has been used to determine particle size distribution, surface topography, texture and to examine the morphology of the fractured of sectioned surface. SEM studies were carried out by using the JEOL JSM. 6380 LA (Japan). Dried ODZ microspheres were place on an electron microscope brass stub and coated with an ion sputter. Picture of ODZ microspheres were taken by random scanning of the stub.

Preparation of Gel:

Accurately weighed quantity of 1g Carbopol 934P was dispersed in 88g of distilled water by continuous stirring for 15-20 min with the help of glass rod in which 10g glycerol was previously added. Mixture was stirred until thickening occurred and neutralized by drop wise addition of 50% w/w triethanolamine. Mixing was continued until a transparent gel appeared.

Incorporation of Microspheres in a Gel:

Microsphere containing Ornidazole incorporated in to the 1% w/w carbopol 934P gel. Mixing well by using an electrical mixer at 25rpm for 2 min to get Ornidazole microsphere incorporated gel.

Evaluation of gel:

Microsphere incorporated gel of Ornidazole were evaluated for the following parameters:

Physical appearance:

Ornidazole incorporated gel were visually inspected for color, clarity, homogeneity, presence of particles and fibers.

Determination of PH¹³:

The pH of the Ornidazole microsphere incorporated carbopol gels were determined by digital pH meter. One gram of gel was dissolved in 25ml of water and the electrode was then dipped in to gel formulation for 30 min until constant reading obtained.

Drug content analysis:

Accurately weighed gel equivalent to 10mg of drug was suspended in 25ml of SVF and the volume was made up to 100ml after proper dilution absorbance was measured using UV-Visible spectrophotometer at 320nm.

Drug Content uniformity¹³

Initially the formulations were tested for homogeneity by visual inspection. To further ensure the homogeneity of drug content in the formulation of the gel, five tubes were sampled from the different locations in the mixer and assayed for the drug content.

Extrudability study:

Extrudability of the prepared gels was based upon the quantity of the percentage of gel extruded from the tube on application of certain tube on application of certain load. More the quantity extruded better was extrudability and the formulation under study was filled in a clean, lacquered aluminium collapsible one ounce tube with nasal tip 5mm opening. It was then placed in between two glass slides and was clamped and the extrudability was determined by weighing the amount of gels extruded through the tip when a constant load of 1kg was placed on the slides and gels extruded was collected and weighed. Percentage of gel extruded was calculated and grades were allotted (++good; + fair).

Spreadability¹³:

Spread ability of ODZ-MG incorporated gels were determined 48h after preparation, by measuring two 20X20cm glass plates after 1min. The mass of the upper plate was standardized at 125g. The spread ability was calculated by using the formula S = m.l/t, where S is spread ability, m is the weight tied to the upper slide, l is the length of the glass slide, and t is the time taken.

Viscosity measurement¹⁴:

A Brookfield (DV-II+) viscometer were used to determine the viscosity in cps of the microsphere incorporated gel. The gel was placed in the sample holder and the suitable spindle selected was lowered perpendicularly in to the sample. The spindle was attached to the viscometer and then it was allowed to rotate at a constant optimum speed at room temperature and readings of the viscosity of the formulation were measured after 2 minutes.

In vitro release studies of Gel¹⁴:

A modified open diffusion cell was used for drug release from the ODZ-MG. Egg membrane was used as the permeation barrier. The membrane was soaked overnight in SVF before the study. 1g of gel was kept carefully between the donor and receptor compartment. The donor compartment as empty and open to the atmosphere but the receptor compartment contained 25ml of pH 4.2 SVF as dissolution medium. The dissolution medium were maintained at 37± 50C and stirred on a magnetic stirrer with a stirring speed of 25rpm. 1ml of dissolution media was withdrawn at predetermined time interval (every 1 hour) and replaced with equal volumes of fresh medium. The absorbance of the samples was analyzed by UV- Spectrophotometer at 320nm.

In vitro Antifungal Activity¹⁴:

Antifungal activity of ODZ-MG and placebo gel was evaluated against Candida albicans j1012 by using cup plate method.

Cup Plate Method:

The composition of Sabouraud’s dextrose agar was taken in a 250ml of conical flask and was dissolved in 100ml of distilled water. The pH was adjusted to 5.6. The medium was sterilized in an autoclave at 15lbs for 15 minutes. After completion of sterilization, the medium was kept aside at room temperature. 0.5ml diluted suspension in Nacl 0.9% were added to 100ml of medium at 47±20℃ and used as inoculated layer. The medium (20ml) was poured in to a sterilized petridish to give a depth of 3-4mm, and was assured that the layer of medium is uniform in thickness by placing petridish on a leveled surface. Petridish was divided in to two sectors. After solidifying the medium at room temperature, with the help of a sterile cork borer, cups of 6mm diameter were punched and scooped out from the petridish. Each bore in different sector was loaded with equal quantity of the placebo gel (gel without the drug) and ODZ-MG. The petridish was then incubated for 24 hours at 370℃. After incubation the zone of inhibition was measured.

