DEVELOPMENT AND CHARACTERIZATION OF DICLOFENAC SODIUM LOADED EUDRAGIT RS100 POLYMERIC MICROSPONGE INCORPORATED INTO IN SITU GEL FOR OPHTHALMIC DRUG DELIVERY SYSTEM

Objective: Purpose of the study to design and formulate Diclofenac sodium (DIC) microsponges.
Methods: With varied polymer: drug ratio DIC loaded microsponges were prepared with Eudragit RS100 polymer by quasi solvent diffusion method. Microsponges evaluated for particle size, entrapment efficiency, drug content, in vitro drug release, Fourier Transform Infrared Spectroscopy (FTIR), Differential scanning calorimetry (DSC) and Scanning electron microscopy (SEM). DIC loaded microsponges incorporated into ocular in situ gel to attained controlled release by microsponge and improved residence time by gelling system. Ocular in situ gel evaluated for pH, drug content determination, gelling capacity, in vitro drug release and sterility study.
Results: DSER4 microsponge formulation having polymer to drug ratio 1:7 showed satisfactory production yield (68.13%), entrapment efficiency (62.86%), drug content (80.73%), requisite particle size (less than 10 µm) (7.52 µm) and in vitro release 87.94% after 6 h. Selected DSER4 formulation was incorporate into in situ gel. Carbopol 940 forms stiff gel at higher pH so used as a gelling agent, whereas Hydroxypropyl Methylcellulose E4M was used as a viscosity-enhancing agent for the formulation of in situ gel in varied compositions. In situ gel formulation IG4 showed sustained release of 76.92% till the end of 8 h and satisfactory gelling capacity so IG4 further evaluated for sterility test. Rheological studies reveal the sol-gel transition of in situ gel occur at the physiological condition to form stiff gel.
Conclusion: Prepared in situ gel formulations showed sustained drug release for a period of 8 h, which is satisfactory for management of ocular pain.

Ophthalmic drug delivery systems have the main problem of fast and substantial precorneal loss, caused by high turnover of tear fluid and further draining of tear fluid form eye [13]. Particulate system suffers from this nasolacrimal drainage. This possibility of short residence time In situ gels, upon installation in ocular cul-de-sac, go through a solgel phase transition to form a stiff gel. Phase transition occurs from droppable low viscous solution to stiff gel by the influence of physicochemical changes i.e. pH, temperature and ions composition, caused by tear fluid. This system allows administration of accurate doses in the form of drop and augmented residence time which in turn improves bioavailability at ocular area [16].
is eliminated by the incorporation of particulate system in situ gel and increases its retention in eye. A lot of attempts have been made on the incorporation of particulate system in ocular in situ gel [14,15]. But lesser studies are carried out on microsponge-loaded ocular in situ gel.
Carbopol is a polyacrylic acid (PAA) polymer, which go through a sol to gel conversion in an aqueous solution with a change in pH above its pKa of about 5.5. However, the high amount of carbopol required to get stiff gel results in highly acidic solutions. Such acidic polymeric solution is unable to neutralize with the buffering capacity of the tear fluid. So to good gelling capacity could be achieved with no further increment in the amount of carbopol in polymer solution; by adding HPMC polymer as viscosity enhancing polymer [17]. HPMC E4M was selected for in situ gel formulation as its frequent use in ophthalmic drug delivery, reliability, superior transparency and less incompatibility [18].
Diclofenac sodium is effectively used for pain management in cases of corneal epithelial defects followed by surgery or accidental trauma, and prevention of intraoperative miosis during cataract surgery, treatment of postoperative ocular inflammations, chronic non-infectious inflammations and for symptomatic relief of seasonal allergic conjunctivitis [19].
The purpose of this study is to attain controlled release through microsponge and improve its residence time by incorporating microsponges in in situ gel. Eudragit RS100 is used as a polymer for the preparation of microsponges. Further microsponges were added to HPMC E4M and carbopol in situ gel.

