FORMULATION AND EVALUATION OF GLICLAZIDE NANOSPONGES

Objective: The objective of the present study was to develop and characterize an optimal stable nanosponges of Gliclazide (GLZ) by using the emulsion solvent diffusion method and aimed to increase its bioavailability and release the drug in sustained and controlled manner. Methods: The GLZ nanosponge was prepared by emulsion solvent diffusion method using different drug-polymer ratios (1:1 to 1:5) Eudragit S100 is used as a polymer. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) estimated the compatibility of GLZ with polymer. All formulations evaluated for production yield, entrapment efficiency, in vitro drug release, scanning electron microscopy (SEM) and stability studies. Results: The DSC and FTIR Studies revealed that no interaction between drug and polymer. The Production yield of all batches in the range of 73.8±0.30 to 85.6±0.32. Batch F3 showed the highest production yield, the entrapment efficiency of batch F3 70.6±0.77. The average particle size ranges from 303±2.36 to 680±2.50 nm. By the end of 10th Conclusion: The results of various evaluation parameters, revealed that GLZ nanosponges would be possible alternative delivery systems to conventional formulation to improve its bioavailability, the emulsion solvent diffusion method is best method for preparation of nanosponges and release the drug in sustained and controlled manner. hour F3 formulation shown highest drug release was found to be 94.40±1.12%. The release kinetics of the optimized formulation shows zero-order drug release. The stability study indicates no significant change in the in vitro dissolution profile of optimized formulation.


INTRODUCTION
Nanosponge is a modern category of material and it is made up of tiny particles with a narrow cavity of few nanometers. These narrow cavities can be filled with various types of substances these tiny particles are having capability due to which it is able to carry both hydrophilic and lipophilic drug substances [1]. Release the drug at specific target site instead of circulate through the body it will more effective for particular given dosage [2,3]. The invention of nanosponges has become significant step towards overcoming the complexity associated with the newly developing systems. The small size and porous nature of nanosponges can bind poorly watersoluble drugs within the matrix and improve their solubility and bioavailability [4]. Nanosponges are able to entrap both hydrophilic as well as lipophilic drug molecules because of their inner hydrophobic cavities and external hydrophilic branching, thereby offering unparalleled flexibility. Nanosponge obtained by using suitable cross-linking agent also by different organic and inorganic materials [5]. The stability of these formulations over a wide range of pH in GI fluids and stable over 130 °C compatible with most vehicles and ingredients. Reducing dosing frequency and increase patient compliance and comfort. This drug delivery system having entrap wide variety of ingredients and reducing side effects, improve stability, increased elegance and formulation flexibility [6]. The effort to improve dissolution and solubility of poorly and practically water insoluble drugs remains one of the most challenging tasks in drug development. Several methods have been introduced to increase dissolution rate and thereby oral absorption and bioavailability of such drugs. Among various approaches, Nanosponges has shown promising results in improving solubility, wettability, dissolution rate of drug and subsequently its bioavailability [7]. The nanosponges can overcome some of the shortcomings of the conventional dosage forms. GLZ is one of the second-generation sulphonylurea, antidiabetic drug which stimulates insulin release. It is used for the treatment of non-insulindependent diabetes mellitus. GLZ is classified under class II according to biopharmaceutical classification system (BCS). The drug shows low, pH dependent solubility. In acidic and neutral aqueous media, GLZ exhibits very poor solubility. This poor solubility may cause poor dissolution and unpredicted bioavailability. Preparation of nanosponges of GLZ to improve the dissolution rate of GLZ and subsequently its bioavailability. The main objective of the study was to increase the amount of dissolved drug molecules at the absorption site by increasing the dissolution rate, since for class II drugs like GLZ, in vivo dissolution rate is ratelimiting step in drug absorption. nanosponges were selected as the method of choice since it would be easier in subsequent formulating. The nanosponges were prepared at various drug-to carrier weight ratios by emulsion solvent diffusion method.

Methods
GLZ nanosponges were prepared by emulsion solvent diffusion method. In this method, two phases used are aqueous and organic. Aqueous phase consists of polyvinyl alcohol and organic phase include GLZ and Eudragit S100. After dissolving GLZ and Eudragit S100 to suitable organic solvent. This phase added slowly to the aqueous phase and stirred for two or more hours and then nanosponges are collected by filtration, washed, and then dried in air at room temperature or in vacuum oven 40 °C for 24 h [8].

Drug-excipient compatibility studies
The drug and excipient compatibility studies were carried out by Fourier transform-infrared spectroscopy (FT-IR). The Potassium Bromide pellets were prepared on KBr press on grounding the solid powder sample with 100 times the quantity of KBr in mortar. The spectra recorded over the wavenumber of 4000 to 400 cm -1

Evaluation studies of prepared nanosponges
. From the results obtained by solubility and dissolution studies, nanosponges which showed better result was selected. For further characterization, nanosponges were performed to access interaction if any between the drug and polymer and also to find out what properties of polymer make them an effective material for solubility and bioavailability enhancement. In present study, the nanosponges of GLZ with Eudragit S100 were characterized by FT-IR, DSC, SEM, in vitro drug release study, production yield, entrapment efficiency etc.

