Journal of Pharmaceutical Sciences xxx (2019) 1-12 Contents lists available at ScienceDirect Journal of Pharmaceutical Sciences journal homepage: www.jpharmsci.org Pharmaceutics, Drug Delivery and Pharmaceutical Technology An Ex Vivo Evaluation of Moxifloxacin Nanostructured Lipid Carrier Enriched In Situ Gel for Transcorneal Permeation on Goat Cornea Shilpkala Gade 1, Krishna Kumar Patel 1, Chandan Gupta 2, Md. Meraj Anjum 1, Deepika Deepika 1, Ashish Kumar Agrawal 1, Sanjay Singh 1, * 1 2 Department of Pharmaceutical Engineering and Technology, IIT (BHU), Varanasi, Uttar Pradesh, India Bombay College of Pharmacy, Kalina, Santacruz, Mumbai, Maharastra, India a r t i c l e i n f o a b s t r a c t Article history: Received 21 November 2018 Revised 4 March 2019 Accepted 2 April 2019 The study was designed to fabricate the moxifloxacin nanostructured lipid carriers (MOX-NLCs) loaded in situ gel for opthalmic application to improve the corneal permeation and retention and also subside the toxic effect associated with intracameral injection of moxifloxacin in endophthalmitis treatment. Initially, Box-Behnken design was used to optimize the various factors significantly affecting the final formulation attributes. MOX-NLCs with particle size 232.1 ± 9.2 nm, polydispersity index 0.247 ± 0.031, zeta potential 16.3 ± 1.6 mV, entrapment efficiency 63.1 ± 2.4%, and spherical shape was achieved. The optimized MOX-NLCs demonstrated the Higuchi release kinetics with highest regression coefficient. Besides this, FTIR, differential scanning calorimetry, and X-ray diffraction results suggested that MOX had excellent compatibility with excipients. Furthermore, the results of ex-vivo permeation study demonstrated 2-fold higher permeation (208.7 ± 17.6 mg), retention (37.26 ± 2.83 mg), and flux (9.57 ± 0.73 mg/ cm2 h) compared with free MOX in situ gel. In addition, MOX-NLCs exhibited normal corneal hydration and did not show any sign of structural damage to the corneal tissue as confirmed by histology. Therefore, the findings strongly suggest that MOX-NLCs in situ gel with higher permeation and retention can be a better alternative strategy to prevent and treat the endophthalmitis infection. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved. Keywords: lipid nanoparticle(s) (LNP) ophthalmic drug delivery drug delivery system(s) nanoparticle(s) factorial design Introduction Endophthalmitis is a purulent infectious inflammatory condition in the intraocular fluid (aqueous and vitreous).1 It mainly arises after intraocular surgeries, particularly cataract surgery and may be owing to hematogenous (i.e., via blood vessels) dissemination from the remote primary source and even may result into loss of vision if left untreated.2 Moxifloxacin (MOX) is a commonly prescribed synthetic fluoroquinolone antibiotic for the prophylaxis after cataract surgery to prevent endophthalmitis or to treat it.3 In the current therapy, it is either given topically or as an intracameral injection in the anterior chamber. Both the administration has its limitation during therapy. Topical administration of MOX solution faces the short retention time in the eye, high lacrimal drainage, and low permeability across Conflict of interest: Authors have no conflict of interest. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.04.005. * Correspondence to: Sanjay Singh (Telephone: þ91-9415290851). E-mail address: ssingh.phe@iitbhu.ac.in (S. Singh). the cornea which collectively contributes to extremely low ocular bioavailability (1%-7%).4 However, corneal endothelial toxicity and toxic anterior segment syndrome is a major concern during the intracameral injection. The existing ocular blood barriers in the eye inflicts the major challenges to the drug delivery, which restrict the access of the drug to the deeper tissues of the eye.5 The ocular blood barriers present in the uvea are namely blood-aqueous barrier (anterior chamber) consisting of the endothelial cells which limits the entrance of hydrophilic drugs into the aqueous humor; blood-retinal barrier made up of retinal pigment epithelium; and the tight wall retinal capillaries which restrict the entry of most of the xenobiotics from blood stream. In addition, human cornea consists of 3 layers including lipophilic epithelium, hydrophilic stroma, and endothelium. Therefore, the drug molecule should possess the higher log p value (2-4)6 for the optimum corneal permeability. All the disease complications (inflammation) along with ocular barriers imposing the major impediment to the diffusion of drug across cornea are foremost cause of drug subtherapeutic level in the aqueous humor.7 To overcome the short washout period, poor retention, and poor penetration of drugs across the cornea, a unique approach with https://doi.org/10.1016/j.xphs.2019.04.005 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved. 2 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 high retention period and permeation across cornea is thereby desired. Currently, nanostructured lipid carrier (NLC) has gained the researchers attention and emerged as a better alternative to the solid lipid nanoparticles, liposome, emulsion, etc.8 