major problem in ocular therapeutics is the attainment of optimal drug concentration at the site of action , which is compromised mainly due to precorneal loss resulting in only a small fraction of the drug being ocularly absorbed . the effective dose administered may be altered by increasing the retention time of medication into the eye by using in situ gel - forming systems . the anatomy , physiology , and biochemistry of the eye render this organ exquisitely impervious to foreign substances . the challenge to the formulator is to circumvent the protective barriers of the eye without causing permanent tissue damage . ophthalmic ointments ensure superior drug bioavailability by increasing the contact time , minimizing the dilution by tears , and resisting nasolacrimal drainage . major disadvantage of ointment , providing blurred vision , due to this it could be used either night time or for treatment on the outside and edges of the eyelids . suspension as ophthalmic delivery systems relies on the assumption that particles may persist in conjunctival sac . precorneal drug loss can be minimal , such as retarding drainage by using diffusion - controlled , nonerodible polymeric insert . the major disadvantage of inserts is the lack of patient acceptance owing to difficult administration . the development of newer , more sensitive diagnostic techniques and therapeutic agents render urgency to the development of more successful ocular delivery systems . the primitive ophthalmic solution , suspension , and ointment dosage forms are clearly no longer sufficient to combat these diseases , and current research and development efforts to design better therapeutic systems are the primary focus of this research work . the aim of the present investigation is to formulate an in situ gel using novel gum system . in situ gel solution increases the residence time and also sustain the release mechanism of the drug . sodium alginate and hpmc - e 50lv was obtained from the nice chemicals , ahmedabad , india . the polymeric solution was prepared by dispersing required quantity of sodium alginate as main polymer and hpmc- e 50 lv , hpmc- k4 m as co - polymers in water using a magnetic stirrer until the polymers completely dissolve . aqueous solution of moxifloxacin hydrochloride was added in to the polymeric solution with continuous stirring . the ph of the solution was adjusted to 6.5 using 0.1 n naoh/0.1 n hcl . the in situ gel formulations are depicted in table 1 . formulation design of in situ gelling system the formulated in situ gel solution was tested for clarity , ph , gelling capacity , and drug content estimation . evaluation parameters of formulations the gelling capacity of the prepared formulation was determined by placing a drop of the formulation in a vial containing 2 ml of freshly prepared simulated tear fluid and visually observed . the time taken for its gelling the samples were analyzed at 37c  0.5c by a circulating bath connected to the viscometer adaptor prior to each measurement.[710 ] the angular velocity of the spindle was increased 1 to 4 and the viscosity of the formulation was measured . the drug content estimation was carried out by diluting 1 ml of prepared formulation in 100 ml of distilled water and analyzed using uv - visible spectrophotometer ( shimadzu uv-1700 pc , shimadzu corporation , japan ) at 290 nm . the in vitro release of moxifloxacin hydrochloride from the prepared formulations was studied using a modified diffusion testing apparatus . the freshly prepared simulated tear fluid ( ph 7.4 ) was used as a diffusion medium . semi permeable membrane , previously soaked in the diffusion medium for overnight , was tied to one end of a specially designed glass cylinder ( open at both ends ) having inner diameter of 3.4 cm . two milliliter of formulation was accurately pipette into the glass cylinder known as donor chamber . the cylinder was suspended in a beaker ( acceptor chamber ) containing 100 ml of diffusion medium so that the membrane just touches the surface of the medium . acceptor chamber was maintained at a temperature of 37  2c with a stirring rate of 50 rpm using magnetic stirrer . about 1 ml of sample was withdrawn at a time interval of 1 hour and replaced with an equal volume of fresh diffusion medium . the aliquots were diluted with the diffusion medium and analyzed at 290 nm using uv spectrophotometer . in a similar manner , 2 ml of pure drug solution ( 0.5% w / v in distilled water ) and 2 ml marketed product ( milflox ) were studied in a similar manner . the standard minimum inhibitory concentration ( mic 2 g / ml ) of control and developed formulations containing moxifloxacin were prepared . a total of 60 ml of nutrient agar media was prepared and sterilized at 15 lb / sq - inch pressure for 18 minutes in an autoclave ; 0.5 ml of microorganism suspension was poured into the above medium which is maintained at temperature of 52c to 58c . after solidification of the media , sterile solutions of moxifloxacin hydrochloride ( standard solutions ) and the developed formulations diluted suitably with sterile distilled water ( test solutions ) were poured in to the cup of sterile nutrient agar petri plates . this was previously seeded