INTRODUCTION:

Ophthalmic drug delivery is one of the most interesting and challenging endeavors for the pharmaceutical scientist. The anatomy, physiology and biochemistry of the eye render this organ exquisitely impervious to foreign substances. The challenge to the formulator is to circumvent the protective barriers of the eye without causing permanent tissue damage. The development of newer, more effective dosage forms and therapeutic agents renders urgency to the development of more successful ocular drug delivery system. ^[1]

From the point of view of patient acceptability, a liquid dosage form that can sustain drug release and remain in contact with precorneal surface for extended periods of time is ideal for ocular delivery. If the precorneal residence time of a drug could be improved from 5 minutes to, say a few hours, then improved local bioavailability, reduced dosing frequency, and improved patient acceptability may be achieved.

Drug delivery systems based on the concept of In situ gel formulation can provide these properties. Such delivery systems are formulated from polymers that exhibit phase transition due to physico-chemical changes in their environment. They can be instilled as liquid drops into the cul-de-sac of eye where they transform into gel or semisolid phase. ^[4,5]

A promising strategy to overcome several problems involves the development of suitable drug carrier systems. The in vivo fate of the drug is no longer mainly determined by the properties of the drug, but by the carrier system, which should permit a controlled and localized release of the active drug according to the specific needs of the therapy. The size of the carrier depends on the desired route of administration and ranges from few nanometers (colloidal carriers), to the micrometer range (microparticles) and to several millimeters (implants).

Solid Lipid Nanoparticles offer several advantages compared to other systems (easy scaling up, avoidance of organic solvents, high content of nanoparticles). These advantages have been discussed in a variety of papers. However, less attention has been paid to the detailed and appropriate investigation of the limitations of this carrier system.

Loteprednol etabonate is a corticosteroid that has been developed as a topical treatment for ocular inflammation. It is a product of so-called ‘soft drug’ design, that is, synthesis of a compound that undergoes predictable metabolism to inactive metabolites after its therapeutic effects have been expressed at or near the site of application. The aim of such drug development is to improve the therapeutic index, i.e. to enhance efficacy while minimizing systemic adverse effects^[^6].

MATERIALS AND METHODS:

Materials:

Loteprednol etabonate (LE), Glyceryl behenate, Glyceryl distearate, Poloxamer, Carbopol. All the chemicals and solvents used are of analytical grade.

Method:

Preformulation study:

DSC study:

DSC study of Poloxamer 407, Loteprednol Etabonate, D-Mannitol and Solid Lipid Nanoparticles were performed on SHIMADU DSC apparatus.

Melting Point:

Melting point of drug was determined by taking a small amount of drug in a capillary tube closed at one end and was placed in melting point apparatus, and temperature at which drug melts was noted.

Preparation of Solid Lipid Nanoparticles of Loteprednol Etabonate:

Solid lipid nanoparticles of Loteprednol etabonate consisting of solid lipid Compritol were prepared by hot homogenization technique. Briefly, the lipid was melt above its melting point and dissolve the loteprednol etabonate in melted lipid and make the molecular dispersion of drug lipid melt in 25 ml glass beaker in different drug: lipid ratio. Then add hot aqueous surfactant solution of pluronic F 127 in drug lipid melt dispersion and stir it on magnetic stirrer for 15 minutes to form a pre-coarse emulsion. Then sonicate this pre-emulsion (2 cycle, 4 min., 80% amp., 0.7 cycle/sec.) using probe sonicator (RR-120, Ralsonics, Mumbai). During sonication, temperature should be maintained above the melting point of lipid. After sonication the hot emulsion was slowly cool down to RT with slow stirring on digital high speed stirrer. The recrystalization of lipid occurred and it formed solid lipid nanoparticles dispersion of Loteprednol Etabonate. Nanoparticulate dispersion was then lyophilized using a cryoprotectant D-mannitol. The ratio of cryoprotectant used compare to lipid was 15: 1.

