Preclinical Assessment of Nanostructured Liquid Crystalline Particles for the Management of Bacterial Keratitis: in-vivo and Pharmacokinetics Study

The research work was driven to develop novel nanostructured liquid crystalline particles of vancomycin for its improved pre-ocular residence time, ocular bio-availability, enhanced targeting, increased permeability, reduced dosing frequency, controlled drug release and reduced systemic side-effects. Formulation was developed by fragmenting cubic crystalline phase of glycerol monooleate, water and poloxamer 407. A four-factor, three-level Taguchi statistical experimental design was constructed to optimize the formulation. Formulations exhibited internal-cubic structure of the vesicles with particle size in the range of 51.11 ± 0.96 nm to 158.73 ± 0.46 nm and negative zeta potential. Ex-vivo transcorneal permeation studies demonstrated that the optimized cubosomes had 2.4-fold increase in apparent permeability co-ecient as compared to vancomycin solution. Whereas, in-vivo studies in rabbits demonstrated that the severity of keratitis was considerably lowered in day 3 with optimized cubosomes. Ocular pharmacokinetic studies evaluated level of drug in aqueous humor and results revealed that the time to peak concentration (T max ) of vancomycin loaded cubosomal formulation was about 1.9-fold higher and mean residence time was 2.2-fold greater than vancomycin solution. Furthermore, histological examination revealed that the corneal layers displayed well-maintained morphology without any stromal swelling, consequently indicating safety of formulation. In conclusion, results manifested that the developed vancomycin loaded cubosomes could be a promising novel ocular carrier and an ideal substitute for conventional eye-drops for the management of bacterial-keratitis.


Introduction
Glycopeptide antibiotic, vancomycin is a tricyclic-glycosylated non-ribosomal peptide.It is used to treat diverse bacterial infections, speci cally gram + ve infections such as species of Staphylococcus.Vancomycin has minimal oral absorption and is delivered by intravenous infusion.It acts by inhibiting polymerization of peptidoglycan layer in cell-wall of bacteria.Speci cally, vancomycin averts incorporation of N-acetylmuramic acid-(NAM) and N-acetylglucosamine-(NAG)-peptide sub-units from being incorporated into the peptidoglycan matrix.Vancomycin forms hydrogen bonds with the terminal D-alanyl-D-alanine moieties of the NAM/NAG-peptides, which impedes glucosyltransferase and the Pphospholipid carrier, therefore averting synthesis and polymerization of NAM/NAG-peptide subunits into the peptidoglycan layer.This inhibition weakens the cell-wall of bacteria and causes intracellular contents to leak-out, eventually causing bacterial cell-death [1].Bacterial keratitis can be described as vision threatening ocular infection predominantly caused by Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa.It presents clinically with corneal in ammation, pain, excessive tearing, corneal edema, ulcers, reduced vision, redness and photophobia.Pre-disposing factors for instance, extended use of contact lenses, eye-trauma, ocular surface-disorders and dry-eyes are correlated with bacterial keratitis.As these factors might alter the defence-mechanism of ocular-surface and allow bacterias' to invade membrane of cornea.There is break-down of corneal-epithelium due to which there is penetration of bacteria.Following the penetration, there is activation of plasminogen to plasmin and bacteria secrets protease, chymase and tryptase that causes corneal necrosis leading to rapid corneal weakening and perforation [2].
Management of bacterial keratitis remains a challenge due to poor corneal penetration of conventional eye-drops and unique anatomical eye-structure.The entry of drug molecule at the site of action is sometimes restricted owing to the unique structure of the eye.Numerous anatomical and physiological constraints such as tear turnover, nasolachrymal drainage, re ex blinking, ocular static and dynamic barriers pose a challenge and impede deeper ocular drug permeation.Corneal epithelium's tight junction acts a selective barrier for entry of small molecules whereas diffusion of macromolecules is prevented by paracellular routes [3].Hence, less than 5% of topically applied dose reaches to deeper ocular tissues.Conventional eye-drops exhibit poor corneal permeability, rapid nasolacrimal drainage, low pre-ocular retention time, rapid precorneal clearance, low ocular bioavailability, frequent administration and rapid drainage due to constant tear ow.To overcome these drawbacks lipid based vesicular drug delivery system such as liposomes, transfersomes, transethosomes, niosomes etc are being developed as novel prospective in ocular drug delivery.But with liposomes there are issues related to physical and chemical stability, oxidative-degradation of phospholipids and poor encapsulation of drugs.They are prone to drug leakage and aggregation of vesicles into larger particles.Niosomes and transferosomes have also reported physical stability issues such as drug-leakage and vesicle-fusion.Therefore, nanostructured liquid crystalline particles have been selected as prospective ocular drug-delivery vehicle [4].
Nanostructured liquid crystalline particles also called as cubosomes are discrete, sub-micron cubic nanoparticles consisting of two congruent non-intersecting hydrophilic channels divided by lipid bi-layer.
Cubosomes are mainly composed of binary system of glyceryl monooleate (GMO) and water, where monooleate acts as precursor for lipid-bilayer.Surfactant provides electrostatic-barrier among particles to avert contact between them, therefore providing steric stabilization to the formulation.Chemically GMO is mono-easter of glycerol and oleic acid.When assembled into liquid crystalline particles, they mimic to the structure of biological-membrane and allow them to amalgamate with highly lipophilic cornealepithelium.They are assembled in three dimensions as honeycomb (cavernous) structure which is structurally similar to biological membrane that enables these carriers to fuse with lipophilic corneal epithelium for increased topical drug delivery [5].Honeycomb structure of cubosomes allows them to retain for prolonged duration on corneal membrane contrast to spherical vesicles, providing increased time-period for drug-loaded particles to permeate through cornea.Cubosomes possess high internal surface-area and presents higher encapsulation as they have higher portion of lipid, consequently exhibits large surface area.They can be harnessed to encapsulate diverse molecules of amphiphilic, hydro-phobic and hydro-philic drugs.They possess high thermodynamic stability, bioadhesive nature, biocompatibility, bio-degradability, protect compounds from physical and enzymatic degradation and sustained drug release behaviour [6].
Therefore, with all the above aspects, Vancomycin HCl loaded cubosomes were developed, optimized and characterized in this investigation.Cubosomes were formulated for prolonged pre-corneal residence time, enhanced targeting, increased permeability, reduced dosing frequency, controlled drug release, reduced systemic side-effects, improved bioavailability and high stability.To assess the e cacy of developed cubosomes, ex-vivo corneal permeation studies, in-vivo studies, ocular pharmacokinetic studies and corneal toxicity studies were performed in rabbit's eyes and compared with marketed eye-drops.

