Abstract: Background and Objective: Biological barriers of eye and typical eye physiology lead to reduced ocular bioavailability of topically administered drugs and for hydrophilic drugs, it is very low. Colloidal drug carriers such as; Solid Lipid Nanoparticles (SLN) increase ocular bioavailability. The motive of present research is to formulate hydrophilic drug (Moxifloxacin) loaded solid lipid nanoparticles demonstrating sustained drug release, possessing efficacy against bacterial keratitis and safe for use on cornea. Materials and Methods: Effect of solid lipid, lipophilic surfactant and hydrophilic surfactant on Entrapment efficiency was studied using 33 Box-Behnken designs. Optimized SLN formulation was evaluated for in vitro characterizations such as particle size, zeta potential and morphological examination by TEM, DSC, in vitro drug release and in vitro antimicrobial efficacy. Ex vivo efficacy and ex vivo ocular irritation study were carried out. Stability studies were also performed for SLN. Results: Lipophilic surfactant (Soya Phosphatidylcholine) and hydrophilic surfactant (Stearoyl polyoxyl-32 glycerides) played an important role to entrap maximum about 57% moxifloxacin. In vitro release study exhibited sustained drug release following Korsmeyer-Peppas model. Ex vivo efficacy study in caprine keratitis model showed significant reduction in bacterial load, 4-logs CFU mL–1 in infected corneas. Histopathology of corneas confirmed reduced bacterial load and showed recovery in corneal epithelium. HET-CAM demonstrated that developed formulation is non-irritant and safe for the use onto cornea. Conclusion: Optimized moxifloxacin loaded solid lipid nanoparticles sustain the drug release, possess anti-bacterial efficacy and is tolerant to eye, so solid lipid nanoparticles can be a good carrier to enhance ocular bioavailability for hydrophilic drugs.
INTRODUCTION
For ophthalmic delivery, topically administered drug is diluted on cornea and a portion is absorbed systemically. In addition to this, typical eye physiology like continuous aqueous humor generation and drainage, lacrimation, blinking, nasolacrimal drainage system and the limited capacity of the cul-de-sac leads to loss of drug. Therefore, only ~10% concentration of recommended dose reaches to the target site in eye1-6. Water-soluble drugs are even more rapidly drained from the site of action after topical delivery7-9. Colloidal drug carriers play a pivotal role to increase ocular bioavailability of topical delivered drug over conventional eye drops by sustaining the drug release and enhancing ocular residence time11-13.
Chronological development of colloidal drug carriers is carried out by researchers to overcome the stumbling blocks of earlier developed drug delivery system14. Emulsions were developed initially for loading lipophilic drugs, but it suffered from serious drawbacks such as; drug partitioning from oil phase to aqueous phase, inability to achieve sustained drug release and physical instability of formulations15. Liposomes were developed to overcome these limitations, but they also had shortcomings like low drug loading, low physical stability and drug expulsion16. Then after developed polymeric nanoparticles were biocompatible and biodegradable offering many advantages over emulsions and liposomes, but still had pitfalls such as localized foreign body reaction due to presence PLGA like polymers, high production cost and difficult industrial scale-up17,18.
Solid Lipid Nanoparticles (SLN) were developed containing lipid matrices which combine the advantages of polymeric nanoparticles, liposomes and emulsions as well as overcome the shortcomings of them19,20. SLN are new generation sub-micron sized lipid emulsions where liquid lipid is substituted by solid lipid. Solid lipids are in solid state at ambient temperature and are melted at higher temperature at the time of formulating the nanoparticles21. SLN offer enormous advantages over other novel drug delivery systems such as controlled nano size, larger surface area, high drug loading and superior physical stability22,23. SLN are non-toxic as they are prepared from physiological and biocompatible lipids. They are commercially preferred because of availability of regulatory approved lipids and easily scalable methods with low production cost. SLN possess ability to entrap lipophilic and hydrophilic drugs.
To put in a nutshell, solid lipid nanoparticles are effective ocular drug delivery system because they improve corneal penetration, corneal absorption, enhance ocular bioavailability, prolong ocular residence time and provide controlled drug release profile24-27. Traditionally lipophilic drugs are successfully entrapped in solid lipid nanoparticles for ocular drug delivery system like Celecoxib28, Itraconazole29 and Voriconazole30. Entrapment of hydrophilic drugs in SLN has been challenging because of low affinity of water-soluble drug towards lipid31. However, SLN have been successfully developed to entrap water-soluble drugs as well after making required modifications in formulation composition32-34.