Solid Lipid Nanoparticles were subjected to centrifugation at 5,000 rpm at 8°C for 10 minutes using ultra centrifuge (REMI). SLNs suspension was decanted and drug pellet was separated. Solid Lipid Nanoparticles suspension was then characterized for vesicle size and percent drug entrapment (PDE). Mass balance was calculated by measuring unentrapped drug in pellet and entrapped one in Solid Lipid Nanoparticles. A flowchart depicting the process is shown in Fig 1. The Solid Lipid Nanoparticles compositions and process parameters were optimized to achieve maximum drug entrapment.^[^7,8]

Figure 1. Steps of Hot homogenization process in preparation of LE loaded SLNs

CHARACTERISATION AND EVALUATION OF SOLID LIPID NANOPARTICLES:

SLNs Size:

The size of Solid Lipid Nanoparticles was measured by dynamic light scattering with a Malvern Zetasizer nano-ZS (Malvern Instruments, Malvern, UK). Diluted SLNs dispersion was added to the sample cuvette and then cuvette is place in zetasizer. Sample is stabilized for two minutes and reading was measured. The average particle size was measured after performing the experiment in triplicate.^[^7]

Zeta Potential:

The zeta potential^[^7] of developed Solid Lipid Nanoparticles was determined using Malvern Zetasizer nano-ZS (Malvern Instruments, Malvern, UK).

Percent Drug Entrapment:

To determine percent drug entrapment (PDE), free and entrapped drug was measured. The free Loteprednol etabonate (un-entrapped) in the SLNs dispersion was separated by controlled centrifugation at low speed method as described by (New, 1990a). Briefly, the Solid Lipid Nanoparticles dispersion was centrifuged at 6000 rpm, 8^ºC for 10 minutes using sigma centrifuge and the SLNs dispersion was removed without disturbing the drug pellet. The drug pellet was dissolved in Methanol and estimated for un-entrapped drug content. Fixed volume of Solid Lipid Nanoparticles dispersion was withdrawn and dissolved in Methanol: CHCl₃ (9:1) mixture and estimated for entrapped drug content.^[^7,8]

Scanning Electron Microscopy (SEM) of SLNs:

The surface morphology of lyophilized Solid Lipid Nanoparticles of Loteprednol etabonate was examined by scanning electron microscopy (JSM-5610LV, JEOL, Japan). Samples were attached to sample stubs using double sided tape, and then viewed using an accelerating voltage of 15 kilovolt at the magnification of 35X to 7,500X.^[8]

pH measurement:

pH is the most important parameter for the ophthalmic preparation and it should remain near to neutral side for patient compliance. pH of the final formulation that was lyophilized Solid Lipid Nanoparticles of Loteprednol Etabonate dispersed in poloxamers solution and carbopol solution was measured using pH meter^[8] (Lab India Instrument, Mumbai).

Preparation of In Situ Gelling Solutions:

Polymer solutions, namely in situ gels, which undergo a phase change from a liquid to a semisolid gel upon exposure to physiological environments, have received extensive interests. The gelation can be triggered by a shift in temperature, as for poloxamers and ethyl (hydroxyl ethyl) cellulose, a shift in pH as for cellulose acetate phthalate and Carbopol, or by the presence of cations as for deacetylated gellan gum and alginate. Here, Solid Lipid Nanoparticles of Loteprednol Etabonate was dispersed into below of in situ gelling solutions ^[9].

Solid Lipid Nanoparticles of LE dispersed in poloxamers solution

Dispersion of SLNs in poloxamers solution:

Poloxamers (Pluronic) are ABA triblock copolymers consisting of hydrophilic poly-oxyethylene (PEO) and hydrophobic polyoxy-propylene (PPO). Aqueous solution of Pluronic F127 at a concentration equal to or greater than 18% forms non-crosslinked hydrogel upon warming to ambient temperature. The sol–gel transition temperature strongly depends on F127 concentration and can be altered by salts. Considering the lachrymal dilution, a relative higher polymer concentration is essential for the F127 solution to form in situ gel.^[^10,11]

Stability Study:

