Design, fabrication and evaluation of clotrimazole loaded polymeric microsphere based in situ ophthalmic gel

The present study was started with aim to design clotrimazole loaded polymeric microsphere based in situ ophthalmic gel for management of corneal fungal infections. The clotrimazole loaded microsphere was fabricated using various percentage of polylactide-co-glycolide polymer by solvent evaporation technique and evaluated for particle size, zeta potential and encapsulation ef(cid:976)iciency. The polyvinyl alcohol was used as stabilizer. The formulation batch which showed good particle size and entrapment ef(cid:976)iciency was selected for further study. The in situ ophthalmic gel of optimized drug loaded microsphere was formulated using various ratios of sodium alginate and hydroxy propyl methyl cellulose. The formulated gel was evaluated with respect to pH, in vitro gelling capacity, clarity, viscosity, in vitro antifungal activity and in vitro transcorneal permeation behavior. The fabricated drug loaded microspheres revealed acceptable particle size, zeta potential and encapsulation ef(cid:976)iciency. The prepared in situ gels were clear and exhibited acceptable pH, rheological properties and in vitro gelation. The drug loaded microsphere based gel revealed superior in vitro antifungal activity against Aspergillus niger than conventional formulation and suf(cid:976)icient drug permeation across goat cornea. Thus, formulated clotrimazole loaded microsphere based in situ gel based systems can be a promising approach for sustained ophthalmic delivery of antifungal agents.

A The present study was started with aim to design clotrimazole loaded polymeric microsphere based in situ ophthalmic gel for management of corneal fungal infections. The clotrimazole loaded microsphere was fabricated using various percentage of polylactide-co-glycolide polymer by solvent evaporation technique and evaluated for particle size, zeta potential and encapsulation ef iciency. The polyvinyl alcohol was used as stabilizer. The formulation batch which showed good particle size and entrapment ef iciency was selected for further study. The in situ ophthalmic gel of optimized drug loaded microsphere was formulated using various ratios of sodium alginate and hydroxy propyl methyl cellulose. The formulated gel was evaluated with respect to pH, in vitro gelling capacity, clarity, viscosity, in vitro antifungal activity and in vitro transcorneal permeation behavior. The fabricated drug loaded microspheres revealed acceptable particle size, zeta potential and encapsulation ef iciency. The prepared in situ gels were clear and exhibited acceptable pH, rheological properties and in vitro gelation. The drug loaded microsphere based gel revealed superior in vitro antifungal activity against Aspergillus niger than conventional formulation and suf icient drug permeation across goat cornea. Thus, formulated clotrimazole loaded microsphere based in situ gel based systems can be a promising approach for sustained ophthalmic delivery of antifungal agents.

INTRODUCTION
Fungal eye infection is extremely rare but major cause of ocular morbidity. Mycotic eye infections are commonly caused by yeast Candida albicans which is the most common cause of endogenous Endophthalmitis (Klotz et al., 2000). The fungi such as Fusarium solani and Aspergillus lavus are also responsible for one-third of all traumatic infectious keratitis. Endophthalmitis is an infection of the inside of the eye i.e. the vitreous and/or aqueous humor whereas keratitis is an infection of the cornea of eye (Müller et al., 2013). Clotrimazole (CLZ), 1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole is well known antifungal drug containing imidazole ring. Conventional ophthalmic drug administration involves local administration of antifungal drug by topical route (Mohanty et al., 2015). However conventional ophthalmic drug delivery is challenging due to barrier properties of cornea and conjunctiva. The unique anatomy and physiology cornea presents constant challenge for formulators to circumvent the protective barriers of eyes. In addition to this, poor ocular residence time of drug due to its loss by lacrimal luid and nasolacrimal drainage are another hurdles for ocular drug delivery (Kaur et al., 2008).
Various strategies like use of nanocarriers, in-situ ophthalmic gelling systems, improving rheological properties of systems were investigated and reported in literatures to increase the ophthalmic bioavailability of drugs by prolonging precorneal residence time of drugs (Das and Suresh, 2011). The water soluble polymers were reported to increase precorneal residence time and improve permeation of drugs across the cornea which eventually improves ocular bioavailability of drugs (Kakkar et al., 2015).
Ophthalmic in-situ gelling systems contain environmentally sensitive polymers that will be structurally altered with the small alteration in parameters like pH, ionic strength and temperature in the environment (Wu et al., 2019). In-situ ophthalmic gel are homogeneous polymeric solution before instillation into the eye; after instillation into eyes the system undergoes sol to gel transition in the ocular cavity to form viscous gels in response to change in environmental condition and gradually releases the drug in ocular cavity.
The present short communication focused on formulation and evaluation of CLZ encapsulated poly(lactic-co-glycolic acid) (PLGA) polymeric microsphere based in situ ophthalmic gel to prolong precorneal residence time of drug. The drug loaded microsphere were fabricated and loaded in in situ ophthalmic gel. The microsphere based gel showed acceptable gelling capacity, clarity, pH and rheological characteristics. The in vitro antifungal activity and in vitro permeation study revealed promising results.