Materials
Vancomycin HCl was obtained as gift sample from Cadila Pharmaceuticals Limited, Ahmadabad, India.Glyceryl Monooleate was procured from Gattefose, France.Poloxamer 407 and dialysis membrane was purchased from Sigma Chemical Company (Sigma-Aldrich Corp., St. Louis, MO, USA).All other solvents used were of analytical grade.Double-distilled water was utilized during the study.

Optimization of formulation variables and process variables
Cubosomes were developed by employing fragmentation method.Distinct concentrations of Glyceryl Monooleate (GMO) ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5% were used.A constant amount of drug was weighed and disseminate in molten blend of GMO and poloxamer 407 (1 % w/v).Similarly, different concentrations of surfactant (poloxamer 407) were taken, 0.1 to 1% and the other parameters were kept constant.Likewise, different stirring time was evaluated to develop the cubosomes so as to optimize the best one.Different time for homogenization was taken as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 minutes.Rest of the parameters such as concentration of GMO, poloxamer 407 and stirring speed was kept constant.Stirring time was optimized with respect to entrapment e ciency, polydispersity index and particle size of cubosomes.Varying stirring speeds from 1000 to 10000 RPM were optimized so that the formulation doesn't occulate or aggregate [7].

Preparation of vancomycin loaded nanostructured liquid crystalline particles
Cubosomes were developed by virtue of fragmentation of glyceryl monooleate and poloxamer 407 bulk cubic phase gels.Primarily GMO and poloxamer 407 were entirely lique ed at 60°C in a hot water bath, and afterward vancomycin HCl was incorporated to amalgamate under ceaseless mixing.To accomplish a homogeneous state, water was included moderately and the blend was vortexed.The cubic phase gel was developed following equilibration for 48 hours at room temperature.Subsequently, the cubic gel was distorted by adding nearly 20 ml water during mechanical stirring.Thereupon, high-pressure homogenizer was utilized to homogenize the developed dispersion at high pressures and cycles to form an opalescent dispersion of cubosomes.The nal dispersion of nanostructured liquid crystalline particles was stored at room temperature [8].