with test organisms ( escherichia coli and staphylococcus aureus ) . after allowing diffusion of the solutions for 2 hours the zone of inhibition ( zoi ) was measured around each cup and compared with that of control . ocular irritation study was performed on optimized formulation in four albino rabbits ( male ) , each weighing about 2 to 3 kg , and 0.1 ml of the optimized sterile moxifloxacin hydrochloride formulation was instilled in to cul - de - sac twice a day for a period of 14 days . sterile optimized ophthalmic formulation was filled in glass vials , closed with gray butyl rubber closures and sealed with an aluminium caps . the vials contain optimized formulation were kept in stability chamber , maintained at 40  2c and 75  5 % rh for one month . samples were withdrawn weekly and estimated for drug content , ph , visual appearance , gelling capacity and in vitro drug release . the polymeric solution was prepared by dispersing required quantity of sodium alginate as main polymer and hpmc- e 50 lv , hpmc- k4 m as co - polymers in water using a magnetic stirrer until the polymers completely dissolve . aqueous solution of moxifloxacin hydrochloride was added in to the polymeric solution with continuous stirring . the ph of the solution was adjusted to 6.5 using 0.1 n naoh/0.1 n hcl . the in situ gel formulations are depicted in table 1 . formulation design of in situ gelling system the formulated in situ gel solution was tested for clarity , ph , gelling capacity , and drug content estimation . the gelling capacity of the prepared formulation was determined by placing a drop of the formulation in a vial containing 2 ml of freshly prepared simulated tear fluid and visually observed . the time taken for its gelling was noted . the samples were analyzed at 37c  0.5c by a circulating bath connected to the viscometer adaptor prior to each measurement.[710 ] the angular velocity of the spindle was increased 1 to 4 and the viscosity of the formulation was measured . the drug content estimation was carried out by diluting 1 ml of prepared formulation in 100 ml of distilled water and analyzed using uv - visible spectrophotometer ( shimadzu uv-1700 pc , shimadzu corporation , japan ) at 290 nm . the in vitro release of moxifloxacin hydrochloride from the prepared formulations was studied using a modified diffusion testing apparatus . the freshly prepared simulated tear fluid ( ph 7.4 ) was used as a diffusion medium . semi permeable membrane , previously soaked in the diffusion medium for overnight , was tied to one end of a specially designed glass cylinder ( open at both ends ) having inner diameter of 3.4 cm . two milliliter of formulation was accurately pipette into the glass cylinder known as donor chamber . the cylinder was suspended in a beaker ( acceptor chamber ) containing 100 ml of diffusion medium so that the membrane just touches the surface of the medium . acceptor chamber was maintained at a temperature of 37  2c with a stirring rate of 50 rpm using magnetic stirrer . about 1 ml of sample was withdrawn at a time interval of 1 hour and replaced with an equal volume of fresh diffusion medium . the aliquots were diluted with the diffusion medium and analyzed at 290 nm using uv spectrophotometer . in a similar manner , 2 ml of pure drug solution ( 0.5% w / v in distilled water ) and 2 ml marketed product ( milflox ) were studied in a similar manner . the drug was allowed to diffuse through a solid agar medium . the standard minimum inhibitory concentration ( mic 2 g / ml ) of control and developed formulations containing moxifloxacin were prepared . a total of 60 ml of nutrient agar media was prepared and sterilized at 15 lb / sq - inch pressure for 18 minutes in an autoclave ; 0.5 ml of microorganism suspension was poured into the above medium which is maintained at temperature of 52c to 58c . after solidification of the media , sterile solutions of moxifloxacin hydrochloride ( standard solutions ) and the developed formulations diluted suitably with sterile distilled water ( test solutions ) were poured in to the cup of sterile nutrient agar petri plates . this was previously seeded with test organisms ( escherichia coli and staphylococcus aureus ) . after allowing diffusion of the solutions for 2 hours the zone of inhibition ( zoi ) was measured around each cup and compared with that of control . ocular irritation study was performed on optimized formulation in four albino rabbits ( male ) , each weighing about 2 to 3 kg , and 0.1 ml of the optimized sterile moxifloxacin hydrochloride formulation was instilled in to cul - de - sac twice a day for a period of 14 days . sterile optimized ophthalmic formulation was filled in glass vials , closed with gray butyl rubber closures and sealed with an aluminium caps . the vials contain optimized formulation were kept in stability chamber , maintained at 40  2c and 75  5 % rh for one month . samples were withdrawn weekly and estimated for drug content , ph , visual appearance , gelling capacity and in vitro drug release . in the present investigation , efforts were made to prepare the sustained release moxifloxacin hydrochloride in situ gel forming ophthalmic solution using polymers such as sodium alginate