Infectious diseases are one of the strongest reasons of deaths in the world due to antibiotic resistance and chemical limitations on synthesis of the new antibiotic molecules35. Bacterial keratitis is infection of cornea which can lead to visual impairment and subsequently vision loss if left untreated36. Major causative bacterial species for bacterial keratitis are Pseudomonas and Staphylococcus. Earlier for the treatment of bacterial keratitis, cephalosporins and aminoglycosides were used at higher concentration and in conjunction with other antibacterial agents. With the advent of fluoroquinolones, monotherapy is capable to treat bacterial keratitis. Hydrophilic model drug, moxifloxacin (MFX) is used in current research. It is a 4th generation fluoroquinolone anti-infective drug indicated for the treatment of bacterial keratitis37. MFX is DNA gyrase and topoisomerase IV inhibitor which binds with the bacterial enzymes and subsequently blocks DNA replication causing bacterial cell death38.
The aim of the present study is to formulate and optimize hydrophilic drug loaded solid lipid nanoparticles showing sustained drug release behaviour and would further increase ocular bioavailability. Optimized formulation would be demonstrating efficacy against bacterial keratitis and be non-irritant to cornea.
MATERIALS AND METHODS
Materials: All key materials required for research work were gifted. Moxifloxacin hydrochloride (MFX) [Centurion Laboratories], soya phosphatidylcholine [Lipoid], glyceryl behenate, glyceryl monostearate, glyceryl distearate and stearoyl polyoxyl-32 glycerides [Gattefosse] and poloxamer 188 [BASF].
Methods
Drug-excipient compatibility: FTIR spectroscopy (Perkin Elmer, Spectrum GX, Waltham, MA, United States) was done for physical mixture of MFX and selected excipients.
| Table 1: Variables and their levels in Box-behnken design for MFX SLN | Levels | Independent variables (%) | -1 | 0 | 1 |
| Solid lipid: Glyceryl behenate | 2.0 | 3.5 | 5.0 |
| Hydrophilic surfactant: Stearoyl polyoxyl-32 glycerides | 2.0 | 3.5 | 5.0 |
| Lipophilic surfactant: Soya phosphatidylcholine | 2.0 | 3.5 | 5.0 |
| Dependant variables (%) | Constraints | ||
| Entrapment efficiency | Maximum | ||
| Table 2: Screening of solid lipids and surfactants: MFX SLN preliminary batches | |||||||
| Solid lipid | Hydrophilic surfactant | ||||||
| Batch numbers | MFX (%) | Name | Concentration (%) | Name | Concentration (%) | Particle size (nm) | Entrapment efficiency (%) |
| MSLN1 | 0.5 | Glyceryl behenate | 2.0 | Polysorbate 80 | 2.0 | 650 | 15.0 |
| MSLN2 | 0.5 | Glyceryl distearate | 2.0 | Polysorbate 80 | 2.0 | 660 | 9.0 |
| MSLN3 | 0.5 | Glyceryl behenate | 2.0 | Polyvinyl alcohol | 2.0 | 700 | 10.0 |
| MSLN4 | 0.5 | Glyceryl behenate | 2.0 | Poloxamer 188 | 2.0 | 850 | 10.0 |
| MSLN5 | 0.5 | Glyceryl behenate | 2.0 | Stearoyl polyoxyl-32 glycerides | 2.0 | 250 | 21.0 |
Formulation of moxifloxacin loaded solid lipid nanoparticles
Screening of solid lipids and surfactants: Solid lipids were screened considering MFX solubility in melted lipid39 and then preparing preliminary batches by solvent diffusion method40.
Design of experiments: Design-expert® (Design-expert 11.1.2.0, State-Ease Inc., Minneapolis, USA) was used. Variables and experiments results are presented in Table 1 and 2, respectively. The research work was carried out at Sigma Institute of Pharmacy, Vadodara, India, from March, 2018- Sept, 2019.