Materials
Clotrimazole (CLZ) was kindly gifted by Glenmark Pharmaceutical, Mumbai, India. Soya lecithin S-90 and S-100 were kindly gifted by Lipoid, Germany. Hydroxy propyl methyl cellulose, sodium alginate, Tween 20, Tween 80 and polyvinyl alcohol were purchased from SDFCL, Mumbai, India. PLGA was purchased from Sigma Aldrich, India. All other reagents and solvents were analytical grade and purchased locally.

Preparation and evaluation of CLZ loaded PLGA microspheres
The Clotrimazole encapsulated PLGA microspheres were fabricated by solvent evaporation technique using various ratios of drug and polymer (Garud and Garud, 2012). Brie ly weighed quantity of drug and polymer were dissolve in dichloromethane (Table 1). The prepare organic phase then slowly added in aqueous dispersion medium containing 0.5% w/v polyvinyl alcohol with continuous stirring at 1000 RPM for 1 hr. The prepared microsphere based dispersion then centrifuged at 5000 RPM to separate unentrapped drug and redisperse in aqueous medium containing stabilizer. The prepared microspheres based dispersion evaluated with respect to particle size, zeta potential and encapsulation ef iciency. The particle size and zeta potential of drug loaded microsphere were measured using Zetasizer Nano ZS (Malvern, Worcestershire, UK).
The encapsulation ef iciency was measured as the percentage of CLZ entrapped in the microsphere. The percentage entrapment ef iciency of CLZ in the CLZ loaded microsphere was determined using the indirect method. The CLZ loaded microsphere dispersions were subjected to ultra-centrifugation at   No gelation -Gelation occur in few minute and retain for few hours + Gelation occur immediately and retain for few hours ++ Gelation occur immediately and retain for extended hours +++ Very stiff gel ++++

Preparation of drug loaded microsphere based in-situ gel
Microspheres based in-situ ophthalmic gel was prepared using various ratios of HPMC K4M and sodium alginate as polymer and copolymer respectively (Makwana et al., 2016). Different ratios of polymers were dissolved in 50 ml water with heating and continuous stirring (Table 2). Once clear homogeneous polymer solution is obtained the resulting solution cool to room temperature. The CLZ microspheres equivalent to 0.5 gm of CLZ (1%w/v) were dispersed and pH of solution adjusted to 6.5 using 0.1 N HCl.

Evaluation of microsphere based in-situ gel
The prepared drug loaded microsphere based insitu gel was evaluated with respect to in vitro gelling capacity, clarity, rheological characteristics, pH determination, in vitro drug permeation behavior and in vitro antifungal activity.
The in vitro gelling capacity of the formulated gel was measured by placing a two drops of the prepared formulation in a petri plate containing 10 molar calcium chloride solution and was observed for gelation (Makwana et al., 2016). The gelation behavior of prepared gel was recorded using scoring system as mention in Table 3.
Clarity of formulated gel was observed by visual observation of in situ gel against a black and white background, with the continuous swirling action.
The rheological characteristic of in gel governs precorneal residence time of drug. Brook ield Viscometer model DVII was used to measure viscosity of formulated gel before and after gelation. The pH of prepared gel was measured using calibrated pH meter.
The permeation of CLZ across goat cornea from the optimized IOG3 formulations was measured out using Franz diffusion cell (Meditech Technologies India Pvt. Ltd.) with 1.76 cm 2 diffusion area. The goat eye was obtained from local slaughterhouse; cornea was removed and used as membrane for assessment of permeation behavior of drug (Khames et al., 2019). The microsphere based gel equivalent to 5 mg of CLZ was placed in donor chamber, and goat cornea was placed in between donor and receptor compartment. The simulated tear luid of pH 7.4 was selected as a diffusion medium in the receptor chamber. The temperature of diffusion medium was adjusted to 32 ± 0.5 • C with the constant stirring at 500 rpm. At periodic intervals the aliquot of diffusion medium was withdrawn and analyzed for drug content using UV spectrometer (V-530, Jasco, Japan) after dilution. The permeation of clotrimazole from IOG 3 formulations were assessed by plotting the graph of cumulative drug permeated (Q) per unit area as a function of time.
The antifungal activity of the prepared microsphere based in situ gel formulations was assessed against standard strain of Aspergillus niger using agar well diffusion technique. Three samples i.e. Vehicle control (distilled water), marketed CLZ formulation and Prepared CLZ microsphere based gel were selected for antifungal activity. Muller-Hinton agar was used as growth medium for growth of fungus. The petri plates containing sterile growth medium was prepared and fungal culture was inoculated to produce 1 × 10 6 CFU/mL. Wells of 12 mm diameter were made in the agar plates after solidi ication of growth medium. Each well was illed with 200 µL of each sample and incubated at 37 • C for 24 h. The inhibition zone diameter was measured using a caliper and compared. The absence of fungal growth around the well indicated fungal growth inhibitory activity of drug.