and hpmc . sodium alginate a novel ophthalmic gel - forming mucoadhesive polymer , which gets converted to gel in the presence of divalent - cations ( calcium ion ) present in the lachrymal fluid ( ph 7.4 ) , was used as the gelling agent . the prepared in situ gel formulations were evaluated for clarity , ph measurement , gelling capacity , drug content estimation , rheological study , in vitro diffusion study . the ph of in situ gel solution was found to be around 6.5 for all the formulations . the formulation should have an optimum viscosity that will allow for easy instillation into the eye , which would undergo a rapid sol to gel transition ( triggered by ion exchange ) as shown in table 2 . rheological evaluation of all the formulation exhibited newtonian flow before gelling and exhibited pseudoplastic flow after gelling in the eye . additionally , the gel formed in situ should maintain its integrity without dissolving or eroding for a prolonged period . rheological studies of formulations formulations f1 to f6 ( before gelling ) formulations f1 to f6 ( after gelling ) from the in vitro results it was observed that percentage release of the drug from the developed formulations f1 ( 93.86% ) , f2 ( 92.78% ) , f3 ( 89.97% ) , f4 ( 82.80% ) , f5 ( 80.49% ) , and f6 ( 78.71% ) as shown in figure 3 . this could be the reason of higher concentration of sodium alginate and hpmc k4 m among the developed formulations . by observing the drug release profile it can be conclude that release is not stagnant even end of 10 hours . formulation f6 showed highest zone of inhibition values against s. aureus ( 28.66 mm ) and e. coli ( 30.99 mm ) , respectively , compared to other developed formulations . comparative in vitro diffusion profile of pure drug , marketed product and f1 to f6 formulations antimicrobial efficacy study was performed on f6 formulation using gram + ve s. aureus and gram -ve e. coli organism . the zone of inhibition of f6 ophthalmic formulation found to be 28.66 and 30.99 mm , respectively , for gram + ve s. aureus and gram -ve e. coli organism . the study indicated moxifloxacin hydrochloride retained its antimicrobial activity when formulated as gel forming ophthalmic system against both selected s. aureus and e. coli . zone of inhibition values of formulation f1 to f6 and pure drug at the concentration of 2 g / ml against staphylococcus aureus and escherichia coli ocular irritation study was performed using healthy albino rabbits after getting prior permission from the institutional animal ethics committee . the eyes of each rabbits were examined at particular time interval after instillation of the optimized formulation ( f6 ) . no ocular damage or abnormal clinical signs to the cornea , iris or conjunctiva were visible . the result of ocular irritation studies indicates that formulations containing all ingredients are non - irritant to rabbit eye . accelerated stability studies were carried out at 40  2c at 75  5 % rh for 1 month using stability chamber . the samples were analyzed periodically on every week , and found that there are no changes in visual appearance , clarity , ph , and gelation . assay values after 1 month of storage are found almost same ( deviating not more than one percent ) . moxifloxacin hydrochloride , a broad spectrum antibacterial agent used in the treatment of ocular infections , was successfully formulated as in situ gel - forming eye drops using sodium alginate as a gelling agent in combination with hpmc as a viscosity enhancing agent . thus , the developed formulation is a viable alternative to conventional eye drops by virtue of its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain drug release . also important is the ease of administration afforded and decreased frequency of administration resulting in better patient acceptance .
background / aim : the aim of the present investigation is to prepare and evaluate in situ gel - forming ophthalmic drug delivery system of moxifloxacin hydrochloride.materials and methods : sodium alginate , a novel ophthalmic gel - forming mucoadhesive polymer , which gets converted to gel in the presence of divalent - cations ( calcium ion ) present in the lachrymal fluid , was used as the gelling agent . 
 hydroxy propyl methyl cellulose ( hpmc ) is a mucoadhesive polymer used as viscosity enhancer . 
 suitable concentrations of buffering agents were used to adjust the ph to 6.5 . 
 all the formulations were sterilized in an autoclave at 121c for 15 minutes . 
 the formulations were evaluated for clarity , ph measurement , gelling capacity , drug content estimation , rheological study , in vitro diffusion study , antibacterial activity , isotonicity , and eye irritation study.results:the developed formulations exhibited sustained release of drug from formulation over a period of 10 hours thus increasing residence time of the drug . 
 the optimized formulations were tested for eye irritation on albino rabbit ( male ) . 
 the formulations were found to be non - irritating with no ocular damage or abnormal clinical signs to the cornea , iris or conjunctiva observed.conclusion:these in situ gelling systems containing gums may be a valuable alternative to the conventional systems .