Stability studies for prepared Solid Lipid Nanoparticles were carried out for up to 2 months and stability was accessed by drug content measurement and particle size of the Solid Lipid Nanoparticles. The factor considered for the study was temperature and humidity on storage as per ICH guideline. The stability study was conducted in at two different temperature conditions, one at room temperature (25+2^°C) and 60+%RH) and other at refrigerated condition(5+3^°C).^[12,13]

Ex Vivo Study:

Dispersion of lyophilized F–SLNs (0.2 % w/v) was prepared in the distilled water. Dispersion was taken in a Neuberger’s counting chamber and observed under Olympus microscope (40X). For the Ex vivo study, goat eye was taken from nearest slaughter house in PBS pH 7.4 under maintained temperature (4 ^°C). The goat cornea was separated with 3 – 4 mm of sclera. The separated goat cornea was stick on the glass slide with help of gum. Three types of formulations were prepared for the evaluation of mucoadhesive strength which include F–SLNs suspension (F-SLNs sus.), F–SLNs in poloxamers solution (F–SLNs P), F-SLNs in carbopol 974 P solution (F-SLNs C). The washing solution (STF) was filled in burette. The glass slide on which cornea was stick, kept under the burette the tip of burette with slight slope. Under this slide one reservoir compartment was kept for collection of washing solution which was passed over the cornea with regulated flow of washing solution. The formulations were instilled on the cornea and left for 1 min for became gel. Then it was washed with washing solution at the rate of 1 ml per 15 min. The washing solution (STF) washed only the F-SLNs particles not adsorbed on the cornea. Collect the washing solution in reservoir apparatus and counted F–SLNs in washing solution as per mentioned above and reported the percentage reduction in F-SLNs counts. The experiment was performed three times for each formulation. ^[14,15]

RESULTS AND DISCUSSION:

Preformulation study:

DSC Study:

Figure. 2 DSC Thermogram of Loteprednol Etabonate

Figure. 3 DSC Thermogram of Loteprednol etabonate loaded Solid Lipid Nanoparticles.

From DSC Study graphs it concluded that no interaction was found between drug and excipients.

SLNs Size

Figure 4. Solid Lipid Nanoparticles Vesicle size

The vesicle size of Solid Lipid Nanoparticles was determined by laser diffraction using Malvern zeta sizer. SLNs prior to sonication had a greater mean size and broader size distribution, however, upon sonication it acquired a narrower range of distribution and a mean Solid Lipid Nanoparticles average size was 141 nm.

Zeta Potential:

Figure 5. Zeta Potential of Solid Lipid Nanoparticles.

Zeta potential was found to be -18.7 mv.

Percent Drug Entrapment:

The mean PDE obtained during the optimization of Solid Lipid Nanoparticles of Loteprednol etabonate by hot homogenization method were reported below. The percent drug entrapment was found to be 60.02 ± 0.143. Reconstituted SLNs size found was 630 nm± 5.367.

Batches with higher PDE were selected for the development of SLNs in situ gelling formulations.

SEM (Scanning Electron Microscopy) photomicrographs:

SEM images are taken at two different magnifications 35 X and 7500 X. Results obtained are as follows.

Figure 6. SEM photomicrographs of Solid Lipid Nanoparticles at 35 X magnification

Figure 7. SEM photomicrographs of Solid Lipid Nanoparticles at 7,500 X magnification

Diffusion Study:

Table 1: Data of Diffusion study

Time (hrs)

Mean Cumulative Percent Drug Diffused across the membrane*

Plain drug suspension

Gel ctg. Plain drug

SLNs suspension

SLNs in gel solution

0.25

10.12±

1.647

9.4±

0.685

24.28±

1.329

13.765±

2.312

0.5

15.22±

0.763

14.68±

1.223

35.655±

1.562

17.835±

2.397

19.93±

0.926

18.32±

0.700

42.535±

1.605

23.535±

1.704

25.68±

1.294

23.46±

1.555

54.545±

1.407

31.26±

0.933

70.86±

1.378

40.64±

1.661

63.975±

1.053

38.11±

0.551

94.36±

0.835

65.85±

1.244

72.5±

1.668

42.975±

1.166

80.95±

0.735

79.765±

0.770

48.585±

1.378

87.72±

0.806

81.75±

0.494

56.57±

0.834

89.075±

0.671

58.98±

0.480

Table 2. In-vitro drug release data of SLNs of LE in Poloxamers solutions

Time (hrs)