Stability studies:

Formulated F8 ODZ-MG incorporated gel was kept at a temperature of 400℃±20℃ and Relative Humidity (RH) 75%RH ± 5%RH for a period of six months. Initial, third month, six months the samples were evaluated for parameters such as color, pH, extrudability, spread ability, viscosity, drug content and in vitro drug Release were analyzed.

RESULTS:

Preformulation study:

Solubility:

The solubility of ODZ in 10mg/ml of solvent was carried out and it reveals that it is freely soluble in methanol, chloroform, and slightly soluble in water.

Determination of λmax:

The λmax for Ornidazole was found to be 320nm

FTIR Studies:

IR spectrum for pure drug and physical mixture of drug polymers were obtained and analyzed for principle peaks at 3199 cm-1 (OH stretching), 3094 cm-1 (CH stretching), 1531cm-1 (NO2 asymmetric stretching), 1364 cm-1 (NO2 symmetric stretching), 1147cm-1 (C-O stretching), 824 cm-1 (NO2 stretching)

Differential Scanning Calorimetry:

DSC curve obtained for pure Ornidazole, Eudragit RS100, Eudragit RL100, and their mixture are shown in the fig.13, 14, 15 and 16 respectively. Pure Ornidazole powder showed a melting endotherm at 93.430C. Physical mixture of Ornidazole and Carbapol 934P showed a melting endotherm at 94.07℃. Combination of Ornidazole and Eudragit RL/RS 100 showed melting endotherm at 93.2℃ and 93.23℃ respectively. These endotherm peaks showed that there is no interaction between the drug and polymer.

Physical characteristics of Microspheres:

Percentage yield and Entrapment efficiency:

Table-1: Percentage yield, Entrapment efficiency and Mean particle size of formulation F1-F8

Formulation code	*Percentage yield (%)**	Entrapment efficiency (%)	Mean particle size (μm)*
F1	83.07 ± 1.0	71.10 ± 0.6	176± 1.4
F2	80.20 ± 0.6	74.4 ± 0.67	287±1.1
F3	77.31 ± 0.71	78.13 ± 0.41	420±1.66
F4	91.03 ± 0.9	71.79 ± 0.45	180±.56
F5	82.93 ± 1.1	78.73 ± 0.71	315±0.59
F6	93.91 ± 0.3	81.13 ± 0.17	458±0.35
F7	82.06 ± 0.58	69.10 ± 0.5	293±0.8
F8	92.03 ± 1.1	80.10 ± 1.1	293±0.8

Actual Drug loading and Theoretical Drug loading of Microspheres

Table-2: Actual drug loading and Theoretical drug loading of formulation F1- F8

Formulation Code	Actual drug loading(%)	Theoretical Drug loading(%)
F1	35.15 ± 0.56	50.00
F2	25.07 ± 0.51	33.33
F3	19.53 ± 0.63	25.00
F4	35.25 ± 0.91	50.00
F5	26.5 ± 0.50	33.33
F6	20.26 ±0.82	25.00
F7	23.13 ± 0.35	33.33
F8	26.77 ±1.1	33.33

*Average of three readings

In vitro drug release studies:

Figure-1: in vitro drug release profile of formulations F7 and F8

DISCUSSION:

The purpose of this study was to design a novel vaginal delivery system composed of Ornidazole microspheres incorporated in a bioadhesive gel. Microspheres were prepared by solvent evaporation method using Eudragit RS-100 and Eudragit RL-100 polymers, with different drug/polymer ratios. Microspheres were subjected to SEM, DSC, FTIR and particle size analysis and evaluated for morphology, drug loading, entrapment efficiency and in vitro drug release. The percentage yield, actual drug loading and entrapment efficiency were found to range between 77.31±0.71% to 93.91± 0.3%, 20.26±0.82% to 35.25±0.91%, and 69.10±0.5% to 81.13±0.17% respectively. The FTIR and DSC spectra showed that there was no chemical interaction between drug and polymer used. SEM revealed that microspheres were spherical with nearly smooth surface morphology with a mean particle size ranging from 176±1.4 to 458± 0.35μm. The formulations F7 and F8 showed higher in vitro drug release rate compared to other formulations. The formulation F7 and F8 was incorporated in the bioadhesive gel made of Carbopol 934P. The formulation F8.ODZ-MG that showed 91.03% drug release at 8 h was then subjected to antifungal activity and stability studies. Stability studies were done as per ICH guidelines for a period of 6 months. Initial and third month studies were done and evaluated for parameters such as pH, drug content, drug content uniformity, extrudability, spreadability, viscosity and in vitro drug release. The results showed that there were no significant changes in the drug content and in vitro drug release. The stability studies will be continued further according to ICH guidelines. It may conclude from the present study that ODZ-MG can use as a novel delivery system for local therapy of vaginal candidiasis.

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Received on 09.03.2021 Modified on 15.10.2021

Research J. Pharm. and Tech. 2022; 15(8):3396-3400.

DOI: 10.52711/0974-360X.2022.00568