Physiologically present biodegradable solid and liquid lipids both are used simultaneously to synthesize the NLCs. It offers superior permeation via passive diffusion through the transcorneal route and retention in addition to the higher drug loading, better stability, excellent biocompatibility, low systemic adverse events, easy scale up, and reduced drug expulsion while storage.9 Additionally, polymeric in situ gels are emerging ophthalmic formulations due to their ease of application, reduced dosing frequency, and increasing patient acceptance. Any modulation in the temperature (T), pH, ions, and ultraviolet irradiation can trigger the gelation in the solution of certain sensitive polymers which offers controlled drug release, higher retention time, and longer washout period. Biodegradable polymers including poly-caprolactone, polyD-Lactic acid, pectin, gellan gum, chitosan, alginic acid, xyloglucan, poly-DL-lactide-co-glycolide, and poly-caprolactone are used to prepare the in situ gel system.10 This study was aimed to develop topical in situ gel containing moxifloxacin loaded nanostructured lipid carrier (MOX-NLCs) for enhancing the corneal permeation, prolonging the washout period, and exhibiting better eye compatibility. Further, MOX-NLCs ex vivo permeation and retention on the freshly excised goat cornea were evaluated. In addition, MOX-NLCsetreated corneas were subjected to histologic studies to find out the probable toxicity of MOX-NLCs on the corneal cells. Optimization of Process Variables Primary Screening of Critical Parameters The fractional factorial (5 factors, 2 levels) design was used for the identification of critical factors affecting the particle size (PS), zeta potential (ZP), and % entrapment efficiency (% EE) of NLC formulation.14 Therefore, solid: liquid lipid ratio (LR), homogenization speed (HS), homogenization time, temperature (T), and surfactant concentration (SC) was evaluated to attain the purpose. Total 16 batches were designed by the software. Using the generated preliminary experimental data, critical factors were identified and selected for further optimization of final NLCs. Optimization by Box-Behnken Response Surface Design The Box-Behnken design with 3-factor 3-levels was selected to create the experimental batches. Meanwhile, the impact of independent variables including HS, SC, and LR was also studied on PS, ZP, and %EE of NLC using Box-Behnken response surface analysis. Total 15 runs with combination of different levels of process and formulation factor were generated by the software. Physicochemical Characterization of MOX-NLCs The descriptive methodology used to perform the PS, polydispersity index (PDI), and ZP analysis.15 Encapsulation efficiency of NLC,13 scanning electron microscopy (SEM),16 atomic force microscopy (AFM),16 differential scanning calorimetry (DSC),17 X-ray diffraction study (XRD)17 are included in the Supplementary Material. Materials and Methods Materials All materials used in the study are listed in the Supplementary Material. Selection of Lipids The solid and liquid lipids were preferred based on the relative solubility of MOX in the different lipid and the relative miscibility of solid and liquid lipid with each other. The separate methodology described in the previous study11,12 was employed to determine the solubility of MOX in different lipids. In Vitro Release Study of NLC In vitro drug release of MOX-NLC was performed in pH 7.4 simulated tear fluid using dialysis bag diffusion method. Overnight soaked dialysis membrane (cut-off molecular weight 12,00014,000 Da) was filled with 5 mL of MOX-NLC and immersed in 100 mL dissolution medium stirring with 100 rpm on magnetic stirrer and maintained at 37 ± 0.5 C. Meanwhile, 2 mL of sample pipette out at the defined interval up to 24 h and replaced with equal volume of fresh simulated tear fluid. Finally, the samples were analyzed in UV spectrophotometer at 292 nm to determine the concentration. Antimicrobial Activity Preparation of Moxifloxacin Loaded Nanostructured Lipid Carriers MOX-NLC was synthesized using hot homogenization ultrasonication method.13 Briefly, 10 mg MOX dissolved in 2 mL chloroform was dispersed in the melt of glyceryl monostearate (GMS) and Capmul MCM mixture (ratios varying from 60:40 to 80:20) maintained at 72 C, 10 C above the lipid’s melting point. Sequentially, the above melt mixture was disseminated in aqueous solution (20 mL) of poloxamer-188 using high speed homogenizer (Ultra Turrax; IKA, Staufen, Germany) for 15 min. Eventually, the resulting primary emulsion was cooled and ultrasonicated (Dr. Hielscher, Buchholz in der Nordheide, Germany) for 3 min at 60% amplitude with the frequency of 0.5 s to obtain the desired MOX-NLCs formulation and then cooled at room temperature. Furthermore, the resulting nanosuspension was centrifuged (20,000 rpm for 30 min) to remove free drug present in the supernatant, and the MOX-NLCs pallets were redispersed in the distilled water to produce final concentration of 2 mg/mL equivalent to MOX. Broth dilution technique was employed to detect the minimum inhibitory concentration (MIC) in 96-well