Preparation of DIC loaded microsponges
For the preparation of DIC-loaded microsponges with ERS polymer; the quasi-emulsion solvent diffusion method was considered. To prepare internal phase ERS polymer and triethylcitrate (1% w/v) dissolved in dichloromethane. To the prepared solution of polymer, DIC was added and homogenously dispersed by ultrasonication to achieve particle size reduction. Polymer: drug ratio was varied as 1:1 to 1:13. The internal phase was added dropwise to 100 ml of 1% w/v PVA solution. This emulsion was stirred for 4hr using magnetic stirrer at 1000 rpm. The DIC loaded microsponges were filtered, washed with double distilled water and then dried for 48 h at room temperature. DIC-loaded microsponges stored for further characterization [5,20].

Determination of Percentage Yield
The percentage yield of obtained microsponges was determined by calculating the percentage of the weight of the DIC loaded microsponges and the collective weight of DIC and ERS polymer. Optical microscopy was used to measure particle size of microsponge by using digital microscope (Motic CV5-2), calibrated with ocular micrometer (AmScope MR400 Microscope calibration slide). Selected microsponge formulation was dispersed in water and few drops were placed on a glass slide. The drop of dispersion was observed under a digital microscope. The mean of particle size for 300 particles was calculated [21].

In vitro drug release from DIC loaded microsponges
Franz diffusion cell was used to carry out in vitro drug release of DIC-loaded microsponges. Freshly prepared STF of pH 7.4 was filled in the receptor compartment of the Franz diffusion cell and with a small magnetic bar STF was constantly stirred at 100 rpm. A dialysis membrane (12 000-14 000 Dalton MW, Hi-media, India) soaked for 12 h in the STF and stretched in between donor and receptor chambers. DIC loaded microsponge dispersed in distilled water and placed in donor compartment; and covered with a coverslip. Samples each of 1 ml, were withdrawn for at predetermined time intervals from the receptor chambers and replaced with fresh STF and sink conditions. In vitro drug release was performed thrice for each formulation; further samples were analyzed by measuring the absorbance at 275.5 nm using UV spectrophotometer [22].

In vitro drug release kinetic study of DIC loaded microsponges
Drug Kinetic model studies of in vitro release data of microsponge formulations were performed to conclude the DIC release mechanism. In vitro release data was fitted to the following models and regression coefficient values (r 2 Where d is the quantity of the DIC remaining in the microsponge at time t and d ) 0 is the initial amount of the DIC in the microsponge. Diffusion mechanism was explained by the diffusion exponent (n) value of Korsmeyer-Peppas equation (d0-d/d0 = Kt n

Fourier transform infrared spectroscopy (FTIR)
). The diffusion exponent value n<0.45 signifies the release follows fickian mechanism. If exponent value indicates 0.5<n<0.8 signifies the release follows the non-fickian mechanism [23].
FTIR spectra of DIC, physical mixture of DIC and ERS polymer and; selected microsponge formulations were recorded in ATR mode with a Perkin Elmer (Model: SPECTRUM BX).

Differential scanning calorimetry (DSC)
Thermal analysis of DIC and the selected microsponge were scanned at a rate of 20 °C/min on a Differential scanning calorimetry (Mettler Toledo, Model: StareSW920, Mumbai) between 40 °C and 320 °C under dynamic nitrogen atmosphere DSC studies were carried out intend to find out any incompatibility.

Scanning electron microscopy (SEM)
A Field Emission Scanning Electron Microscope (FEI Nova NanoSEM 450) operated at 3.00 kV and magnification of ×500 and ×5000; was used to study the morphology of microsponges. Selected microsponge need to coat with gold-palladium alloy for 45s under an argon atmosphere before observation.

Formulation of DIC loaded microsponges containing ocular in situ gel
Ocular in situ gel formulations containing varied concentrations of Carbopol 940 and HPMC E4M were prepared as per the composition summarized in table 1. Accurately weighed HPMC E4M was dispersed in 50 ml of distilled water and carbopol 940 was dispersed over this solution and kept to hydrate 12h. The solution was stirred with an overhead stirrer. DIC-loaded microsponges (equivalent to 0.1 gm DIC) were dispersed in distilled water and added to the polymeric solution. Benzalkonium chloride was mixed to the prepared solution. Required amount of distilled water was added to make up the volume to 100 ml. Prepared in situ gel formulation thoroughly agitated and sterilized in an autoclave at 121 °C for 20 min [24,25].