Production yield
The production yield (PY) can be determined by calculating the initial weight of raw materials and final weight of nanosponges.
Production yield = Practical mass of nanosponges Theoretical mass(polymer + drug) × 100 The percentage yield of different batches was determined by weighing the nanosponges after drying [9].

Drug entrapment efficiency
To calculate the entrapment efficiency accurately weighed the quantity of nanosponges (10 mg) with 5 ml of methanol in a volumetric flask was shaken for 1 min using vortex mixer. The volume was made up to 10 ml. Then the solution was filtered and diluted and the concentration of GLZ was determined spectrophotometrically at 229 nm [9].
Loading efficiency = Actual drug content in nanosponge Theoretical drug content × 100

Determination of drug content
To study the amount of drug incorporated in the nanosponges, GLZ was extracted from the nanosponges by dissolving them in 25 ml Methanol. The resulting solution was filtered through a 0.45-micron membrane filter. The GLZ content in the methanolic extracts was analyzed spectrophotometrically by using a UV-Visible spectrophotometer (UV Jasco V630) at a wavelength of 229 nm, against methanol as blank [10].
Where, Wa = Actual drug content and Wt = Theoretical drug content

Determination of particle size
The size of particles is maintained during polymerization for the formation of free-following powders having fine aesthetic attributes. Particle size analysis of loaded and unloaded nanosponges performed by laser light diffractometry or Malvern zeta sizer. The cumulative graph is maintained or plotted as particle size against time to study effect of particle size on drug release [11].

In vitro drug release studies
In vitro drug release studies were performed in triplicate using USP Paddle method at 50 rpm and 37±0.2˚C in 900 ml of phosphate buffer (pH 6.8) [12]. 100 mg of the formulated nanosponges is used for each experiment. Samples were taken at appropriate time intervals for 1 h interval for 10hr. The samples were measured spectrophotometrically at 229 nm. Fresh dissolution medium was replenished each time when sample is withdrawn to compensate the volume.

Fourier-transform infrared analysis
Fourier-transform infrared (FTIR) spectra of pure GLZ and pure polymer (Eudragit), and nanosponges of drug GLZ was taken to access interaction if any interaction between drug and polymer in mixtures. The scanned mixture by using an FTIR (Jasco-460 plus). The FTIR spectra of mixtures were compared with that of the pure drug and Polymer to assess any change in the principle peaks of spectra of pure drug and polymer [13].

Differential scanning calorimetry
Differential scanning calorimetric (DSC) studies of pure GLZ, pure polymer (Eudragit S100) and nanosponges of drug GLZ with Eudragit S100 was performed to assess what changes happen during the heat change and thermal behavior of the drug is also determine. The pure drug sample shows sharp endothermic peak at melting point of drug. The samples were kept on DSC reference pan and DSC curves were obtained by differential scanning calorimeter (DSC 60; Shimadzu) at a heating rate of 10 °C/min from 0 to 300 °C in nitrogen atmosphere [14].

Scanning electron microscopy studies
Scanning electron microscopy (JSM-5200, Tokyo Japan) was used to analyze particle size and surface topography was operated at 15kV acceleration voltage. A concentrated aqueous suspension was spread over a slab and dried under vacuum. The sample was shadowed in a cathodic evaporator with a gold layer 20 nm thick. Photographs were elaborated by an image processing program and individual NP diameters were measured to obtain mean particle size.
Nanosponges that showed the best results in the solubility and dissolution studies were subjected to scanning electron microscopy (SEM) studies to confirm the changes mounting made during the formation of nanosponges. Samples were prepared by powder onto a brass stub using graphite glue and coated with gold under vacuum before use. Images were recorded at the required magnification at an acceleration voltage of 10 KV using a scanning electron microscope [15].

Stability studies
Accelerated stability studies: -40 °C±2 °C/75%RH±5%RH. As per ICH guidelines, the samples for stability analysis must be exposed to an environment of 40C ±2˚C/75%RH±5 % RH for a period of 6 mo. As per the standard protocol, the samples must be analysed at 0, 1, 2, 3-and 6-months' time points. Accelerated stability studies were performed for the final optimized formulation. Samples were analysed at 1,2 mo' time points [16].

Preformulation studies
Calibration curve of GLZ

Production yield
The production yield (PY) can be determined by calculating the initial weight of raw materials and final weight of nanosponges.
The percentage yield of different batches was determined by weighing the nanosponges after drying. The percentage yields of different formulations were found to be in the range of 73.8±0.30%-85.6±0.32% as shown in below table 2.