plate against Staphylococcus aureus strain. MOX solution and MOX-NLCs (equivalent to 32 mg MOX concentrations) was sequentially diluted in the 96-well plate preoccupied with 100 mL LB medium and inoculated with 100 mL of 0.5  10 6 CFU S. aureus to obtain the 8, 4, 2, 0.5, 0.25, 0.125, 0.0625, 0.0312, and 0.0156 mg/mL final MOX concentration. After 24 h incubation at 37 C, the absorbance at 570 nm was measured using microplate reader (SYNERGY/HTX; BioTek Instruments, Inc., Winooski, VT) to determine the MIC. Furthermore, agar diffusion technique was carried out to validate the result of broth dilution method and antimicrobial efficacy of MOX-NLC. In this method, formulation (equivalent to MIC) was allowed to diffuse through solidified agar medium inoculated with S. aureus suspension for 24 h at 37 C. After 24 h, the zone of inhibition was calculated by means of Vernier caliper and compared with control. The whole protocol was followed according to previous study.18 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Stability Studies The optimized MOX-NLCs were studied for it stability up to 90 days at 30 ± 2 C/65% ± 5% RH. The stored samples were analyzed at regular intervals for any variation in PS, PDI, ZP, and %EE and compared with initial data. Furthermore, one-way ANOVA (Dunnett post test; compared all time point with initial data i.e., 0 days) was performed for each parameter individually to determine the significance difference. Preparation of MOX-NLC Loaded In Situ Gel Sodium alginate (1% w/v) and hydroxypropyl methylcellulose (0.5% w/v) were dissolved in the water on magnetic stirrer. Subsequently, the final MOX-NLCs suspension was dispersed in the above polymeric solution to produce the MOX-NLCs in situ gel suspension (equivalent to 2 mg/mL of MOX).19 Finally, the pH was adjusted to 6.4 with the help of 0.1N NaOH/0.1N HCl after addition of the buffering and osmolality agents. Characterization of MOX-NLC Loaded In Situ Gel Clarity and Gelling Capacity The prepared formulation was visually checked for clarity,19 and then gelling capacity of formulation was measured in terms of gelling time by instilling the 50 mL of the in situ gel in a vial having 2 mL of simulated tear fluid and observed visually.20 Measurement of Viscosity of In Situ Gel Brookfield viscometer (AMETEK Brookfield, Middleborough, MA) with spindle No 63 was used to determine the viscosity of in situ gels.21 Viscosities of in situ gel solutions were measured before and after gelling at varying angular velocities (50 rpm & 100 rpm) maintained at 37 ± 1 C. The objective of varying the spindle speed was to determine the flow behavior of gel. Particle Size and PDI Analysis of Gel PS and PDI of gel were determined by photon correlation spectroscopy using Delsa Nano C, PS analyser after 10 times dilution of in situ gel. Texture Profile Analysis Texture profile analysis was performed using a TA-XT2® Texture Analyzer (Stable Micro Systems, Surrey, UK) to find out the mechanical property of gel.21 In texture profile analysis mode, a back extrusion 35 mm probe was compressed twice with 0.1 g trigger force into the samples at the rate of 1 mm s 1 to a depth of 30 mm allowing delay period of 5 s between 2 compression cycles. Subsequently, from the obtained resultant force-time plots, the values of the adhesiveness, cohesiveness, and hardness were determined. Collection and Processing of Goat Eye Freshly excised goat eyes procured from the butcher’s house were immediately stored in Krebs ringer solution. Further, the eyes were dissected to obtain the corneal tissue pieces (3 cm2) and were stored in Krebs ringer solution under 80 C to keep the eye preserved.22 In Vitro Release of In Situ Gel In vitro release of in situ gel was studied on Franz diffusion cell using the overnight soaked (in pH 7.4 simulated tear fluid) cellulosic dialysis membrane (M.wt 12,000-14,000 Da.). The pH 7.4 simulated tear fluid was used as the release medium in receptor 3 compartment. The whole release apparatus was maintained at 37 ± 1 C with continuous stirring on hot plate magnetic stirrer. The 1 mL aliquot was removed on specified time interval and replaced with fresh medium. Eventually, the withdrawn sample was diluted suitably and analyzed with UV spectrophotometer at 292 nm to determine the released drug concentration. Ex Vivo Corneal Permeation and Retention Study Ex Vivo Transcorneal permeation studies were carried out on Franz diffusion cell (internal area 1.77 cm2) using the freshly procured goat cornea.23 The Central Animal Ethical Committee, Faculty of Medicine, Institute of Medical Sciences, Banaras Hindu University approved the given protocol for ex vivo corneal permeation study. The excised cornea clipped in between receptor and donor compartment of a Franz diffusion cell in such a way that inner epithelial surface was opposite to the receptor compartment. Here, the receptor compartment was occupied with the simulated tear fluid (pH 7.4) as release medium. MOXNLC loaded in situ gel/MOX loaded in situ gel equivalent to 1 mg MOX was instilled in the donor cell over cornea. The release medium in receptor compartment was maintained at 37 C and