Taguchi statistical experimental design for optimization of VA-loaded cubosomes
Taguchi method of design of experiment was employed for the optimization of the formulation, which uses standard orthogonal arrays for forming a matrix of experiments in such a way so as to extract the maximum important information with minimum number of experiments.The fundamental principle of this method serves as screening lters which examine the effects of many process variables and identify those factors which have major effects on process using a few experiments.It included four factors (formulation variables) i.e. lipid concentration (%v/v), surfactant concentration (%v/v), stirring time (sec) and stirring speed (rpm).All the factors were assigned three levels i.e. low, medium and high as depicted in Table 1.Based on the number of factors and their levels, L 9 (3 4 ) orthogonal array was employed as shown in Table 2. Optimization was done on the basis of 2 techniques i.e. "smaller the better" and "larger the better" and signal to noise ratio (S/N ratio).The signal to noise ratio takes the mean as well as the variation between results.Smaller the better is usually chosen signal to noise ratio for un-desirable characteristics like "defects" when the desired ideal-value is zero, for example size of particles, PDI.
Whereas, larger the better is chosen when desired-ideal value is larger, for example entrapment e ciency [9].

Drug Entrapment E ciency
Drug entrapment e ciency of VA-loaded cubosomes were determined by calculating the percentage of amount of drug entrapped by cubosomes.The developed formulations were evaluated by centrifugation technique, in this method cubosomes were centrifuged at 12000 rpm for 30 minutes employing refrigerated centrifuge (Remi, C-24BL, New Delhi, India).The resultant suspension was then segregated and supernatant was accumulated.The absorbance of supernatant was analyzed spectrophotometrically at absorption maxima (λ max) 281 nm by using UV-visible spectrophotometer (UV-1800, Shimadzu, Japan) [14].The % EE was calculated by using the formula: % EE = Amount of drug in supernatant x 100 ___________________________ Initial amount of drug added

In-vitro Drug Release Studies
The release of vancomycin form cubosomes was demonstrated by employing Franz Diffusion (FD) cell.Dialysis membrane of molecular weight cut off 12000-14000 Da (Spectra Pro, Spectrum Chemical Manufacturing Corp., USA) and pore size was 2.4 nm.Surface area of the dialysis membrane for the release of drug was 3.14 cm 2 .Receiver compartment was lled with simulated tear uid (STF) having pH 7.4 as medium.Dialysis membrane was positioned in the FD-cell and in the donor compartment cubosomal preparation was placed [15].FD-cell was thermostatically maintained at 37°C ± 1°C under constant magnetic stirring.5 ml of sample were drawn from receiver chamber at pre-determined interludes and concurrently replenished with equivalent quantity of buffer to perpetuate sink conditions.
The withdrawn samples were appraised for cumulative percentage drug release by using UV-visible spectrophotometer at 281 nm and graph was plotted [16].

Ex-vivo Transcorneal Permeation Studies
Ex-vivo transcorneal permeation studies were carried out on rabbit cornea utilizing FD-cell.Cornea and 4-5 mm of scleral tissue was cautiously extracted and rinsed with cold saline to eliminate protein adhered to the tissue.Freshly isolated cornea was mounted in-between donor and receptor chamber in such a manner that the epithelial side positioned towards donor chamber and endothelial side towards receptor chamber.Simulated tear uid of pH 7.4 was added in the receptor chamber and the temperature was thermoregulated at 37°C ± 1°C with persistent magnetic agitation.Aliquot of optimized VA-cubosomal formulation and VA-solution was introduced on the corneal surface for correlative examination.Form the receptor compartment, 1ml of sample was drawn out at pre-established schedule and afterwards replaced with equivalent volume of STF [17].The samples were investigated by UV-visible spectrophotometer at 281 nm.The drug permeation % was determined by following; Permeation % = Amount of drug permeated in receptor x 100 _________________________________ An initial amount of drug in donor

Corneal Hydration Studies
The effect of the VA-loaded cubosomes and VA-solution on corneal hydration was determined.In brief, at the end of the experiment, the scleral tissue was removed from cornea; its epithelial surface was wiped with lter paper and weighed (initial weight).The cornea was then soaked in 1ml of methanol, dried overnight at 90°C, and reweighed ( nal weight) [18].From the difference in weight, corneal hydration (%) was calculated as follows: Corneal hydration (%) = Initial weight -Final weight / Initial weight ×100

Bioadhesion studies
Bioadhesion studies were performed to ascertain the potential of developed formulation to cling with corneal surface for protract duration.Rabbit eyes were exercised and cornea was extracted and cleansed.Extricated cornea was kept on petri-dish containing STF with mucosal side of the cornea outwards.Aliquot of formulation was applied to the cornea followed by a droplet of water soluble dye.The ocular tissue was kept hydrated by perpetuating supply of STF.Bioadhesion was visually perceived for its integrity at designated interludes up to 24 hours [19].

