INTRODUCTION:

Ocular diseases are mainly treated topically by application of drug solutions administered as eye drops for a localized action on the surface or in the interior of the eye¹. These conventional dosage forms account for 90% of the available ophthalmic formulations². This may be due to the simplicity and convenience of such dosage forms. A major problem in ocular therapeutics is the attainment of an optimal drug concentration at the site of action. Poor bioavailability of drugs from ocular dosage form is mainly due to the tear production, non-productive absorption, transient residence time, and impermeability of corneal epithelium. Only 1-3% of the applied drug in eye drops penetrates the cornea and reaches the intraocular tissues with the rest of the dose undergoing transconjunctival absorption or drainage via the nasolacrimal duct before transnasal absorption. This results in loss of drug into the systemic circulation and provides undesirable systemic side effects^3-4. It is important to minimize the systemic absorption and enhance ocular bioavailability of drug. This problem can be addressed by the use of suitable carrier systems.

Niosomal vesicular system is one of the potential approaches, which may be suitably used⁵. Niosomes are unilamellar or multilamellar vesicles capable of entrapping both hydrophilic and hydrophobic drugs. Niosomes help in providing prolonged and controlled action at the corneal surface and preventing the metabolism of the drug by enzymes present in the tear/ corneal surface. Drug enclosed in the vesicles allows for an improved partitioning and transport through the cornea. Moreover, vesicles offer a promising avenue to fulfill the need for an ophthalmic drug delivery system that has the convenience of a drop and will localize and maintain drug activity at its site of action⁶. From a technical point of view, niosomes are promising drug carriers as they possess greater stability. Other advantages are, simple method for the routine and large scale production of niosomes without the use of unacceptable solvents; they bear low toxicity because of their nonionic nature; unlike phospholipids, handling of surfactants requires no special precautions and conditions; they exhibit flexibility in their structural characterization, e.g. in their composition, fluidity and size; it may improve the performance of the drug via better availability and controlled delivery at a particular site; and they are biodegradable, biocompatible, and non-immunogenic⁷.

In the present study, it is aimed to prepare nonionic surfactant vesicles for ocular use containing diclofenac sodium with better release characteristics and longer duration of action. The diclofenac sodium was used in the present study due its safety and efficacy in the treatment of post operative inflammation in patents who have under gone cataract extraction, and temporary relief of pain and photophobia in patients under gone corneal refractive surgery^8-9.

MATERIAL AND METHODS:

Materials:

Diclofenac sodium was obtained from Novaratis Limited, India, as a gift sample. Span 60, cholesterol, chloroform, sodium acetate and sodium chloride were purchased from Loba chem. Pvt ltd, Mumbai. Methanol, ethyl acetate, potassium di-hydrogen phosphate and disodium hydrogen phosphate were purchased from S.D fine chem. Ltd, Mumbai, India. Sodium hydroxide from Nice chem. Ltd, Mumbai. Xylocaine from Astrazenica Ltd, India. Distilled water from Leo scientific, Erode,T.N, India and HPLC water was purchased from Qualigens, India.

Preparation of Diclofenac sodium niosomes

The lipid film hydration method was used to prepare niosomes^10-11. Surfactant: cholesterol in a molar ratios of 100: 30, 100:40, 100: 50, 100:60, 100: 70 and 100: 100 with diclofenac sodium were dissolved in chloroform/ methanol (2:1, v / v) in a 100 ml round bottom flask. A thin lipid film was formed under reduced pressure in a rotary flash evaporator at 60º C. After the removal of last trace of organic solvent from the round bottom flask, the film was then hydrated by 10 ml of phosphate buffer saline pH 7.4 at 60º C for one hour. The prepared niosomal suspensions was mechanically shaken for one hour using horizontal mechanical shaker at 60 rpm and 40ºC leading to the formation of multilamellar niosomes. The niosomal suspension was left to mature overnight at 4ºC.

Characterization of diclofenac sodium niosomes

Entrapment efficiency

Entrapment efficiency of diclofenac sodium niosomal suspension prepared by lipid film hydration method was determined by dialysis method¹². The prepared niosomes were filled into dialysis bags and the free diclofenac sodium dialyzed for 24 h into 100 ml of phosphate buffer saline pH 7.4. The absorbance of the dialysate was measured at 285 nm against phosphate buffer saline pH 7.4 and the absorbance of the corresponding blank. Niosome was measured under the same condition. Amount of entrapped drug was obtained by subtracting amount of unentrapped drug from the total drug incorporated.