stirred constantly using a magnetic stirrer. Predefined aliquot was pipetted out during the study from the receptor compartment at definite time interval up to 12 h and analyzed for MOX content at 292 nm in a UV/Visible spectrophotometer after appropriate dilutions with tear fluid. Furthermore, the treated corneal tissue was washed thrice using the phosphate buffer saline to remove loosely adhered formulation and free drug. Recovered tissue was homogenized using homogenizer (IKA) in phosphate buffer saline to recover the MOX retained in the corneal tissue in each sample. Finally, the homogenized corneal tissues were centrifuged at 5000 rpm at 4 C for 10 min. The supernatant was collected and analyzed spectrophotometrically at 292 nm after proper dilution to determine the amount retained in the tissue after permeation study. The permeation flux across the cornea was calculated using the formula: Permeation Flux ¼ Slope of the amount permeated vs time graph Internal Area of Diffusion cell Hydration of Cornea The cornea without sclera tissue was treated with MOX, placebo, and MOX-NLCs in situ gel for 12 h to determine the corneal hydration. The cornea treated with 0.9% w/v saline was used as control. Each treated cornea was weighed, saturated with methanol for 5 min, and stored overnight at 80 C for complete drying. After drying, the cornea was reweighed, and % hydration was calculated.24 Histopathologic Study Corneal tissue was treated with different formulation group including MOX, placebo NLCs, MOX-NLC in situ gel, and 0.9% w/v saline (taken as control) for 12 h. The treated corneal tissue was cut in to 40 mm transverse section using Leica cryotome (Leica, Wetzlar, Germany) to prepare glass smears, and the transverse sections were dehydrated using graded solutions of ethanol. Finally, the transverse sections of corneal tissues were stained with hematoxylin and eosin dye, and the stained sections were observed under inverted microscope at the magnification 40 and analyzed.25 4 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Result and Discussion Effect on Particle Size Lipid Screening As shown in Table 1, the PS of all 15 batches varied from 180.5 ± 10.1 to 387.8 ± 16.5 nm. Based on the nonsignificant lack of fit test (F value 0.47 and p value 0.732) and lowest PRESS value 3155.78, quadratic model was preferred to explain the effect. The following polynomial equation was generated from ANOVA for evaluating the factor's effect on PS using Design expert 7.0 software. The miscibility of 2 different lipids and the drug solubility in the lipids affect the characteristic like entrapment efficiency, loading capacity, and stability of NLCs12 which are critical prerequisite for pharmaceutical applications. Out of 4 liquid lipid, MOX had maximum solubility in Capmul (1.5 mg/mL) compared with capric glyceride (0.6 mg/mL), capric triglyceride (0.68 mg/ mL), and Hariol (0.49 mg/mL). Similarly, glyceryl monostearate melt maximally dissolved the MOX (1.3 mg/mL) than stearic acid and behenic acid, etc. Therefore, based on the results of preliminary solubility data of MOX in different lipids, glyceryl monostearate (solid lipid) and Capmul (liquid lipid) were selected for NLCs formulation. Furthermore, the solubility of MOX in lipid mixture (Capmul and GMS) was enhanced slightly to 1.8 mg/mL. Meanwhile, the selected lipid produced the homogenous mixture (ratio 80:20, 70:30 and 60:40) without separation compared with other combinations. Separation of oil droplets was tested by filter paper test method. Optimization of MOX-NLCs Total 15 experimental batches depicted in Table 1 were designed using Box-Behnken design with 3-factors 3-levels to obtain the optimized level of independent variables for preparing the MOX-NLCs with desired responses. At the same time, the best suitable model for illustrating the impact of various variables on formulation parameter was determined on the basis of nonsignificant lack of fit model and lowest predicted residual error sum of squares value (PRESS value) for different models. Moreover, based on the model suggested, a polynomial equation correlating the effect of various formulation factors on responses was generated by ANOVA test. The positive and negative coefficient value for particular variables in this equation describes the direct and indirect effect on responses. Interestingly, the statistical analysis of data in hands suggested that quadratic model was best model for describing the effect of variables on PS and % EE (Table 2). ANOVA of responses and factors for quadratic model produced the equation in terms of A, B, C, AB, AC, BC, A2, B2, C2, where, A, B and C represents HS, surfactant concentration and LR, respectively. PS ¼ þ222:97 49:03*A 23:05*B þ 27:9*C þ 12:75*A*B 1:40*A*C þ 6:30*BC þ 60:24*A2 þ 20:99*B2 0:56*C2 (1) It is clear from the Equation 1 and Figures 1a-1c that HS and SC had inverse relation ( ve coefficient), while solid to liquid LR had direct relation (þve coefficient) with the PS. The equation has larger magnitude for A (HS) 49.03 and B (SC) 23.05, whereas the A2 and B2 is þ60.24 and 20.99, respectively. Both values are larger compared with the