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.Brie y, fertilized eggs acquired from poultry farm were incubated for 3 days at 37 ± 0.5 o C and 55 ± 5% relative humidity [20].On day 3, albumin (3ml) was drawn-out from egg utilizing sterilized approach and hole was fastened with para n 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 speci ed as per scoring scheme.No visible hemorrhage = 0 (Nonirritant), 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].

Anti-microbial studies
Comparative anti-microbial activity of VA-cubosomes and VA-solution were determined utilizing agar welldiffusion 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 121 o C 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 ow unit.Subsequently, in the inoculated solidi ed 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 37 0 C. The diameter of inhibition zone was calculated and compared with control [23].

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 VAcubosomes were blended with red blood cells of mice and perceived with inverted microscope at magni cation 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].

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 asks encompassing 10 ml of soybean-casein digest medium and uid thioglycollate medium.The inoculated media was blended with medium and was incubated at 20 0 C-25 S. aureus was grown aerobically in 10 mL of brain heart infusion broth (HiMedia, India) at 37 o C 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 10 7 -10 8 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.Signi cance was determined by applying unpaired t-test [31].

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 uids.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 26gauge needle.The aqueous humor samples were collected in glass tubes and stored at -20 o C [32].Study was repeated in triplicate and samples were examined utilizing UV-spectrophotometer.The pharmacokinetic parameters such as area under the concentration-time curve (AUC 0→t ) and peak aqueous humor concentration (Cmax) were calculated from appropriate graphs using the linear trapezoidal rule (Kaleidagraph, Synergy Software) [33].

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 xed with 8% v/v formalin solution.Corneal tissues were dehydrated with an alcohol gradient and placed in melted para n 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].

Stability Studies
The aim of stability studies was to determine that the developed formulation has preserved its e cacy 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 ± 1 0 C, 25 ± 2 0 C, 40 ± 2 0 C, 60 ± 2 0 C, 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 e ciency, particle size distribution, polydispersity index, pH, in-vitro drug release and phase separation [35].

Optimization of Formulation Variables and Process Variables
For optimization of formulation variables and process variables different characterization parameters were considered such as particle size, polydispersity-index and entrapment e ciency.In the optimization of lipid concentration, an increment in particle size and decreased drug entrapment was perceived on increasing the ratio of drug to lipid.On increasing the lipid concentration from 0.5-5% v/v, the size of the cubosomes increased from 46.24 ± 0.58 nm to 273.55 ± 0.43 nm.The formulations with concentration of 1%, 1.5% and 2% glyceryl monooleate demonstrated best appropriateness for ocular delivery.On increasing the concentration of lipid from 0.5% − 2.5% v/v, increased entrapment was observed as large number of cubosomes were formulated which led to higher drug entrapment.Increasing the quantity of glyceryl monooleate may have resulted in faster solidi cation of cubosomes.But on further augmentation of lipid from 3-5% v/v, drug entrapment was decreased which might be due to increase in viscosity that may hinder diffusion of drug in lipid matrix [36].
The concentration of surfactant was optimized so as to acquire cubosomes of small size with highest drug entrapment.0.1%, 0.5% and 1% w/v concentration of surfactant was considered as optimization concentration as it led to low PDI, low particle size of 46.24 ± 0.58, 82.31 ± 0.26 and 68.22 ± 0.85 nm with better entrapment e ciency of 87.28%, 86.92% and 89.25%.Poloxamer 407 provided steric stabilization in opposition to accumulation of cubosomes particles by securing its poly-propylene oxide that causes impediment in polar-region or at exterior of glyceryl monooleate bi-layer.
Cubosomes were synthesized at distinct stirring time and 2, 2.5 and 3 minutes was the optimized stirring time.Stirring speed of homogenization was varied from 1000 to 10000 rpm.Stirring speed of 4000, 5000 and 6000 rpm were optimized as small particle size and high drug entrapment was attained.Higher shear was acquired on escalating the stirring speed that decreased the particle size [37].The FT-IR spectrum of pure GMO displayed characteristics peaks in the range of 3423 − 765 cm − 1 .