Entrapped drug (mg)

Percentage entrapment = --------------------------------- X 100

Total drug added (mg)

In-vitro drug release study

In-vitro release pattern of niosome dispersion was carried out in dialysis bag method⁶. 2mg equivalent of 0.1% of niosome disperesion was taken in dialysis bag and the bag was placed in a beaker containing 100 ml simulated tear fluid, pH 7.4 phosphate buffer saline. The beaker was placed over magnetic stirrer and the temperature was maintained at 37±1º C. Aliquots of the dialysate were taken at predetermined time and replenished immediately with the same volume of fresh simulated fluid. The sink condition was maintained through out the experiment. The withdrawn samples were diluted suitably and analyzed for drug content using UV spectrophotometer at 285 nm keeping phosphate buffer pH 7.4 as blank.

Zeta potential analysis

The presence of surface charge in vesicular dispersion is critical. Aggregation is attributed to the shielding of the vesicle surface charge by ions in solution and there by reducing the electrostatic repulsion. Vesicle surface charge can be estimated by measurement of particle electrophoretic mobility and is expressed as the zeta potential. The study was conducted using zeta potential probe (model DT 300)¹³.

Rheological character

The viscosity of the prepared niosome suspension was studied using Brookfield viscometer LVDV-E. Accurately measured, 6.7 ml of the niosomal suspension was introduced into the SC4 series small sample chamber -13R. The spindle 18 at 100 RPM and shear rate 1.32 N (Sec^-1) were used¹⁴.

Physical stability

Physical stability study was carried out to investigate the leaching of drug from niosomal suspension during storage⁶. The surfactant, cholesterol in molar ratio of 100:60 was sealed in 20 ml glass vials and stored in refrigerated temperature (2-8⁰C) for a period of 60 days. Samples from each batch were withdrawn after the definite time intervals and the residual amount of drug in the vesicles was determined by dialysis method.

RESULTS AND DISCUSSION:

Six niosomal formulations of diclofenac sodium using span 60 and cholesterol were prepared with different molar ratios. The selection of surfactant, cholesterol and the ratios were based on the report of Yongmei Hao et al, 2000¹². The prepared niosomal suspensions were further characterized. The prepared nonionic surfactant vesicles were examined for vesicle formation under optical microscope and photographed at a magnification of 400x by means of fitted camera⁶. Fig. 1 showed that the niosomes were spherical in shape and the particles were slightly larger in size. This may be due to span 60 which has longer saturated alkyl chain and generally will produce larger vesicles¹⁵.

Entrapment efficiency was studied for all the formulations to find the best in terms of entrapment efficiency. The surfactant, cholesterol in molar ratio of 100:60 produced 79.67% due to the length of alkyl chain of surfactant which is crucial factor in permeability and influences the HLB value of the surfactant mixture which by its turn directly influences the drug entrapment efficiency. The lower the HLB of the surfactant the higher will be the drug entrapment efficiency and stability¹⁶. Moreover, span 60 has the highest phase transition temperature¹⁷. The larger vesicle size may also contribute to the higher entrapment efficiency¹⁵. The entrapment efficiency of surfactant: cholesterol in molar ratio of 100:30 and 100:40 were 48.79 % and 59.02 % respectively, Fig. 2. These results may be explained by the fact that an increase in cholesterol content resulted in an increase of microviscosity of the membrane indicating more rigidity of the bilayers¹⁵. Cholesterol has the ability to cement the leaking space in the bilayer membranes¹⁸.

Fig. 1 Photomicrograph of diclofenac sodium niosome (400x) in molar ratio of 100:60.

Fig. 2 Entrapment efficiency of diclofenac sodium niosomes.

However, the entrapment efficiency of surfactant: cholesterol in molar ratio of 100:50, 100: 70 and 100: 100 were 68.93 %, 61.80 % and 58.13 % respectively. The increase of cholesterol content 100:30 to 100:60 increases the entrapment efficiency but further increase of cholesterol reduces the entrapment efficiency. This could be due to the fact that cholesterol beyond a certain level starts disrupting the regular bilayered structure leading to loss of drug entrapment¹⁹.