baseline factor (223); therefore, the HS and SC significantly affected the PS. At the same time, the effect of HS and SC was increased more than proportionally as the values of HS and SC was increased. On the other hand, the C (LR) magnitude þ27.9 representing that PS will increase on increasing the LR, but the value of C2 is 0.56, so will have negligible effect on the PS. However, Figures 1a and 1b clearly show that on further increasing the HS up to maximum, PS increased perhaps due to droplet recoalescence or Ostwald ripening. Besides that, surfactant leads to decreased interfacial tension and according to Laplaces pressure theorem, as the interfacial tension decrease the disruption of droplet into fine particles increase. Effect on Entrapment Efficiency The quadratic model was the best model to precisely demonstrate the effect of variables on the %EE. The quadratic model with lowest 344.5 PRESS value and nonsignificant lack fit test (10.11, F value and 0.09, p value) generated the following polynomial equation based on the ANOVA test for responses. EE ¼ þ69:23 1:00*A þ 5:35*B þ 9:88*C þ 0:55*AB 0:40*AC þ 2:20*B*C 4:47*A2 10:57*B2 2:32*C2 (2) Table 1 Summary Table of Box-Behnken Design Exp. Run Actual Values Batch HS SC LR PS F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 15,000.00 10,000.00 15,000.00 20,000.00 20,000.00 15,000.00 15,000.00 15,000.00 10,000.00 10,000.00 15,000.00 20,000.00 15,000.00 10,000.00 20,000.00 0.50 1.00 1.50 1.00 1.00 1.00 1.50 1.00 1.00 1.50 1.00 1.50 0.50 0.50 0.50 80:20 80:20 80:20 80:20 60:40 70:30 60:40 70:30 60:40 70:30 70:30 70:30 60:40 70:30 70:30 293.7 356.7 257.8 255.8 211.4 233.4 180.5 211.9 306.7 318.6 223.6 246.1 241.6 387.8 264.3 Mean ± SD, n ¼ 3. Dependent Variables ZP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 16.7 21.9 14.2 10.7 11.2 9.8 10.1 17.3 18.6 17.4 13.2 14.1 9.8 16.5 10.3 13.8 16.8 21.4 17.8 18.1 16.4 22.6 17.9 15.9 20.4 16.7 22.4 14.5 12.8 13.5 EE ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.2 1.4 2.1 1.7 1.3 1.9 2.5 1.4 2.1 2.3 1.8 2.7 1.7 1.3 1.5 56.7 75.3 73.6 71.5 50.4 68.5 51.6 70.1 52.6 58.6 69.1 58.7 43.5 50.8 48.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.2 4.9 5.7 3.1 4.1 5.3 3.7 4.8 3.6 4.2 4.2 4.3 2.9 3.3 4.1 5 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Table 2 Precisely Collected Data of Lack of Fit Test and Model Summary Statistics for Suggested Quadratic Model Responses PS EE Lack of Fit Test Sum of Square Degree of Freedom Mean Square F Values p Values Remarks 164.65 21.34 3 3 54.88 7.11 0.47 10.11 0.7323 0.0913 Suggested Suggested Std. Dev r2 Adjusted r2 Predicted r2 PRESS 8.90 2.13 0.9913 0.9813 0.9756 0.9475 0.9306 0.7161 3155.78 344.52 Model Summary Statistic PS EE The values of entrapment efficiency obtained in the range of 43.5 ± 2.9 75.3 ± 4.9% are depicted in Table 1. From the Equation 2, it was deduced that the entire factors cumulatively contributed the similar extent of effect on %EE, the qualitative effect was different. The HS with magnitude 1 (A) and 4.47 (A2) had inverse relation and decreased the %EE on increasing the HS (from 10,000 to 20,000). Conversely, SC with þ5.35 (B), 10.57 (B2), and LR with þ9.88 and 2.32 (C2) shown in Equation 2 had direct relation Suggested Suggested with %EE. The data in hands and Figures 1d-1f concluded that EE increased with increase in the fraction of liquid lipid (from 80:20 to 60:40) and SC apparently due to high solubility of MOX in lipids and more voids produced in the NLCs due to different polymorphic structure of the 2 different lipids that can accumulate more drug and the presence of sufficient SC to cover the newly formed particles that prevent the drug expulsion and ultimately form the stable nanoparticles. Figure 1. 3D plot elaborating the effect of various factors on the PS (a, b, c) and %EE (d, e, f) of MOX-NLCs. 6 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Process and Variable Control SEM Analysis Various diagnostic tools were analyzed to determine the process control including plot between the predicted versus actual response values, residual versus predicted values, and residual versus run values and many more. Figure 2 elucidated that all the process and formulation variables were well controlled as all the values for PS and %EE falls within the normal range. The shape and surface characteristics of the optimized batch of MOX-NLC were evaluated by scanning electron microscopic analysis (Fig. 4a). The SEM micrograph revealed the spherical shape and uniform distribution of particles, which was also evident from the narrow PDI obtained in the photon correlation spectroscopic analysis. Selection of Optimized Formulation Atomic Force Microscopy HS and SC, having highest magnitude in polynomial, equation were the critical parameters to optimize the PS, while LR and SC proved to be the important factor to obtain the maximum EE. Based on the extent of variable’s effect on PS and EE, the constraint was applied to factors level to achieve the set of optimized or predicted factors level, PS and %EE (Table 3). Optimized MOX-NLCs with predefined response were derived from numerical optimization technique using