Characterizations of MFX SLN
Entrapment efficiency, particle size and zeta potential: Indirect method (measuring unbound drug concentration)41, as shown in the following Eq. 136:
| (1) |
Dynamic light scattering technique was used by Malvern Zetasizer (Malvern Instruments Ltd, Nano ZS, Worcestershire, UK).
Differential scanning calorimetry: Thermal analyzer system (Pyris-1, Perkin Elmer, Waltham, MA, USA) was used.
In vitro release study: Dialysis bag method42 was used. Dissolution media was simulated tear fluid43. Welch's unpaired t-test was applied by GraphPad Instat, 3.10 (GraphPad Software Inc. USA). Release kinetics was evaluated using Kinet DS 3.0 (Kraków, Poland).
In vitro antimicrobial efficacy study: Agar well diffusion method was used44.
Ex vivo efficacy study: Caprine bacterial keratitis model45 was used. Goat eye balls were collected were Corneas were disinfected, cultured and incubated at 37°C at in CO2 incubator (Thermo Scientific, USA). Infection was induced Pseudomonas aeruginosa (105 CFU mL–1). Viability of the cells was assessed periodically visually and by keratome sectioning followed by H-E staining. After 24 h of infection, test samples were added. Bacterial load was measured, macrophotographs were captured and histopathology (Leica DM 750 light microscope using its LasEZ software (Leica Microsystems, India) was done. Results were analyzed by using ANOVA and Fisher’s LSD (Graph Pad Prism 6 software, USA). Box and Whiskers plot was prepared.
| (2) |
Ex vivo ocular irritation study: Hen’s Egg Test Chorioallantoic Membrane (HET-CAM) was used46. Viable chicken eggs were incubated to grow for 8 days. Egg shell was broken to open air cell after saline treatment, CAM was ready for the treatment. Samples were applied.
Stability study of MFX SLN: As per ICH Q1A-R2, stability was evaluated.
RESULTS AND DISCUSSION
Drug-excipient compatibility: Pure drug, MFX possesses reference peak of key function groups such as; C=O and COOH at 1709 cm–1, C=O and phenyl rings at 1620-1520 cm–1, O-H at 3530-3472, C-N at 1166 cm–1, C-H bending47 at 991-802 cm–1 and COO– at 772-721 cm–1. Figure 1 graphical peaks showed that all wavenumber peaks of samples before and after one month accelerated stability incubation matched with that of pure drug reference FT-IR spectra/peaks. So, it is evident that MFX is compatible with selected excipients of SLN formulation.
Formulation of moxifloxacin loaded solid lipid nanoparticles
screening of solid lipids and surfactants: Figure 2 represents solubility of MFX in lipids. The MFX demonstrated highest solubilizing potential in glyceryl behenate and glyceryl distearate. Results of preliminary batches of SLN executed using shortlisted solid lipids considering MFX solubility and using various hydrophilic surfactants are presented in Table 3.
MFX entrapment efficiency was in the range of 9.0-21.0% and particle size also had a wide range of 250-850 nm. Out of two solid lipids, glyceryl behenate was found to be more effective with 15.0% entrapment efficiency compared with 9.0% for the batch prepared with glyceryl distearate. As far as hydrophilic surfactant is considered, stearoyl polyoxyl-32 glycerides looked convincing as it resulted in 21.0% entrapment efficiency with 250 nm particle size. Hydrophilic Lipophilic Balance (HLB) value of poloxamer 188, polyvinyl alcohol, polysorbate 80 and stearoyl polyoxyl-32 glycerides is 29, 18, 15 and 11, respectively. It was observed that more hydrophilic surfactant prepared the SLN of larger size and less entrapment efficiency may be because of higher miscibility of hydrophilic drug such as; moxifloxacin and hydrophilic surfactant and subsequent less entrapment.
It is obvious that when hydrophilic drug is loaded in SLN, it tends to rapid partitioning into external aqueous phase due to its hydrophilic nature during manufacturing which leads to lower entrapment efficiency. Recent study reveals use of lipophilic surfactant such as phosphatidylcholine and lecithin can improve the entrapment of hydrophilic drug48.
Design of experiments
Effect of independent variables on entrapment efficiency: Statistical analysis of results of Design of Experiments (DOE) provided useful information and assured the application of statistical software, Design Expert®. Results of 33 Box-Behnken design are presented in Table 2.