Drug-Excipient Compatibility Studies
Spectra of GMO exhibited broad sharp peak at 3423 cm − 1 that could be attributed stretching of O-H group, peak at 2926 cm − 1 were ascribed to CH 2 stretching and the band at 1597 cm − 1 indicated presence of C-C bonds.Sharp peak at 1384 cm − 1 designated nitrate absorption band and peak at 618 cm − 1 were mainly due to C-H bending [40].
Pure poloxamer 407 exhibited characteristic broad peak at 3425 cm − 1 due to O-H group stretching, 2889 cm − 1 peak indicated C-H stretching vibration, sharp peak at 1596 cm − 1 corresponded to C-C stretching and sharp peak at 1384 cm − 1 indicated nitrate absorption band.Peak at 1113 cm − 1 corresponded to C-O stretching vibration and slight peak at 766 cm − 1 was related to C-N stretch of aromatic and peak at 618 cm − 1 were due to C-H bending [41,42].
Combination of vancomycin HCl and excipients exhibited absorbance in the range of 3419 cm − 1 to 617 cm − 1 .From the FTIR spectra it was observed that there was compatibility between drug, GMO and poloxamer 407.The combination exhibited similar functional groups with extremely slight shift demonstration outstanding compatibility and no evidence of interaction between physical mixture of drug and excipients [43].

Particle Shape and Surface Morphology
VA-loaded cubosomes were envisaged for particle shape and surface morphology through transmission electron microscopy.TEM photomicrographs illustrated nearly cubic organization with well-dispersed individual particles as shown in Fig. 2. Cubosomes were uniformly distributed without any aggregation that could be accredited to optimum combination of drug, excipient, stirring-time and stirring-speed.Large concentration of surfactant adsorbed on exterior of cubosomes acted as overlaying covering to stabilize cubosomes surface.Whereas low surfactant quantity caused enlargement in particle-size due to expansion in inter-facial pressure that caused accumulation of particles [44,45].

Particle Size Distribution and Polydispersity Index
Dynamic light scattering (DLS) measurements were employed to assess size distribution and polydispersity index of developed cubosomes.Particles size values were in between 51.11 ± 0.96 nm to 158.73 ± 0.46 nm, whereas PDI was found to be in the range of 0.275 ± 0.53 to 0.525 ± 0.48.Usually, molecular size should be less than 200 nm for easy permeation across corneal membrane.The developed formulation had molecular size smaller than 200 nm that ensures adequate bioavailability, good uniformity and signi cant potential for effortless permeation.The outcome divulged that particlesize of cubosomes was inversely-proportional to increase in quantity of surfactant [46].High poloxamer quantity could have reduced surface tension of lipid-phase and aqueous phase that might have reduced particle size.High poloxamer amount might have reduced steric hindrances.On minimizing the amount of surfactant, particle size was expanded because of decreased inter-facial stability that was due to inadequate concentration of poloxamer that caused agglomeration of cubosomes [47].Particle size distribution and polydispersity index of formulation have been tabulated in Table 3.

Zeta Potential Measurement
Zeta potential measurement was analyzed to ascertain the surface charge of cubosomes that is of great signi cance for anticipating prolonged stability.Zeta potential revealed that vancomycin cubosomes carried negative charge with mean values of -21.53 ± 0.29 to -30.36 ± 0.41 as represented in Table 3.
-40mv to + 40mv is considered as ideal stability range of zeta potential for formulations.High zeta potential manifests high electric repulsion between dispersed particles which averts agglomeration and provides good stability and dispersion quality [48].Negative charge could be due to existence of fatty acid in glyceryl monooleate.Non-ionic surfactant might have caused negative charge because of hydroxyl ions anchored on exterior of cubosomes and association of hydroxyl group with aqueous medium.Poloxamer 407 provided good steric stability as it stabilized the formulations by steric hindrances that avoided aggregation [49].

Drug Entrapment E ciency
Drug entrapment e ciency was evaluated to determine concentration of drug incorporated in VAcubosomes.Entrapment e ciency was found between 72.181% ± 0.64 to 91.624% ± 0.42 as shown in Table 3.Strong a nity between drug and lipid in cubosomes might have acquired the drug into its inverted-type self assembled nanostructure.Cubic crystalline structure promotes high drug entrapment as they have higher portion of lipid that presents large surface area.Entrapment e ciency was determined by the concentration of glyceryl monooleate and poloxamer [50].The outcome indicated that with the increase in concentration of lipid and surfactant, the entrapment was also increased.This could be attributed to increased viscosity of the formulation, as the concentration of lipid was increased cubosomal dispersion was solidi ed faster that could have prevented the diffusion of the drug to external phase medium.On further escalating the concentration of lipid, reduction in entrapment was perceived which could be due to immoderate viscosity of cubosomal dispersion that obstructs dispersion of drug from the binary lipid matrices [51].