Determination of visual appearance and clarity
The appearance and clarity ocular in situ gel formulations were evaluated by observing gel in front of black and white background in fluorescent light inside a bright, lightened cabinet.

Determination of pH
The pH of in situ gel formulation was determined using a pH meter (Labindia, Mumbai, Model: GMPH) which was calibrated before use with the buffered solution at pH 4 and 7. Three readings were taken for every sample and the average of three samples was calculated.

Drug content determination
In situ gel formulation was added to 100 ml volumetric flask. To this add 60 ml of pH 7.4 phosphate buffer was added and shaken for 4 h. and filtered. Filtered solution was further diluted to 100 ml with 7.4 phosphate buffer. Analyzed using UV spectrophotometer (UV1Jasco, Japan, Model: V-530) with phosphate buffer (pH 7.4) as blank. The drug content of the microsponges was calculated.

Determination of gelling capacity
To vial containing 2 ml of freshly prepared STF (maintained at 37 °C), drop of the in situ gel was added. The drop of in situ gel was observed and the time taken by drop for gelation was noted also the time taken for the gel formed to dissolve was noted [12]. Gelling capacity of formed in situ gel was noted as follows.

( ̶ ) No gel formation (+)
The gel formation occur after few minutes and dissolves quickly (++) Instantaneous gel formation and resides for few hours (+++) Instantaneous stiff gel formation which resides for long period of time

Rheological studies
Brookfield digital viscometer (DVII+PRO) was used to measure the viscosity (in cps) of the prepared in situ gel formulations. The spindle (T-D) was rotated at 20 rpm. Three readings were reported 30 sec after putting the motor on and an average calculated.

In vitro drug release from In situ ocular gel containing DIC loaded microsponges
The in vitro drug release from the in situ ocular gel was carried out as per procedure described above under 'In vitro drug release from DIC loaded microsponges'. The in situ ocular gel was placed on donor compartment. To the top of the in situ gel 250 μl of the STF was added to simulate the small amount of tear secreted on the eye surface and covered with a coverslip. 1 ml of samples were taken out at time intervals 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h with timely maintenance of sink condition. Samples were analyzed by using UV spectrophotometer [22]. In vitro drug release studies performed thrice for each in situ gel formulation.

In vitro drug release kinetic study of in situ ocular gel containing DIC loaded microsponges
Kinetic study of drug release data was performed ocular in situ gel as described above under 'In vitro drug release kinetic study of DIC loaded microsponges'.

Sterility study
The sterility study was performed according to Indian Pharmacopoeia [26]. In situ ocular gel formulation was directly inoculated aseptically into fluid thioglycolate medium (20 ml), and incubated for 14 d at 30 °C to 35 °C. Separately, In situ ocular gel formulation was directly inoculated aseptically into soyabean-casein digest medium (20 ml) and incubated for 14 d 20 °C to 25 °C. Microbial contamination was tested with a Fluid thioglycollate medium and the presence of fungi tested with soybean-casein digest medium with incubation under aerobic conditions. The tests were performed in triplicate under aseptic conditions to avoid accidental contamination. The positive and negative controls were performed for the examination of growth promotion and sterility, respectively.

Production yield (%), drug content (%) and entrapment efficiency (%)
With increase in polymer: drug ratio there is increment in production yield, drug content and entrapment efficiency. Production yield varied between 27.50% and 72.50%. At low DIC concentration production yield was found extremely low (27.50%). Drug content varied from 27.27% to 90.15% (table 2).

Particle size analysis
Particle size is an important parameter in ocular administration of particular system. Particle size range from 20.39±4.21 µm and 0.78±0.11 µm. Particle size decreased as an increase in Polymer: drug ratio. This finding is in accordance with literature [2].