Drug entrapment efficiency
The drug entrapment efficiency found in the range of 62.8±0.22 to 73.1±0.39. The increases concentration of polymer the drug entrapment efficiency also increases. Average of three values±SD n=3 Average of three values±SD n=3

Determination of drug content
The various batches of the nanosponges were subjected for drug content analysis. The powdered nanosponges (30 mg) were dissolved in adequate quantity (100 ml) of phosphate buffer PH 6.8 then filter. The UV absorbance of the filtrate was measured using a UV spectrophotometer at 229 nm.
The drug content of different formulation was found to be in the range of 77.21±0.17 to 90.62±0.25 as shown in below table 4. Average of three values±SD n=3

Particle size analysis
The average particle size was obtained in range of 303±2.36 nm to 680±2.50 nm. The change in the concentration of polymer results in variation of particle size of nanosponges.
The average particle size of formulation batch F3 showed minimum particle size i.e. 418±2.23 nm while formulation batch F5 showed maximum particle size i.e. 680±2.50 nm. An increase in the concentration of polymer leads to increase in the particle size of nanosponges.  Average of three values±SD n=3

Modeling of dissolution profile
In the present study, different release kinetic equations (zero order, first order, Higuchi equation and Korsmeyer-peppas equation) were applied to interpret the release rate of drug from nanosponges. The data of in vitro release were fitted to these models and equations to explain the release kinetics of GLZ from nanosponges [17].  The in vitro release profile of the formulations indicates that the rate of drug release was higher for formulations, among all the formulations F3 shows more amount of drug release with the medium amount of polymer ratio. The plot of time vs cumulative % drug release in the zero-order kinetic model the regression coefficient value shows linearity as shown in (fig 3). The slopes and regression coefficient values (R 2 ) of various mathematical models for optimized batch F3, 0.9927, 0.9711, 0.9475, 0.7425 for zero order, first order, Higuchi and Peppas model respectively. The good linearity observed with the zero-order and regression coefficient value is higher. Hence this formulation is best fitted into zero-order release model.

Stability studies
Optimized formulation was subjected to stability studies as per ICH guidelines. Various parameters such as drug content, and in vitro drug release were measured before and after 30, 60, days of stability. Results of stability studies are shown in below table 8.
Results of stability studies showed there is no significant change in the above mentioned parameter after elevated temperature and humidity conditions during stability studies. Thus, it can be proved from the stability studies that the prepared formulation is stable and not much affected by elevated humidity and temperature conditions Stability studies.

DISCUSSION
The calibration curve of GLZ by UV method showed the linearity in absorbance and coefficient of regression found to be 0.9994. The IR studies shows that there is no interaction found between drug and excipients. All the characteristic peaks of GLZ were present in the spectrum of drug ( fig. 7) and prepared GLZ nanosponges ( fig. 10), indicating compatibility in drug and polymer the spectrum confirmed that there is no significant change in the chemical integrity of the drug [18]. The DSC thermogram pure GLZ shows sharp endothermic peak at 174C , which is its melting point as it melts with decomposition [19]. Such a sharp endothermic peak indicates that GLZ used was in the pure crystalline state. The thermogram of prepared GLZ nanosponges 1:3 ( fig. 14) displayed the nanosponge structure is amorphous as no sharp melting peak is shown.  [20]. The average particle size of formulation batch F1 showed minimum particle size i.e. 303±2.36 nm while formulation batch F5 showed maximum particle size i.e. 680±2.50 nm. An increase in the concentration of polymer leads to increase in the particle size of nanosponges. The SEM of the prepared nanosponges revealed a porous nature of GLZ nanosponges ( fig. 15). Drug release data for the nanosponge formulation fitted into various release kinetic equations to find out order of drug release. The various release kinetic curves are shown in fig. 3-7. The correlation coefficient showed that the release profile followed the zero-order drug release (R 2

CONCLUSION
=0.9927) from these results it is apparent that the regression coefficient value closer to unity in case of zero order kinetic model. The data indicate more linearity when plotted by the zero-order kinetic model. Hence, it can be concluded that the major mechanism of drug release follows zero-order kinetics [21].
Nanosponge is the best novel drug delivery system. For improving the dissolution and bioavailability profile of poorly water-soluble drug. BCS class-2 drug are more suitable drug candidate for formulated into nanosponges. Nanosponges are able to encapsulate variety of various types of drug molecules. Nanosponges prepared by using emulsion solvent diffusion method are simpler and production cost of method is less. Nanosponge prepared by emulsion solvent diffusion method shows good entrapment efficiency ranged between 62.8±0.22 to 73.1±0.39 and optimum drug release is 94.40%. The compatibility of drug and polymer determine by FT-IR, and DSC study. The result of IR and DSC shows drug and polymer are compatible to each other. The nanosponge formulation shows zero order drug release. The outcome of the study concluded that Eudragit S100 are employed as polymer for oral drug delivery system. Nanosponges of gliclazide increases solubility and dissolution rate of drug. The emulsion solvent diffusion method is best method for preparation of nanosponges.