In-vitro Drug Release Studies
The release of vancomycin from developed cubosomes was carried-out utilizing FD-cell for 24 hours.The release of drug-loaded formulations ranged from 79.43 % ± 0.68 to 92.32% ± 0.29 for 24 hours.The invitro release pro le of VA-cubosomes manifested that the formulations demonstrated initial burst release up to 14.42% ± 0.54 and sustained release up to 92.32% ± 0.29.One probable rational for burst release could be attributed to spreading of free-drug adsorbed onto cubosomes particle surface which could easily diffuse [52].Whereas, drug encompassed into particle core would release over extended time due to rm entrapment of drug by lipid layer.This pattern of slow release could be ascribed to sedated expulsion of drug from inner-water channels in the cubic phases.Both the properties of initial-burst release and sustained drug release were bene cial in ocular delivery of drug.Initial-burst was favorable for ameliorating permeation of the drug and provided rapid onset drug action, however sustained-release provided action of drug over a protracted time-span.The sustained-release pattern of drug release up to 24 hours would facilitate reduced dosing frequency [53].In-vitro drug release of vancomycin from cubosomes is depicted in Fig. 3.

Ex-vivo Transcorneal Permeation Studies
Transcorneal permeation studies of VA-cubosomes and VA-solution were evaluated through FD cell on rabbit cornea.On the basis of small particle size, PDI, surface morphology, entrapment e ciency and invitro drug release, formulation F8 was selected as the optimized formulation.The permeation studies demonstrated that a linear-relationship between accumulative permeated vancomycin and time could be perceived.The apparent permeability co-e cient (P app ) of VA-loaded cubosomes and vancomycin solution was 4.48 ± 0.62 x 10 6 and 1.83 ± 0.84 x 10 6 µg/s/cm 2 respectively.Steady state ux (Jss) was found to be 1.45 ± 0.26 x 10 3 and 0.67 ± 0.59 10 3 µg/cm 2 /h.Compared with vancomycin solution, VAcubosomes demonstrated nearly 2.4-fold increase in permeability co-e cient and 2.1-fold increase in steady state ux.Signi cant higher corneal permeation of optimized formulation could be due to strong mucoadhesive property and nano-dimension of nanostructured liquid crystalline particles.Enhanced permeation of optimized cubosomes via paracellular route could be due to presence of surfactant that could have led to disturbance in tight junction of epithelial cells of cornea.Ameliorated drug permeation could also be due to structural similarity between bi-continuous lipid bi-layer of cubosomes and corneal epithelial cells that might permit membrane fusion, facilitating direct drug absorption into corneal cells [54].Transcorneal permeation pro les of VA-loaded cubosomes and VA-solution are graphically represented in Fig. 4.

Corneal Hydration Studies
The corneal hydration level of normal mammalian cornea is between 75%-83% whereas 83-92% hydration level denotes damage to cornea.The corneal hydration value of the optimized formulation and vancomycin solution was between 75 to 82%, which indicated that the formulation did not cause any harm to the corneal tissue.Corneal hydration level of VA-cubosomes was 81.68% ± 0.73 and VA-solution was 79.53% ± 0.29.

Bioadhesion studies
The purpose of the bioadhesion studies was to perceive the nature of developed cubosomes adherence to cornea tissues to resolve the drainage issue contrast to vancomycin solution.To improve the visibility of the formulation, indicator dye was used.It was observed that the intensity of color diminished progressively over 24 hours time period.This indicated the adherence of cubosomal formulation to corneal surface over the entire duration of study.The optimized formulation F8 remained intact in the corneal surface for 24 hours and that could be attributed to the optimum concentration of lipid and surfactant that prolonged the bioadhesion time.

Ocular tolerance test
HET-CAM assay was employed to assess predictive model for in-vitro irritation by perceiving vascular damage, hemorrhage, hyperemia, coagulation and computing irritation-score.Arteries, veins and capillaries are encompassed in CAM that gives identical reaction as in ammation induced in corneal tissue of rabbits during draize-eye irritancy test.With normal saline, 0 mean score was observed [55].Optimized VA-cubosomes was subjected as test and showed 0 mean score up to 12 hours and 0 score was found up to 24 hours.Results denoted that formulation was non-irritating in nature and could be well-tolerated and was safe for application to eyes.It could be accredited to bio-compatible and nonimmunogenic nature of cubosomes.Positive control reported hemorrhage and coagulation followed by lysis that con rmed severe irritant nature with mean score of 3 [56].Figure 5 represents photographs illustrating results of HET-CAM test.

