2.2.4.10. Ocular tolerance test
Hen’s Egg Test/Huhner-Embroynen Test on Chorioallantoic membrane (HET-CAM) is substitute method to draize test done on rabbit eyes. In this investigation ocular irritancy, vascular response, hemorrhage and injuries to conjunctiva are observed for an ophthalmic product. Fresh fertilized hen’s egg weighing 50–60 g were acquired from poultry farm. Briefly, fertilized eggs acquired from poultry farm were incubated for 3 days at 37 ± 0.5oC and 55 ± 5% relative humidity [20]. On day 3, albumin (3ml) was drawn-out from egg utilizing sterilized approach and hole was fastened with paraffin sterilized with 70% alcohol. On day 10 of incubation, at equator 2 x 2 cm of egg shell was removed to create a window [21]. Normal saline (0.9%, negative control), sodium hydroxide (0.1 N, positive control) and VA-cubosomes (test formulation) were administered directly to the surface of CAM. At pre-established interims, CAM was observed for vascular responses/injuries and scores were specified as per scoring scheme. No visible hemorrhage = 0 (Non-irritant), observable membrane discoloration = 1 (mild irritant), structures covered slightly because of membrane discoloration/hemorrhage = 2 (moderately irritant) and structures covered completely owing to membrane discoloration/hemorrhage = 3 (severe irritant) [22].
2.2.4.11 Anti-microbial studies
Comparative anti-microbial activity of VA-cubosomes and VA-solution were determined utilizing agar well-diffusion technique along with cup-plate technique. There studies were performed on Staphylococcus aureus and Pseudomonas aeruginosa. Media was composed by blending agar and nutrient broth and sterilized by autoclave at 15 lb/sq-inch at 121oC for 60 minutes. In two distinct petri-dish, nutrient agar media was poured and was inoculated with S. aureus and P. aeruginosa in sterile conditions under laminar air flow unit. Subsequently, in the inoculated solidified nutrient-agar plates, two wells of 8 mm were created with the assistance of sterile cork-borer. The wells were loaded with vancomycin solution as control and developed cubosomes as test and then incubated for 24 hours at 370C. The diameter of inhibition zone was calculated and compared with control [23].
2.2.4.12 Isotonicity evaluation
Isotonicity of ophthalmic preparation has to be maintained to avert tissue damage and corneal irritation. Isotonicity assessment was performed by hemolytic method. The developed formulation of VA-cubosomes were blended with red blood cells of mice and perceived with inverted microscope at magnification of 45X. The concentration of hypotonic solution was (0.45% w/v NaCl), hypertonic solution (3% w/v) and isotonic solution (0.9% w/v NaCl). Aforementioned method was replicated for isotonic solution (negative control), hypotonic solution (positive control) along with hypertonic solution to observe bursting, swelling and cremation effect on red blood cells [24].
2.2.4.13 Sterility Studies
Sterility is a crucial requirement for ophthalmic preparation as contamination may cause ocular infections. Sterility studies are performed to detect the existence of pathogens in the developed ocular formulations [25]. Analysis was executed on the optimized cubosomal formulation by direct inoculation technique as described in Indian Pharmacopoeia 2010. In this study, 2 ml of VA-cubosomes were transferred to two distinct flasks encompassing 10 ml of soybean-casein digest medium and fluid thioglycollate medium. The inoculated media was blended with medium and was incubated at 200C-250C for soyabean-casein digest media and 300C-350C for fluid-thioglycollate medium for 14 days. After end of 14 days, the incubated flasks were scrutinized for any indication for growth of microorganisms [26].
2.2.4.14 Drug Release Kinetics Studies
For determination of drug release kinetics of developed formulation, various kinetic models were implemented. Data acquired from in-vitro drug release studies was fitted into different kinetic models such as Korsmeyer-Peppas model (log percentage drug release v/s log time), Higuchi’s model (percentage of drug release v/s square root of time), first order equation (log percentage of drug remaining v/s time) and zero order equation (cumulative amount of drug release v/s time). For respective models, correlation co-efficient was evaluated [27].
2.2.4.15 In-vivo studies
Rabbits were provided by Central Animal House, Amity University, Noida, India. The study was approved by University Animal Ethical Committee with registration number 1327/PO/ReBi/S/10 /CPCSEA. All institutional and national guidelines for the care and use of laboratory animals were followed. In-vivo studies were performed on rabbit’s eyes which were induced with bacterial keratitis. In this investigation, twelve New Zealand albino rabbits weighing 2–3 kg with no sign of ocular inflammation and gross abnormalities were utilized. Rabbits were categorized into 3 groups of 4 rabbits. Group I (control) was instilled with sterile saline solution, group II was topically administered with optimized VA-cubosomes and group III administered with VA-solution. In group III, 100 µl of optimized cubosomes were introduced twice a day and in group II vancomycin solution four times a day [28].