the desirability approach (desirability plot with predicted level and responses; Fig. 3). The values of statistically predicted optimized levels of different variables along with predicted responses are provided in Table 3. Further, keeping the optimized factor's level in mind the optimized MOX-NLCs batch was prepared and found that prepared MOX-NLCs had almost similar PS 232.1 ± 9.2 nm and % EE 63.1 ± 2.4 as predicted. Besides that, it had 0.247 ± 0.03 PDI and 16.3 ± 1.6 mV ZP. Though MOXNLC had low ZP; however, poloxamer also provides the steric stabilization to prevent the agglomeration.26 Atomic force microscopy (Fig. 4b) of NLCs was performed to take 2-dimensional and three-dimensional views and also to measure average height, roughness, diameter, and skewness of MOXincorporated NLCs. Average height of NLCs was found to be 149 ± 10.2 nm which was equivalent to the diameter obtained in the SEM. Average height of NLCs did not vary significantly than SEM data, indicating that particles were spherical in shape. In Vitro Release Study of NLC In vitro release studies performed in dialysis bag are shown in the Figure 4c. Unlike the 97.5 ± 8.7% immediate release of drug from pure MOX within 8 h, release from MOX-NLCs was slow where 36.3 ± 3.2% MOX released within 6 h followed by up to 82.3 ± 7.8% sustained release in 24 h. The initial fast release could be attributed to the surface-adsorbed MOX on the nanoparticles; however, the slow diffusion of MOX from the lipid matrix of NLCs resulted in Figure 2. Process diagnostic plots between predicted versus actual (a and d); residual versus predicted (b and e); and residual versus run responses (c and f) showing that responses for PS and EE were within the range with controlled process variables. 7 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Table 3 Summary of Predicted and Experimental Values of PS and EE at Model Optimized Levels of Process Variables Batches Predicted Experimental Process and Formulation Variables Responses HS (rpm) SC (%w/v) LR PS (nm) EE (%) PDI ZP (mV) 13,565 13,600 1.08 1.10 2.76 2.75 238.4 232.1 ± 9.2 64.2 63.1 ± 2.4 e 0.247 ± 0.03 e 16.3 ± 1.6 sustained release of MOX.27 Further, the kinetic study of release data revealed that the drug release followed the diffusion mechanism as the Higuchi kinetics had best regression coefficients (r2, 0.911) indicating that MOX release from MOX-NLC was directly proportional to square root of time.28 Similarly, the “n” value was below 0.5 in Peppas model supporting the Fickian diffusion of MOX from NLCs. lyophilized MOX-NLC thermogram perhaps due to the molecularly dissolved state of MOX or may be due to presence of amorphous state of MOX in the NLCs. Interestingly, the molecular dissolved state in NLCs might be the outcome of somewhat higher solubility of MOX in the different lipids. Hence, the findings of DSC study deduced that MOX was either present in the molecularly dissolved or amorphous form in the lipid matrix. FTIR Study XRD Study As shown in Figure 5a, FTIR spectra of MOX had the prominent specific band at 1760 cm 1 corresponding to C¼O stretching, 1320 cm 1 due to C-N stretching, and 1622 cm 1, 1518 cm 1, and 1451 cm 1 corresponding to aromatic C¼C stretching. Moreover, an aromatic C-H bending for substituted benzene was found at 1875 cm 1. The MOX also contains spectral peak for most reactive primary amine group at 3539.35 and 3459.6 cm 1. Furthermore, all these peaks were also present intact in the physical mixture (MOX, GMS, Capmul, and poloxamer-188) and MOX-NLCs demonstrating the excellent physicochemical compatibility of drug and excipients and no sign of interaction. The reduced intensity of scattered pattern shown in Figure 5c obtained in XRD findings also support the fact that crystallinity of MOX was diminished in the MOX-NLCs. The MOX had many characteristic intense diffraction (2q) peaks at 10.26 , 14.65 , 17.15 , 17.57, 20.53 , 23.769 , 24.238 , 27.628 , 29.308 , 34.28 , 38.795 , 43.32 , and 49.43 . Moreover, physical mixture of MOX and excipient demonstrated the majority of peaks with reduced intensity, but at the same time, most of the diffraction peak diffraction pattern was disappeared in the MOX-NLCs diffraction pattern suggesting the presence of homogeneously dispersed molecular form of MOX in NLC’s lipid matrix. The relative reduction of diffraction intensity may also be either due to reduced crystal structure or change in the crystal orientation.30 DSC Study DSC data elucidate the degree of crystallinity based on the melting enthalpy of different samples. Similarly, the shift in the drugs melting point gives the idea about physicochemical compatibility among the drug and the other component of formulation.29 It is evident from the Figure 5b that the DSC thermogram of MOX had endothermic peak at 239.9 C corresponding to the melting temperature, whereas the same peak was absent in Stability Studies The statistical analysis (one-way ANOVA) of stability data obtained at different time period for MOX-NLCs archived in Table 4 demonstrated insignificant variation in the PS, PDI, and EE after 90 days incubation. Furthermore, the ZP of the MOX-NLCs was sufficient to stabilize the formulation owing to poloxamer-188 Figure 3. Desirability plot of predicted values of PS (a) and EE (b) based on the optimized processing and formulation factors. 