Mean Cumulative Percent Drug Diffused across the membrane*

Plain drug suspension

Gel ctg. Plain drug

SLNs suspension

SLNs in gel solution

0.25

10.12±

1.647

8.65±

1.246

24.28±

1.329

11.46±

1.231

0.5

15.22±

0.763

12.56±

1.452

35.655±

1.562

15.54±

2.361

19.93±

0.926

17.44±

0.69

42.535±

1.605

22.42±

1.754

25.68±

1.294

24.35±

1.535

54.545±

1.407

30.36±

0.863

70.86±

1.378

38.92±

1.321

63.975±

1.053

36.37±

0.681

94.36±

0.835

65.32±

0.746

72.5±

1.668

41.52±

1.524

79.37±

1.256

79.765±

0.770

44.76±

1.217

85.32±

0.81

81.75±

0.494

48.31±

0.765

89.075±

0.671

51.23±

0.521

Table 3. Stability Data

Sampling Time (month)	Observation	Average % Drug retain (% Assay)	Average particle size (nm)
0	White free flowing powder	99.17	608.2
Room condition (25^oC ± 2^oC, 60 ± 5% RH)
1	No change in color	98.85	625.3
2	White to pale yellow & some aggregation	98.61	644.6
Refrigerated condition (5+3^oC )
1	No change in color with free flow	99.05	615
2	No color change & no aggregations	98.83	630.7

From the % assay determination of the Solid Lipid Nanoparticles stored at different temperature conditions, it was found that there is no significant decrease in the % assay of the drug in the Solid Lipid Nanoparticles, but in case of the particle size analysis, there was slight increase in the particle size after two month of storage at room temperature, while at refrigerated condition, there was no significant change in the % assay and particle size. The higher particle size at room temperature storage may attribute to the aggregation of lipid particles and after long period due to the recrystalization of lipid. Slight change in color of Solid Lipid Nanoparticles was observed after two months in both conditions but at room condition, the color change observed within 2 months. Thus it was concluded that the optimum temperature of storage of the Solid Lipid Nanoparticles is refrigerated condition (2-8 °C).

Ex Vivo Study:

Table 4. Data of Cumulative % reduction in Count.

Sr. No.	Time (min.)	Cumulative % reduction in count *
Sr. No.	Time (min.)	F –SLNs suspension	F – SLNs P	F – SLNs C
1	15	57.64 ±1.234	21.23 ±2.654	15.32 ±1.620
2	30	92.37 ±2..263	34.84 ±1.351	24.89 ±2.300
3	60	-	72.56 ±2.945	48.26 ±3.208
4	90	-	89.42 ±3.014	66.75 ±2.374
5	120	-	-	87.67 ±0.350

Figure 8. Plot showing cumulative % reduction vs time

CONCLUSION:

The Solid Lipid Nanoparticles were prepared by hot homogenization method using Glyceryl behenate (Compritol 888 ATO) lipid matrix. The prepared Solid Lipid Nanoparticles were assessed for physical properties, in-vitro drug release study, stability study and Ex-vivo study. For mucoadhesive preparations, Loteprednol Etabonate loaded Solid Lipid Nanoparticles were dispersed in thermosensitive gelling solution (Poloxamers solution). The findings of this investigation was also confirmed by its ex-vivo study on goat’s cornea where it was found that drug LE with Carbopol 934 shows good mucoadhesive property as compared Poloxamer. Further the detail in vivo studies are required to be done in order to see the effect of formulation in reducing the eye inflammation in at least two or more animal models followed by an extensive clinical evaluation.

ACKNOWLEDGEMENT:

I am thankful to God for providing enough strength for my research work, my loving parents for their support and managing trustee parul arogya seva mandal and Multimedics organisation for providing facilities for this research wok.

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Received on 23.05.2014 Accepted on 15.06.2014

Asian J. Pharm. Res. 4(2): April-June 2014; Page 78-83