Preparation and evaluation of CLZ loaded microsphere
The CLZ loaded microspheres were successfully fabricated using solvent evaporation technique. The particle size and zeta potential of drug loaded microsphere were measured using Zetasizer Nano ZS (Malvern, UK). Table 4 displays particle size, zeta potential and encapsulation of drug loaded microspheres. The particle size and zeta potential of formulated microspheres were in the range of 2870-8915 nm and -19.7--45.6 mV respectively. An increased average particle size was observed with increase in drug: polymer ratio. This could be attributed due to increase in amount of polymer in dispersion medium which could be dif icult to wet by stabilizer. The high negative value indicated better stabilization of microspheres in dispersion medium.
The CLZ entrapment ef iciency in polymeric microspheres was in the range of 52.71 -70.89%. The encapsulation ef iciency was observed to be increase with increase in polymer concentration. This could be attributed due to availability of more concentration of polymer to encapsulate CLZ. The optimized microsphere dispersion (F3) was used for formulation of in situ ophthalmic gel using various ratios of polymer and copolymer. The particle size and zeta potential of optimized formulation (F3) are represented in Figures 1 and 2 respectively.

Evaluation of microsphere based in-situ ophthalmic gel
The in vivo performance of in situ gelling system is governed by two major characteristics i.e. gelling capacity and rheological characteristics (Makwana et al., 2016).
The in situ gelling system formulated using different ratios of HPLC and sodium alginate was assessed for gelling capacity and viscosity. Gelling capacity of prepared in situ gel formulations are highlighted in Table 5. According that IOG 3 revealed immediate gelation and for extended period of time. Thus formulation IOG 3 with acceptable consistency was considered for further studies. All the prepared batches of in situ gel were clear with absence of turbidity and impurities. Newtonian low before gelling and pseudoplastic low after gelling was major indings of rheological studies. There was increase in the viscosity after gelling. The pH of in situ gel was found to be in range of 6.49-6.60 which was acceptable for ophthalmic drug delivery (Table 5). The formulation IOG3 has pH 6.53, which confer its suitability for ophthalmic administration.
In present study, the permeation of CLZ across goat cornea was assessed to evaluate the effectiveness of the prepared formulation. Effective drug permeation across cornea along with the acceptable antifungal activity are major characteristics of effective ocular antifungal drug delivery system (Khames et al., 2019). The transcorneal permeation study revealed acceptable drug permeation across goat cornea from in situ gelling system as represented in Figure 3. The study was performed for 4 hrs due to viability of cornea. The encapsulation of CLZ in PLGA polymer matrix provided sustained drug release. Loading of drug encapsulated microspheres into the in situ gelling vehicle represented another factor that could assist in sustained drug release.
The zone of fungal growth inhibition is represented in Figure 4. The marketed CLZ formulation (sample 2) showed zone of inhibition 17 mm whereas microsphere based in situ gel (sample 3) showed zone of inhibition 20 mm. Thus developed microsphere based in situ gelling system exhibited superior antifungal activity as compared to marketed formulation. However in-vivo antifungal activity is necessary to assess in order to prove ef icacy of developed formulation.

CONCLUSIONS
Polymeric microsphere based in-situ gel of CLZ was successfully developed using solvent evaporation technique to overcome drawbacks associated with conventional ophthalmic drug delivery and for ophthalmic drug delivery for extended period. The selected technique was simple, reproducible and rapid; which led to the formation of microsphere with acceptable drug entrapment ef iciency. The diverse drug-polymer ratio revealed varied particle size, zeta potential and entrapment ef iciency. Among the all prepared drug loaded microspheres, the F3 formulation was selected for further study on the basis of its superiority in terms of particle size, zeta potential and entrapment ef iciency. The microsphere based in-situ gel was formulated to improve precorneal residence of drug. The microsphere based gel showed acceptable gelling capacity, clarity, pH and rheological characteristics. The in vitro antifungal activity and permeation study revealed promising results. Thus, microsponge based in-situ gel developed and investigated in present study was seems to be promising with respect to management of ocular fungal infections and to improve precorneal residence time of CLZ.