8 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Figure 5. Drug excipient compatibility study. (a) FTIR spectra; (b) DSC thermogram; and (c) XRD diffraction pattern of MOX-NLCs and different other samples. Figure 4. Characterization of optimized MOX-NLCs. (a) HR-SEM image, (b) AFM image, and (c) In vitro release profile of MOX-NLCs. steric stabilizing property which can easily compensate the missed electrostatic repulsion.26 when studied on agar plate swabbed with S. aureus bacteria. This may be due to higher penetration, sustained release, and enhanced uptake of MOX-NLCs by the microorganism. The findings of antimicrobial study indicated that MOX-NLCs possessed the enhanced antimicrobial potential. Antimicrobial Efficacy Test Characterization of MOX-NLC Loaded Gel The results of broth dilution methods revealed that MOX and MOX-NLCs has same MIC of 0.125 mg/mL. Though, they had similar MIC against S. aureus, Figures 6a and 6b revealed that MOX-NLCs exhibited higher zone of inhibition compared with MOX alone Clarity The in situ gel was visually observed for clarity against black & white background and found to be clear. Clarity is a quality control test to reduce number of the large particles in the formulation S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 9 responsible for cross-linking of alginate polymer and thus gel formation. Table 4 Stability Data of MOX-NLCs Parameters 0 Days 30 Days 60 Days 90 Days Particle size (nm) PDI EE (%) 232.1 ± 9.2 237.3 ± 12.5a 247.6 ± 14.8a 258.6 ± 10.8a 0.247 ± 0.03 63.1 ± 2.4 0.261 ± 0.03b 61.7 ± 2.5c 0.275 ± 0.04b 60.4 ± 3.1c 0.298 ± 0.06b 59.6 ± 3.2c Mean ± SD; n ¼ 3. The one-way ANOVA was performed to determine the significance difference. a Represents nonsignificant difference of PS at different time point versus 0 d. b Represents nonsignificant difference of PDI at different time point versus 0 d; similarly. c Represents nonsignificant difference of EE at different time point versus 0 d. which may cause irritation and tear flow and hence the loss of drug from ocular surface. Gelling Capacity The in situ gel formed gel matrix within fraction of seconds in presence of tear fluid due to replacement of monovalent Naþ of sodium alginate with Caþ2 ions present in tear fluid, which is Measurement of Viscosity of In Situ Gel The findings of rheological studies are shown in Table 5. Besides increase in the viscosity of MOX-NLCs of in situ gel from 246.4 ± 9.67 to 461 ± 8.78 cps on gelling with tear fluid, the viscosity of control gel was also enhanced on addition of MOXNLCs in gel before as well as after the gelling. Moreover, the viscosity of in situ gel decreased with respect to increasing spindle speed from 461 ± 8.78 cps (50 rpm) to 291 ± 9.72 cps (100 rpm). These findings inferred that in situ gel had shear thinning property (Pseudoplastic; non-Newtonian flow); conversely, the MOX-NLCs supplemented in situ gel (Sol form) showed Newtonian flow. PS and PDI of In Situ Gel The PS distribution of gel system was investigated to determine the probable interaction of MOX-NLCs and gel components in terms of particle aggregation. The PS of MOX-NLC after incorporation in gel was 234.6 ± 8.7 nm with 0.301.0 ± 0.04 PDI whereas, PS of NLC Figure 6. S. aureus zone inhibition on agar plate by pure MOX (a) and MOX-MLCs (b); whereas, (c, d, e, and f) represents the histologic images of untreated cornea; placebo gel treated MOX free NLC in situ gel, and MOX-NLCs in situ gel treated cornea, respectively. 10 S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Table 5 Rheological and Mechanical Properties of Control Gel and MOX-NLCs In Situ Gel Gel Properties Control (sol) Control (gel) MOX-NLCs In Situ Gel (sol) MOX-NLCs In Situ Gel (gel) Viscosity (cps) Cohesiveness Adhesiveness (N-mm) Hardness (N) 135.4 ± 9.89 0.82 ± 0.09 14.22 ± 1.12 3.97 ± 0.44 398.6 ± 7.90 0.93 ± 0.12 27.58 ± 2.26 7.33 ± 0.53 246.4 ± 9.67 0.78 ± 0.08 14.39 ± 1.45 3.66 ± 0.46 461 ± 8.78 0.83 ± 0.07 28.12 ± 1.92 7.56 ± 0.69 (Mean ± SD; n ¼ 3). suspension was 232.1 ± 9.2 nm with 0.247 ± 0.03 PDI. Therefore, attained data supported the fact that incorporation of MOX-NLCs in the gel did not affect the nanoparticles attribute adversely and had shown stable nanoparticle. Texture Profile Analysis The mechanical properties of control in situ gel and MOX-NLC loaded gel before and after gelling are depicted in the Table 5. The results showed significant change in all the properties of MOXNLC loaded in situ gel before and after gelling. The cohesiveness was increased from 0.78 ± 0.08 g to 0.83 ± 0.07 for MOX-NLCs in situ gel; however, adhesiveness and hardness were increased from 14.39 ± 1.45 to 28.12 ± 1.92 N-mm and from 3.66 ± 0.46 to 7.56 ± 0.69 N, respectively. Moreover, the results of mechanical property depicted in Table 5 clearly indicated that addition of MOX-NLCs to in situ gel did not have significant effect on the hardness, cohesiveness, and adhesiveness compared with the control in situ gel. Interestingly, interaction