Anti-microbial studies
The anti-microbial e cacy of optimized cubosomal formulation and vancomycin solution was executed against Staphylococcus aureus and Pseudomonas aeruginosa.VA-cubosomes displayed higher growth inhibitory effect as compared to control.The anti-microbial properties exhibited that the diameter of zone of inhibition against S. aureus for VA-cubosomal formulation was 36 mm and against P. aeruginosa was 30 mm which was signi cantly higher than VA-solution.Higher zone of inhibition acquired by synthesized cubosomes indicated that formulation had optimum viscosity that resulted in sustained drug release that maintained minimum inhibitory concentration for extended time-period [57,58].Pictorial presentation of anti-microbial studies is depicted in Fig. 6.

Isotonicity evaluation
Isotonicity test was performed to examine whether the tone of cell would be in uenced by entry of water from the instilled solution as instance of hypo-tonic solution or egress of water as instance of hyper-tonic solution.Hypertonic and hypotonic formulations have a tendency to cause irritation, pain and tissue damage to eyes.Ideally ophthalmic formulations should be isotonic in nature so that they don't cause discomfort, irritation or swelling.Microscopic observations illustrated that blood cells treated with hypertonic solution seemed shrunk whereas hypotonic solution treated blood cells were swollen.
Although, RBCs treated with formulated cubosomes and isotonic solution were unaltered and didn't show lysis or crenation of blood cells.The results implied that VA-cubosomes were isotonic with lachrymal uid and blood and were found to be non-irritating with no ocular damage [59].Microscopic images for isotonicity evaluation have been depicted in Fig. 7.

Sterility Studies
For sterility studies, uid thioglycollate medium was employed to detect aerobic bacteria while soya-bean casein digest was used to detect anaerobic fungi and bacteria.Sterility testing corroborated that there was absence of turbidity and no bacteriostatic and fungistatic activity was perceived after incubation for 14 days.Therefore, the optimized VA-cubosomes were found sterile and free from microorganisms [60].

Drug Release Kinetics Studies
Formulation F8 was optimized on the basis of drug release studies to determine drug release kinetic.Data acquired from in-vitro release pro le was tted into diverse kinetic models.The results manifested that the optimized cubosomal formulation was predominantly elucidated by rst order (R 2 = 0.9831, K = 0.048), followed by higuchi's equation (R 2 = 0.9762, K = 19.045)and zero order (R 2 = 0.8724, K = 3.683).
The corresponding plot for korsmeyer-peppas equation indicated good linearity (R 2 = 0.9817).The release exponent 'n' was found to be 0.6032, that indicated non-ckian or anomalous transport.In non-ckian release diffusion, rate of vancomycin release from cubosomes were governed by more than one process i.e. diffusion controlled and swelling controlled release.

In-vivo studies
The optimized VA-cubosomal formulation and VA-solution were subjected to in-vivo studies and treatments effects were compared.Criteria taken for response to drug therapy were redness, lacrimal Histopathological studies con rmed the results obtained from in-vivo studies.The cross-section of ocular tissues of rabbit were treated with optimized VA-cubosomes, sodium dodecyl sulfate and phosphate buffer saline as illustrated in Fig. 10.Histopathological studies were performed to corroborate the existence of normal corneal tissues by retaining their morphology without causing impairment to tissues.
In positive control, epithelial layer was grievously damaged with absolute separation from bowman's layer at diverse regions of corneal epithelium.Distorted cells were evidently observable with signi cant corneal swelling.Whereas, corneal tissues incubated with normal saline and VA-cubosomes demonstrated no cytotoxicity and intact epithelial layer with no changes in corneal cross-sections.The corneal layers displayed well-maintained morphology without any stromal swelling, consequently indicating safety of formulation [66].

Stability studies
Stability studies were performed over a period of 6 months and formulations were stored at varied temperatures 5 ± 1 0 C, 25 ± 2 0 C, 40 ± 2 0 C, 60 ± 2 0 C, and 75 ± 5% RH to analyze the stability.The optimized cubosomes were observed for entrapment e ciency, particle size distribution, polydispersity index, pH, invitro drug release, precipitation and phase separation.The formulation exhibited satisfactory results and remained stable during 6 months.There were no changes in the physical appearance and no precipitation and phase separation was observed.Slight changes were perceived in the formulation at 60 0 C. Table 7 illustrates slope and degradation constant.