To establish the correlation between independent variables and entrapment efficiency, various mathematical models like linear, 2FI and quadratic were analyzed for test of fit. Linear model was considered based on good fit of regression coefficient. ANOVA was performed and model F-value of 16.68 implies the model is significant.
Polynomial equation of linear model obtained from software is presented as below:
| (3) |
Significance of factor is indicated when p<0.05 and non-significance when p-value is >0.05. B (Stearoyl polyoxyl-32 glycerides) term was significant with p-value of 0.00015. Positive value of coefficient in polynomial equation indicates that response increases with increase in the value of factor and negative value indicates vice versa.
Contour plots and 3-dimensional Response surface plots were prepared (Fig. 3 and 4). These plots are utilized in studying the interaction effect of the independent variables on the responses as well as are useful to study the effects of three factors on the response at one time.
| Table 3: Results of 33 Box-behnken design of experiments | |||||
| Factor 1 | Factor 2 | Factor 3 | Response 1 | ||
| Run | Batch numbers | Glyceryl behenate (%) | Stearoyl polyoxyl-32 glycerides (%) | Soya phosphatidylcholine (%) | Entrapment efficiency (%) |
| 1 | MSLN6 | 3.5 | 2.0 | 5.0 | 24.1 |
| 2 | MSLN7 | 5.0 | 3.5 | 2.0 | 42.4 |
| 3 | MSLN8 | 2.0 | 3.5 | 5.0 | 36.4 |
| 4 | MSLN9 | 3.5 | 2.0 | 2.0 | 24.6 |
| 5 | MSLN10 | 2.0 | 2.0 | 3.5 | 17.6 |
| 6 | MSLN11 | 5.0 | 2.0 | 3.5 | 19.4 |
| 7 | MSLN12 | 5.0 | 3.5 | 5.0 | 43.8 |
| 8 | MSLN13 | 3.5 | 5.0 | 5.0 | 56.4 |
| 9 | MSLN14 | 2.0 | 3.5 | 2.0 | 41.2 |
| 10 | MSLN15 | 5.0 | 5.0 | 3.5 | 57.5 |
| 11 | MSLN16 | 3.5 | 5.0 | 2.0 | 48.6 |
| 12 | MSLN17 | 2.0 | 5.0 | 3.5 | 52.4 |
| Fig. 1(a-b): | Drug-excipient compatibility study: FT-IR Spectra before stability incubation |
| Fig. 2: | Screening of lipids: MFX solubility in solid lipids |
| Fig. 3: | Effect of independent variables on entrapment efficiency: Contour plot |
| Fig. 4: | Effect of independent variables on entrapment efficiency: 3D response surface plot |
| Fig. 5(a-b): | (a) Optimized formulation and (B) Values of desirability |
Contour and 3D plots showed that on increasing the concentration of solid lipid, glyceryl behenate (A) and lipophilic surfactant, soya Phosphatidylcholine (C) from 2.0-5.0%, there was not significant impact on entrapment efficiency and it slightly increased. While on increasing the concentration of hydrophilic surfactant, stearoyl polyoxyl-32 glycerides (B) from 2.0-5.0%, entrapment efficiency was significantly enhanced.
Optimization: Optimization was done in Design Expert® software to get optimum values of independent variables to achieve desired dependant response. For instance, criteria were provided to use the independent variables in the range of 2.0-5.0% concentration. Target of MFX entrapment efficiency was set to maximum keeping duplicate solution filter to 1. The formulation composition containing of glyceryl behenate-3.66%, stearoyl polyoxyl-32, glycerides-4.99% and soya phosphatidylcholine-4.97% was found to provide 57.5% Entrapment efficiency considering higher combined desirability. Figure 5a contains values of independent variables and response for optimized formulation. Figure 5b represents corresponding values of desirability.
| Fig. 6: | Effect of independent variables on desirability as Contour plot |
| Fig. 7: | Effect of independent variables on desirability as 3D Response surface plot |
| Fig. 8: | DSC |
Contour plot and 3D response surface plot are shown in Fig. 6 and 7, respectively which represent the impact of independent variables on desirability. MFX SLN batches were manufactured using optimized concentration of glyceryl behenate, stearoyl polyoxyl-32 glycerides and soya phosphatidylcholine in triplicate and observed entrapment efficiency was 57.0% which is comparable with the software generated predicted value validating the formulation optimization carried out by Design Expert® software.