of Ca2þ ions with in situ gel had favorable effect on concerned mechanical properties including cohesiveness, adhesiveness, and hardness. Where, higher values of adhesiveness and hardness were due to increased viscosity. The higher values for adhesiveness favor the higher adhesion to the surface and thus prolong the retention time on corneal surface. However, the hardness had moderately high value which favors the easy removal from the tissue surface. Unlike the hardness and adhesiveness, lower cohesiveness is preferred which renders less irritancy and easy spreadability on ocular surface.26 In vitro Release of In Situ Gel The in vitro release profile of MOX and MOX-NLC in situ gel is shown in Figure 7a. In vitro release of in situ gel of MOX-NLCs was slow but had similar pattern to the in vitro release of NLCs. MOX in situ gel almost completely released (96.5 ± 6.23%) the drug in 12 h. On the contrary, MOX-NLCs in situ gel reflected the slow steady release and released only 68.83 ± 5.61% of MOX in 24 h. The higher viscosity of in situ gel imposed the slow diffusion of MOX within the polymeric matrix and slowed down the release from in situ gel, and thereby the release of MOX-NLC from in situ gel was slow compared to the in vitro release of MOX-NLCs. Figure 7. In vitro release and ex vivo permeation data of MOX-NLCs in situ gel. (a) In vitro release pattern; (b) ex vivo permeation profile; (c) cumulative amount of drug release from MOX-NLC loaded gel; (d) flux of MOX-NLCs across the goat cornea. S. Gade et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-12 Ex Vivo Permeation Study Comparative ex vivo permeation was determined to evaluate the permeability potential of MOX-NLCs supplemented in situ gel on the goat cornea. The complete permeation profile in Figure 7b indicated the total amount of MOX permeated across the cornea at each time point. The MOX-NLCs in situ gel exhibited 2 times more permeation across the freshly excised goat cornea 20.87 ± 2.53% in 12 h than pure MOX in situ gel which showed only 10.48 ± 1.19% permeation. Similarly, the total fraction of drug permeated and the flux of MOX-NLCs in situ gel across cornea was 208.70 ± 17.65 mg and 9.57 ± 0.73 mg/cm2 h, respectively (Figs. 7c and 7d), which was significantly higher than MOX in situ gel. At the same time, the amount of MOX retained in the corneal tissue was 21.78 ± 2.34 mg for MOX in situ gel & 37.26 ± 2.83 mg for MOX-NLCs in situ gel (Fig. 7c). The increased permeation and retention of MOX-NLCs across the cornea was perhaps as a result of the depot formation in close proximity of cornea which released the drug in sustained fashion and also due to enhanced endocytosis of NLCs by corneal epithelium. Corneal Hydration Higher corneal hydration is a marker of disruption or toxicity to the corneal tissue and therefore needed to be evaluated. The normal corneal hydration level lies in between 75% to 80%, and significant variation in this level signifies the alteration in the tissue structure. Interestingly, the corneal hydration of the MOX-NLCs and MOX in situ gel were 79.68 ± 3.7% and 77.13 ± 4.1%, respectively, which was reversible. The hydration within the normal range proved that the MOX-NLCs did not have any toxic effect on the cornea. Histopathologic Study Histopathologic study of eye was conducted by exposing eye to the formulation, and no inflammation was observed in the image of inverted microscope. The images 6C, 6D, 6E, and 6F represent the untreated, placebo in situ gel treated, MOX free NLCs in situ gel treated, and MOX-NLCs in situ gel treated cornea. Conclusion MOX-NLCs were efficiently prepared using hot homogenization technique and successfully incorporated in in situ gel. The MOXNLCs showed improved antibacterial activity in agar plate zone inhibition method. The incorporation of MOX-NLCs in in situ gel improved the viscosity and mechanical properties essential for the enhanced retention and permeation of MOX. Furthermore, the results of ex vivo permeation study supported the hypothesis where MOX-NLC in situ gel showed significantly higher permeation flux, cumulative permeated, and retained amount of MOX compared with pure MOX in situ gel. Meanwhile, the results of % hydration and histopathologic study on cornea suggested that formulation had not altered the normal corneal structure or had no corneal toxicity. Thus, the data in hands collectively deduced the conclusion that the prepared formulation is a better alternative to the current endophthalmitis antibiotic therapy. Acknowledgment The financial support to carry out the research was provided by Indian Institute of Technology, (BHU), Varanasi, India, as the Research Support Grant. Author also acknowledges the CIFC, IIT 11 BHU, Varanasi for carrying out the XRD, SEM, and AFM study. Further, author acknowledges the Bharati Vidyapeeth College of pharmacy, Navi Mumbai for carrying out the DSC analysis. The author also indebted to MHRD, Govt. of India for providing monthly fellowship to Ms. ShilpkalaGade. References enz MC, Arias-Puente A, Fresnadillo-Martinez MaJ, Carrasco-Font C. 1. 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