In vitro drug release of DIC loaded microsponges
In vitro drug release is graphically presented in fig. 1. Formulation DERS4 (87.94%) and DERS5 (90.73%) showed relatively comparable drug release as compared to DERS3 (79.41%) at the end of 6 h. Study revealed that the highest cumulative drug release after 6 h is 90.73%.

Kinetic analysis of the in vitro release data of DIC loaded microsponge formulations
The release of DIC in both DIC-loaded microsponges formulations DERS3 and DERS4 showed more adherence to Higuchi's diffusion kinetic model, with regression coefficient (r 2 )value 0.9730 and 0.9655, respectively (table 3).   Microsponge formulation DERS4 was selected as having requisite particle size, sustained release and satisfactory Production yield, Entrapment efficiency and Drug content. DERS4 was further studied for FTIR, DSC and SEM.
) value 0.9883. The diffusion exponents (n) of all microsponge formulations have a value more than 0.5 but less than 0.8. This indicates that the DIC release mechanism from microsponges is non-Fickian diffusion.

Fourier transform infrared spectroscopy (FTIR)
Drug-polymer compatibility in the selected microsponges DERS4 was evaluated by FTIR. The principle IR peaks of DIC appeared at wavenumbers of 3361.34 cm -1 (NH starching of secondary amine groups), 1302.12 cm -1 (C-N stretching), 1503.46 cm -1 (C=C ring stretching) and 1548.76 cm -1 (C=O stretching of the carboxyl ion) and 757.12 cm -1 (C-Cl stretching) [27]. The characteristic DIC peaks were present in the physical mixture as well as in the DIC-loaded microsponges DERS4 ( fig. 2) with no significant changes in frequencies.

Differential scanning calorimetry (DSC)
The thermal behavior of the DIC, and selected microsponge DERS4 are presented in (fig. 3).
DSC thermogram of DIC exhibited endothermic peaks, initial peak due to loss of water followed by the other sharp endothermic peak at 291.97 °C, indicating to melting point of DIC in crystalline form; reflecting DIC purity [9]. The DSC curves of DERS4 formulation exhibited suppressed peak of the DIC at 280.40 °C.

Scanning electron microscopy (SEM)
Morphology DIC loaded microsponges were studied by SEM analysis. The representative SEM images of microsponge (DERS4) are shown in ( fig. 4 and 5).

Formulation and characterization of in situ ocular gel containing DIC loaded Microsponges
In situ ocular gel found clear with suspended microsponge particles. The pH of in situ gel solution was found in range 6.03 to 6.13.
The formulation have an optimum viscosity at Non-Physiological conditions that will give ease in instillation to eye in the form of drop, which go through a quick sol to gel transition (triggered by pH) at physiological conditions (37 °C and 7.4 pH) as shown in table 4.
The drug release kinetic of all in situ gel formulations suggests Higuchi kinetic model (table 5) and diffusion exponent showed nonfickian diffusion mechanism.

Sterility study
The study results of the sterility study are summarized in table 6, the prepared ocular in situ gel formulation passed the sterility test.
There was zero growth found in fluid thioglycollate medium and soyabean casein digest medium.