Characterizations of MFX SLN entrapment efficiency: For determining entrapment efficiency, precipitation of SLN in acidic condition supported for to calculate entrapped MFX easily and quickly as larger (micron) sized aggregates were formed in acidic medium from nano-sized particles which were easily separated by ultracentrifugation.
Entrapment efficiency for design of experiments was in the range of 17.6-57.5%. Based on statistical analysis and optimized formula it was revealed that lipophilic surfactant such as soya phosphatidylcholine and hydrophilic surfactant such as stearoyl polyoxyl-32 glycerides play an important role to entrap hydrophilic drug. Almost 57% MFX entrapment in SLN as a hydrophilic model drug makes SLN as a good carrier for water-soluble candidates. Use of lipophilic surfactant such as phospholipids to improve entrapment of hydrophilic drugs is reported earlier by Liu et al.49. Literature also supported that higher concentration of hydrophilic surfactant is required to increase entrapment efficiency of hydrophilic drugs (Chloramphenicol)50.
Particle size, polydispersity index (PDI) and zeta potential: Particle size distribution result of MSLN18, 245.4 nm confirmed nanosized particles in MFX SLN formulation. Lesser PDI of 0.164 proved that prepared nanoparticles have uniform and narrow size distribution51. Surface charge (Zeta Potential) of MFX SLN optimized formulation was estimated to 28.0 mV reflecting physical stability. Higher value of zeta potential, either positive or negative side, preferably above +20 mV or -20 mV indicated stable nanoparticles formulation when kept in storage for long time due to very low chances of aggregation between the nanosized particles52. Cationic charge may be attributed to higher concentration of lipids as well as lipophilic surfactant and amine group of MFX.
pH: pH of MFX SLN was 7.3±0.05 (mean±SD, n = 3) which is correlated with neutral pH of tear fluid. So, the SLN formulation would be safe to use on the eye surface for the treatment of bacterial keratitis.
Differential scanning calorimetry: DSC thermograms (Fig. 8) of pure drug, MFX, solid lipid (Glyceryl behenate), hydrophilic surfactant (Stearoyl polyoxyl-32 glycerides) and lipophilic surfactant (Soya Phosphatidylcholine) showed respective endothermic peaks at their melting range temperature. DSC thermogram of MFX SLN did not show melting peak of moxifloxacin at around 240°C. This indicated that MFX was not in crystalline state in SLN, rather was in amorphous state and completely entrapped in solid lipid nanoparticles.
| ||||||||||||||||
| Fig. 9(a-b): | (a) In vitro release study and (b) Drug release kinetics models |
| Fig. 10: | In vitro antimicrobial efficacy study: Zone of inhibition |
Similar results were reported by Cavalli et al.53, mentioning rapid quenching of microemulsion does not allow the drug to crystallize. In present solvent diffusion method for preparation of SLN, MFX is dissolved in organic phase and quickly organic phase was transferred to aqueous phase maintaining 75°C. After that resultant o/w emulsion is rapidly cooled down to 25±5°C to get solid lipid nanoparticles. So MFX is completely entrapped in lipid matrix of solid lipid and lipophilic surfactant and does not get a chance to crystallize.