S. aureus was grown aerobically in 10 mL of brain heart infusion broth (HiMedia, India) at 37oC for 18 hours. Microorganisms were collected by centrifugation (Remi, C-24BL, New Delhi, India) at 5000 rpm for 10 mins and washed with phosphate buffer saline (PBS) pH 7.4 and suspended in 2 mL PBS. Keratitis was induced by inoculating 100 µL S. aureus suspension (containing nearly 107-108 colony-forming units) in rabbit’s eyes by intrastromal injection with 29-gauge needle. Manifestations of keratitis were developed after 24 hours of microbial inoculation into eyes [29, 30]. Rabbit’s eyes were evaluated for clinical signs of bacterial keratitis such as redness, mucoid discharge, lacrimal secretion, corneal ulcer and swelling of eyelid. The grading scale was followed on the scale from 0 to 4 as 0 (absent), 1 (mild), 2 (moderate), 3 (severe) and 4 (extensive). The grading of clinical signs were performed at intervals of 0, 24, 72 and 120 hours. The formulations were instilled into eyes of rabbit following the complete development of infection. The eyes were perceived for the recuperation of infected eyes on each successive day by aforementioned categorization till complete recovery from keratitis. Comparative analysis of developed VA-loaded cubosomes with vancomycin solution was done and the treatment results were compared. Significance was determined by applying unpaired t-test [31].
2.2.4.16 Ocular pharmacokinetic studies
The rabbits were randomly divided into the following two groups: a group treated with vancomycin solution and a group treated with VA-cubosomes. Ocular absorption of VA-cubosomes was assessed on aqueous humor fluids. Pupils of rabbits were dilated with topical instillation of 0.25% tropicamide. Rabbits were anesthetized through-out the investigation using injection of sodium pentobarbital (30 mg/kg) injected into marginal ear-vein. The rabbits were administered with 100 µl of optimized cubosomal formulation in the lower conjunctival sac of the right eyes and VA-solution in left eyes. Aqueous humor samples (0.15 ml ≈ 50–80 µL) were withdrawn at different time intervals at 0.25, 0.5, 1, 2, 3, 4, 5 and 6 hours by anterior chamber paracentesis utilizing 1 ml insulin syringe attached with 26-gauge needle. The aqueous humor samples were collected in glass tubes and stored at -20oC [32]. Study was repeated in triplicate and samples were examined utilizing UV-spectrophotometer. The pharmacokinetic parameters such as area under the concentration–time curve (AUC0→t) and peak aqueous humor concentration (Cmax) were calculated from appropriate graphs using the linear trapezoidal rule (Kaleidagraph, Synergy Software) [33].
2.2.4.17 Corneal Toxicity Studies (Histopathology Studies)
Histopathology studies were done to corroborate the results obtained from in-vivo studies on developed cubosomes and also to scrutinize corneal structure and integrity. Rabbits were euthanized with injection of sodium pentobarbital and then eyeballs were removed. Rabbit corneas was cautiously exercised and incubated with optimized VA-cubosomes (test sample), sodium dodecyl sulfate 0.1% w/v (positive control) and phosphate buffer saline (negative control) for 2 hours. Following incubation, rabbit corneas were rinsed with phosphate buffer saline (pH 6.4). Subsequently, corneas were instantaneously fixed with 8% v/v formalin solution. Corneal tissues were dehydrated with an alcohol gradient and placed in melted paraffin and then hardened to form block. Cross-sections of blocks < 1mm were slit and stained with haematoxyline and eosine. The slides were cover-slipped and assessed under microscope [34].
2.2.4.18 Stability Studies
The aim of stability studies was to determine that the developed formulation has preserved its efficacy and productivity with the progression of time, humidity and temperature. Comprehensive stability data was collected for shelf-life determination and slope and degradation constant of VA-loaded cubosomes. The protocols for determination of stability studies were in accord as per ICH guidelines. For evaluation for stability, the developed cubosomal preparation was subjected to distinct humidity and temperature conditions. Optimized VA-cubosomes were kept in screw capped amber colored glass vials and stored at 5 ± 10C, 25 ± 20C, 40 ± 20C, 60 ± 20C, and 75 ± 5% relative humidity for a period of 6 months. The samples were drawn-out at different interims viz. 0, 30, 60, 90 and 180 days and investigated for entrapment efficiency, particle size distribution, polydispersity index, pH, in-vitro drug release and phase separation [35].