When the release study was carried out with all formulations, most of the formulations were found to have a linear release and the formulations were found to provide approximately 60 % release within a period of 10 h. The formulation which has high cholesterol ratio (100:70) was found to sustain the drug release, Fig. 3. Cholesterol is known to abolish the gel to liquid phase transition of niosome systems which could be able to effectively prevent leakage of drug from niosomes. The slower release of drug from multi-lamellar vesicles may be attributed to the fact that multilamellar vesicles consist of several concentric sphere of bilayer separated by aqueous compartment²⁰. The formulations like 100:30, 100:40, 100:50 and 100:60 were found to give the release of 56.39 %, 63.93 %, 69.60 % and 79.34 % respectively over a period of 10 h, the higher release from the formulation 100:60 may be because of its moderate cholesterol content. Formulation 100:100 having the highest cholesterol content showed the lowest release over the period of 10 h provided a release of 63.27 %. From the above study, the surfactant, cholesterol in the molar ratio of 100:60 was selected for the further study. Linear regression analysis for the release data was done to determine the proper order of release²¹. Zero-order, first order and Higuchi diffusion controlled model equations were applied to all in-vitro release results⁶. The results showed the release of diffusion controlled mechanism.

The surfactant: cholesterol in the molar ratio of 100:60 was subjected for the zeta potential analysis which has practical application in the stability of systems containing dispersed particles since this potential governs the degree of repulsion between adjacent, similarly charged, dispersed particles. Zeta potential probe (DT- 300) was used for the study and the value was +27. If the zeta potential value is less the attractive forces exceed the repulsive forces, and the particles come together and will lead to flocculation/ aggregation¹³. But, this study concluded that there was no attractive force in niosomal suspension.

The niosomal suspension of surfactant: cholesterol in molar ratio of 100:60 was found to have an optimum viscosity of 1.26 cps to prolong the residence time in the eye, compared to solutions. The viscosity of ophthalmic products is most important parameter because It is generally agreed that an increase in vehicle viscosity increases the residence time in the eye, although there are conflicting reports in the literature to support the optimal viscosity for ocular bioavailability products formulated with a high viscosity are not well tolerated in the eye, causing lacrimation and blinking until the original viscosity of the tear is regained¹⁴. Drug diffusion out of the formulation into the eye may also be inhibited due to high product viscosity. Finally, administration of high viscosity liquid products tends to be more difficult. There fore the viscosity of 100:60 ratio with 1.26 cp will not produce lacrimation or blinking or blurred vision.

Physical stability was carried out to investigate the leaching out of the drug from niosomal suspension at refrigerated temperature. The efficiency of the span 60, cholesterol in molar ratio of 100: 60 after storage for the period of 60 days was 77.43%. When the samples were analyzed at specific time intervals, the percent retained were 94.42 at 15^th day, 90.75 at 30^th day and 86.84 at 45^th day. The system in this study showed that vesicles are stable enough to store under refrigeration temperature with least leakage. The leakage of drug from formulation may be due to its higher surfactant content and least cholesterol level which formed leaking vesicles⁶. Also the results indicate that approximately 85.0 % of diclofenac sodium was retained in niosomal formulations for a period of 45 days.

CONCLUSION:

Post operative inflammation in patents, temporary relief of pain and photophobia in patients under gone corneal refractive surgery requires a continuous and chronic administration of drug in eye. Conventional dosage forms of diclofenac sodium will not meet/satisfy above needs. Niosomal suspension formed with span 60: cholesterol in a molar ratio of 100:60 showed highest drug entrapment of 79.67% ± 1.53 and produced 79.34 % ± 1.04 drug release in-vitro over the period of 10 h. Zeta potential, +27, conformed the stability. Viscosity was 1.29 cp which showed suitable consistency for ocular preparation and did not produce blurred vision or drainage. By these facts, study can be concluded by saying niosomes formed with span 60: cholesterol in the molar ratio of 100:60 is a promising approach to improve the bioavailability of diclofenac sodium for an extended period of time.

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Received on 20.03.2009 Modified on 18.05.2009

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 710-713