| Fig. 11(a-c): | Ex vivo efficacy study, (a) Macrophotographs, (b) Histopathology and (c) Box and Whiskers Plot of bacterial load (*p<0.05, ANOVA, Fisher’s LSD): (I) Negative control: Healthy cornea (48 h after dissection) (ii) Positive control: Infected cornea before treatment (24 h) (iii) Cornea treated with MFX solution (24 h after treatment) (iv) Cornea treated with MFX SLN (24 h after treatment) |
| Table 4: Results of Ex vino ocular irritation study | ||||||||||||
| Irritation score (Mean±SD, n = 3 eggs), time (min) | ||||||||||||
| Positive control 0.1N NaOH | Negative control 0.9% NaCl | MFX solution | MFX SLN | |||||||||
| Effects | 0.5 | 2 | 5 | 0.5 | 2 | 5 | 0.5 | 2 | 5 | 0.5 | 2 | 5 |
| Lysis (A) | 5±0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Haemorrhage (B) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5±0 | 0 | 0 | 0 | 0 |
| Coagulation (C) | 9±0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Cumulative irritation | ||||||||||||
| Score for 5 min (A+B+C) | 14±0 | 0 | 5±0 | 0 | ||||||||
| Table 5: Results of stability study of MFX SLN | |||||
| Refrigerated temperature (5±3°C) | Room temperature (25±2°C and 60±5% RH) | ||||
Zero day (initial) |
1 month | 3 months | 1 month | 3 months | |
| Z-average (nm)±SD, n = 3 | 245.4±10.2 |
249.3±11.6 | 251.7±13.8 | 250.8±12.8 | 252.4±9.9 |
| Entrapment efficiency (%) ±SD, n = 3 | 57.0±3.2 |
54.6±4.0 | 54.0±3.5 | 53.1±3.1 | 54.2±4.0 |
| ζ-potential (mV)±SD, n = 3 | +28.0±1.5 |
+28.5±2.0 | +28.0±1.0 | +28.5±1.0 | +29.0±1.5 |
In vitro release study: Graphical presentation of in vitro release profile of MFX SLN is provided in Fig. 9a. SLN showed initial burst release of about 40% in 30 min which may be attributed to water solubility of moxifloxacin hydrochloride leading to rapid release of unentrapped MFX. Initial burst release of SLN loading hydrophilic drug is reported as well. Then after drug release behaviour was sustained which lasted for almost 24 h. While it was observed that complete MFX was released from MFX aqueous solution in just 4 h. Statistically significant difference in MFX (%) release was found at all the time points. When in vitro drug release profile results of MFX SLN were fitted into release kinetics models, considering highest value of correlation coefficient (r2), Korsmeyer-Peppas model was considered as the best fit model54. The value of release exponent n, 0.22 indicated Fickian diffusion mechanism of drug release55. The r2-values of each model is presented in Fig. 9b.
In vitro antimicrobial efficacy study: MFX SLN and MFX solution diffused into the agar and inhibited germination and growth of test microorganism which is represented by Zone of Inhibition (ZOI). ZOI of MFX SLN was significantly higher than MFX solution. Negative control (distilled water) did not show the ZOI. Samples were run in triplicate. Figure 10 presented the graphical results. Antimicrobial efficacy of moxifloxacin is increased as compared with plain aqueous solution after 24 h of instillation when it is loaded in solid lipid nanoparticles.
Ex vivo efficacy study
Bacterial load of corneas: Macrophotographs of corneas of each group are presented in Fig. 11a. Positive control shows the progression of infection at 24 h of incubation after induction of infection as compared to negative control which is healthy cornea. Corneas after 24 h of treatment with MFX solution and MFX SLN exhibits comparative less haziness compared with positive control. Bacterial load in the cornea after 24 h of infection was 2.96×109. Bacterial load decreased significantly to about 4-logs in both corneas treated with MFX solution and MFX SLN. Box and Whiskers plot generated by GraphPad Prism 6 software shows the bacterial load of Pseudomonas aeruginosa (CFU mL–1) of each group (Fig. 11c). At 5% significance level (p<0.05, ANOVA, Fisher’s LSD), bacterial load between Positive control and MFX SLN is statistically significant, p<0.05 (0.0337). At the 5% significance level (p<0.05, ANOVA, Fisher’s LSD), bacterial load between Positive control and MFX solution are also statistically significant, p-value-<0.05 (0.0337). Bacterial load results also reflect that entrapment of MFX into solid lipid nanoparticles does not impact efficacy of MFX in ex vivo corneal bacterial keratitis model.