DISCUSSION
DIC-loaded microsponges were prepared by the quasi emulsion solvent diffusion method using ERS polymer. In literature, this method is considered to be simple, replicable, rapid and solvent toxicity also got eliminated [3]. With the increase in drug concentration in polymer: drug ratio; production yield, drug content and entrapment efficiency increases. As more drug are available for entrapment leading to high amount of drugs in microsponges leading to increased values of production yield, drug content and entrapment efficiency. The lower values of entrapment efficiency (15.00%) could be due to dissolution of some fraction of DIC in PVA solution during the emulsification process [20]. With an increase in level of DIC (besides drug dissolution), more drugs available for entrapment in ERS polymer matrix. As a result entrapment efficiency increases with increment in polymer: drug ratio.
High PVA concentration and high stirring speed yield small particle size which is requisite for this study. In the quasi emulsion solvent diffusion method, organic phase i.e. dichloromethane diffuses out to yield particles in which drug particle surrounded by polymer. At high polymer: drug ratio less polymer amounts surrounds the drug causing decreased particle sizes of microsponges [2].
As in the polymer: drug ratio the concentration of drug increased, polymer units surrounding drug units is less than at low. During phase transition process less polymer got bound to the drug giving smaller size microsponge. Particle size of less than 10 µm is required for ocular administration [11]. Hence microsponge formulations above 10 µm were not further investigated. Microsponges have particle size range from 5-300 μm. So microsponges below 5 μm were also excluded from further studies.
Sustained release of drug was as a result of incorporation of drug in polymer Matrix [28]. Drug release is inversely related to the Polymer: drug ratio. High Polymer: drug ratio yield smaller particles having more active surface area which coming in contact with diffusion media. Also smaller particle exhibit shorter path length drug need to travel through microsponge pores into the diffusion media [8]. Drug release rates were increased with an increasing amount of DIC in microsponge. At a high Polymer: drug ratio, the corresponding polymer amount is lesser. As more drug got released more void channels formed which allows more diffusion media to enter and in turn increases release rate.
FTIR studies confirms that no interaction between drug and ERS polymer. The thermogram of microsponge formulation showed suppressed peak signify uniform dispersion of DIC in polymeric matrix and altered crystallinity of DIC. Absence of any other significant endothermic peak, in the melting point range of DIC (melting point found as 286.26 °C) indicates that absence of any interaction between the DIC and ERS polymer, and verify the aptness of microsponge preparation technique [27].
SEM results indicated that DERS4 microsponges were tiny, porous and spherical. Surface integrity of microsponge got retained. Microsponges particles are free from aggregates. This might be due to aqueous solution of PVA which is used during the quasi emulsion solvent diffusion method. PVA reduces surface charge on the particle giving aggregate free microsponges [29].
In the present investigation, incorporation of DIC-loaded ERS polymeric microsponges was performed into in situ gel formulated using polymers Carbopol 940 and HPMC E4M. Carbopol 940 and HPMC E4M were used in combination as alone high concentration of carbopol 940 yield a highly acidic solution, which is not suitable for ocular administration (pH of lacrimal fluid 7.4) [30]. So moderate concentration of Carbopol 940 (0.1% w/v) along with viscosityenhancing polymer HPMC E4M (0.2-0.8 % w/v) [31] was used to formulate in situ gel. The pH of formulation would raises instantly to pH 7.4 (an ideal ocular pH) after instillation into eye due to pH of lacrimal secretion. Thus, the formulations were considered to be suitable for ocular administration and may not cause any irritation to ocular tissues upon instillation [25].
At Physiological conditions formulation form stiff gel which increases residence time of microsponges incorporated in gel. In situ gel formulation contains HPMC E4M and carbopol 940 which are known to gel reversibly on elevation of temperature and pH respectively [31]. Hence at physiological conditions viscosity of formulation increases to form a stiff gel, as both pH and temperature increases.
Formed stiff gel should display good integrity and not to get dissolve and erode for extended period of time. Formulations IG2 and IG3 exhibited satisfactory "++" grade of gelling capacity [12].
In situ gel formulation IG3 showed sustained release with a satisfactory "++" grade of gelling capacity. Hence, IG3 formulation was taken for further Sterility test. Sterility study results proved the efficiency of autoclaving for sterilization of ocular in situ gel.

CONCLUSION
With the present study, the potential of DIC loaded microsponges as drug carriers for ocular delivery was investigated. Microsponges showed good practical yield, entrapment efficiency, drug content and suitable particle size for ocular administration. Controlled release of up to 6 h observed through DIC loaded microsponges. DIC loaded microsponges successfully incorporated in in situ gels. The clarity, pH, drug content, gelling capacity and rheological studies of DIC loaded microsponges containing ocular in situ gel formulations were found to be acceptable. Further, in situ gel formulations showed sustained drug release for a period of 8 h, which is satisfactory for management of ocular pain.