Conclusion
In the present study, novel nanostructured liquid crystalline particles were developed using glyceryl monooleate and poloxamer 407 by fragmentation method.The prospect of cubosomes as carrier for topical administration was established by the results acquired, demonstrating drug penetration.FTIR results revealed that there was no interaction between the drug and excipient of the formulations.TEM con rmed cubic organization with well-dispersed individual particles.HET-CAM assay denoted that the formulation was non-irritating in nature and could be well-tolerated and was safe for application to eyes.Ex-vivo studies revealed signi cantly high corneal permeation of optimized formulation.Whereas, in-vivo studies demonstrated that the intensity of keratitis was reduced after 72 hours and after 120 hours the symptoms were signi cantly relived.Ocular pharmacokinetic studies evaluated drug level in aqueous humor and results revealed that AUC, T max, C max and MRT of cubosomal formulation was considerably higher than vancomycin solution.Histological evaluation revealed that VA loaded cubosomes demonstrated no cytotoxicity and corneal layers displayed intact epithelial layer without any deleterious    Transcorneal permeation pro les of VA-loaded cubosomes and VA-solution using excised rabbit corneas.

FT-IR was
done to analyze estimate any physico-chemical in uence on drug and its excipients and to establish that chosen excipients were compatible with drug.FT-IR spectroscopy of vancomycin HCl, glyceryl monooleate, poloxamer 407 and physical mixture of drug-excipients are depicted in Fig.1.Pure vancomycin manifested its characteristics sharp peak at 3418.51 cm − 1 that was ascribed to N-H stretching, 1505.76 cm − 1 due to C = C stretching, 1383.18cm − 1 indicated OH phenol group, C-O-C stretching at 1230.26 cm − 1 and 1062.47 cm − 1 aliphatic amine for C-N stretching were in accordance with standard vancomycin FT-IR data[38,39].

3. 20 .
Shelf Life Determination (Arrhenius plot) Shelf-life determination was by Arrhenius plot.Value of log K was obtained − 4.2485 by extrapolation of Arrhenius plot.Optimized VA-cubosomes exhibited shelf-life in range of 3.5 years.Consequently, an arbitrary shelf-life of 3 years could be assigned to cubosomal formulation.

Figure 1 FT
Figure 1

Table 1
Experimental control factors and their levels for VA-cubosomes.
[27].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 tted 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), rst 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-e cient was evaluated[27].2.2.4.15In-vivo studiesRabbits 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 in ammation and gross 0C for soyabean-casein digest media and 300C-350C for uid-thioglycollate medium for 14 days.After end of 14 days, the incubated asks were scrutinized for any indication for growth of microorganisms [26].2abnormalities 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].

Table 3
Compiled characterization of Vancomycin loaded Cubosomes.
secretion, mucoid discharge, eye-lid swelling and corneal ulcer.Eyes of rabbits were examined and recorded for ocular damage or abnormal signs to conjunctiva, iris or cornea and systemic side-effects[61].Scores of cubosomal formulation and VA-solution were compared and results indicated that optimized formulation was e cacious in mitigating manifestation of keratitis as depicted in Table4.After 120 hours, the score of the redness in group II was 2.927 ± 0.59 whereas in group III was considerably lower 0.09 ± 0.0.Lacrimal secretion and mucoid discharge in group II was 2.032 ± 0.63 and 1.649 ± 0.19 whereas in group III it was 0.07 ± 0.0 and 0.08 ± 0.0 respectively.Intensity of keratitis was reduced after 72 hours by the optimized cubosomes and after 120 hours the symptoms were signi cantly relived.Images during treatment of infected rabbit eyes at different time intervals are depicted in Fig.8.Rate of frequency of administration was also lowered as compared to solution.Remarkable better results were acquired with vancomycin cubosomes in reducing the severity of keratitis[62].Grading of parameters of keratitis was done and the results of un-paired t-test between 2 groups demonstrated that cubosomal formulation showed signi cantly superior response as shown in Table5.The time to peak concentration (T max ) of cubosomal formulation was about 1.9-fold higher and mean residence time was 2.2`-fold greater than VA-solution[63].The transcorneal penetration of vancomycin might be increased due to interaction between cubosomes and cornea in presence of glyceryl monooleate.Moreover, prolonged mean residence time and T max could be attributed to close adherence of cubosomes to ciliary muscle and conjunctival sac that would provide subsequent absorption and surface-lipid exchange effect[64].That might form drug depot at corneal area and would provide more drug to anterior chamber of eyes.Increased ocular-bioavailability could be ascribed to 3 factors: enhanced drug-loading capacity, permeation capability of drug through corneal membrane and ocular contact-time of cubosomes.These factors corroborated intimate contiguity in-between drug and epithelial mucosa, averting tear-washout and therefore correlated with sustained-release of drug and protracted drug-retention[65].Concentration-time pro les of tobramycin in aqueous humor is graphically represented in Fig.9, whereas pharmacokinetic parameters are depicted in Table6.

Table 7
Slope and Degradation constant of VA-cubosomes.