Histopathology: Histopathology of corneas is presented in Fig. 11b. Positive control of the infected corneas revealed disrupted epithelium as infection progressed compared with negative control. Cornea treated with MFX solution and MFX SLN showed slight recovery of epithelium. Since bacterial keratitis is chronic infection, moxifloxacin treatment recommended for at least 7 days. Histopathology was done after 1 day treatment (24 h). Observing at the slight recovery of corneal epithelium after 1 day, it is presumed that after dosing for 7 days with MFX SLN, epithelium would be recovered comparable to healthy cornea.
| Fig. 12(a-d): | Ex vivo ocular irritation study: HET-CAM (a) Positive control, (b) Negative control, (c) MFX solution and (d) MFX SLN |
In addition to this, there might be limitation of ex vivo model to study histopathology of cornea and after in vivo administration of MFX SLN would help in complete corneal recovery after bacterial keratitis infection. Histopathology results also indicated high bacterial load in untreated corneas while the bacterial load was almost similar in corneas treated with MFX solution and MFX SLN. The ex vivo explant model has been used for wide spectrum of applications such as evaluating drug efficacies in drug eluting contact lenses56 as well as for trans corneal permeation of drugs57.
Ex vivo ocular irritation study: Animal welfare concerns and efforts to develop newer toxicity models which have higher correlation with human have resulted into emergence of ex vivo models to study skin and eye irritation avoiding direct use and sacrifice of animals58. HET-CAM is a rapid, sensitive and inexpensive method to study the ocular irritation of pharmaceutical formulations where test formulation is applied directly onto the CAM. Chorioallantoic membrane of the chick embryo can be resembled with in vivo rabbit eyes because CAM is a complete tissue including veins, arteries and capillaries which responds to injury and exhibit inflammatory process, a process similar to that induced in the conjunctival tissue of the rabbit eye59. Cumulative irritation scores of the test formulations are presented in Table 4. Considering the results, MFX SLN does not show irritation to CAM. Macrophotographs of representative egg from each group after 0.5, 2 and 5 min are depicted in Fig. 12. Thus, ex vivo ocular irritation study assured that MFX SLN formulation is non-irritant to cornea and safe at provided dosage after topical ocular administration.
Stability study of MFX SLN: Results of MFX SLN stability study at refrigerated temperature and room temperature are presented in Table 5.
Particle size was in the range of 245.4-252.4 nm over one month and three months at both stability conditions. Optimized MFX SLN formulation was physically stable for three months which was evident by not significant change in value of zeta potential, +28.0 - +29.0 mV. MFX entrapment efficiency slightly decreased from 57.0-53.0% indicating no significant change during storage at specified atmospheric conditions. It can be inferred that developed solid lipid nanoparticles formulation loading moxifloxacin is physically and chemically stable for at least three months at refrigerated temperature and room temperature.
CONCLUSION
Solid lipid nanoparticles formulation, optimized by 3-factor, 3-level Box-Behnken design established the correlation between formulation variables and quality attribute. Optimized formulation successfully entrapped 57% moxifloxacin which is promising for hydrophilic drug. In vitro release study exhibited sustained release behaviour and indicated Fickian diffusion mechanism for drug release. In vitro antimicrobial efficacy study showed efficacy against Gram-positive and Gram-negative bacteria. Bacterial load of infected got cornea was significantly reduced when optimized MFX SLN was applied topically as confirmed by ex vivo efficacy study and Histopathology. Hen’s Egg test where test formulation was applied on chorioallantoic membrane of chicken egg revealed that developed formulation is tolerant to eye. Considering the all the characterizations, moxifloxacin loaded solid lipid nanoparticles emerges as an efficient colloidal drug carrier for the treatment of bacterial keratitis via topical ophthalmic route of administration.
SIGNIFICANCE STATEMENT
Present research work reveals the optimized formulation combination of solid lipid, lipophilic surfactant and hydrophilic surfactant which is beneficial for successful entrapment of hydrophilic drug into solid lipid nanoparticles. This study will help the researcher to entrap hydrophilic drugs into SLN as a carrier being a cost-effective and sustained release formulation preserving the drug efficacy. This research will provide the reference to the budding researchers for lab scale ex vivo models to assess efficacy and toxicity which would eventually prevent exhaustive use of animals in the research with regulatory constraints and time delay. Considering this study as a basis, further entrapment of hydrophilic drug can be increased in SLN formulation by employing advanced lipids and presented rapid ex vivo evaluations.
ACKNOWLEDGMENT
Authors acknowledge the support of Centurion Laboratories Ltd. (India), Lipoid (Germany), Gattefosse (France) and BASF in providing gratis samples of API and Excipients required for Research work. Authors are also thankful to Himedia (India) for providing Dialysis membrane.