INTRODUCTION:

Therapeutic efficacy of any drug depends upon the four fundamental pathways of drug in the body such as absorption, distribution, metabolism and excretion. Failure in any of these processes leads to failure in therapeutic efficacy. A promising strategy to overcome any problems encountered in these by the development of a suitable drug colloidal carrier system. Among the colloidal carrier systems solid lipid nanoparticles have many advantages and limited disadvantages as compared to conventional colloidal carrier systems. Solid lipid nanoparticles (SLN) have gained attention as carriers for the preparation of a wide variety of poorly water-soluble drugs due to their biodegradable and biocompatible properties and low toxicity¹.

The use of SLNs is a striking improvement because the solid matrix of the lipids presents high flexibility in controlling the drug release and protects the encapsulated drugs from gastric degradation.

SLNs are generally composed of biodegradable and biocompatible solid lipid as solid core, coated by nonhazardous surfactant/co-surfactant as the outer shell. Use of solid lipids increases drug absorption mainly through enhanced drug dissolution and solubilization in the intestinal-milieu, improved lymphatic-transport, enhanced gastrointestinal permeability, and decreased gastric-emptying rate. Particle size and PDI are key characteristics and are critical parameters in the stability and fabrication of SLNs. These characteristics mainly depend upon particles composition and different fabrication techniques^2-4.

The main objective of current research work is to prepare felbamte loaded solid lipid nanoparticles, using glyceryl monostearate and glyceryl monooleate as the lipid matrices and tween 80 as stabilizer with a view to improve the dissolution rate of felbamate which would increase the biological activities. Given that solid lipid nanoparticles, as an alternative colloidal carrier (transport) system, have the ability to improve the solubility/permeability of lipophilic drugs, they may also enhance the drug absorption.

MATERIALS:

Felbamate was received as gift sample from Aurobindo Pharma, Hyderabad. Glyceryl monostearate [GMS] (CDH Pvt. Ltd., Mumbai), Glyceryl monooleate [GMO] (Otto Chemicals, Mumbai), Polysorbate 80 (Sisco Research Laboratories, Chennai), Chloroform and Methanol (Rankem, Chennai), Dialysis Membrane 50 – LA 387 (Himedia, Mumbai) were purchased from the local market. All the reagents used were of analytical grade.

METHODS:

Preparation of Felbamate loaded solid lipid nanoparticles:

Felbamate (API) loaded solid lipid nanoparticles were fabricated by hot homogenization followed by ultrasonication method [5-6]. API and monoglyceride were dissolved in a mixture of methanol and chloroform (2:1). Organic solvent mixture was then slowly removed by evaporation in rotary flash evaporator. Resulting embedded lipid layer was then molten by heating to 5°C above the melting point of the lipid. In another vessel, an aqueous phase was prepared by mixing the tween 80 (Stabilizer) with distilled water (sufficient to produce 30 ml) and then heated to same temperature as oil phase. Then this hot aqueous phase was added to the oil phase and homogenization was performed (at 2500 rpm and 70°C) using a mechanical stirrer for 30 minutes. The coarse oil in water emulsion so obtained was sonicated using probe soincator for 25 minutes. Felbamate loaded SLN was finally obtained by allowing the hot nanoemulsion to cool to room temperature, and was stored at 4°C in the refrigerator. The composition of the different formulation has been given in Table 1. In all SLN formulations the lipid concentration was kept constant (as 6% w/v)

Table 1: Composition of felbamate loaded solid lipid nanoparticles

Name of the Ingredient	F1	F2	F3	F4	F5	F6
Glyceryl monostearate	6% w/v	6% w/v	6% w/v	NA	NA	NA
Glyceryl Monooleate	NA	NA	NA	6% w/v	6% w/v	6% w/v
Polysorbate 80	1% w/v	1.5% w/v	2% w/v	1% w/v	1.5% w/v	2% w/v

Characterisation of Solid Lipid Nanoparticles of Felbamate:

Particle size, poly dispersity index and zeta potential:

Prepared solid lipid nano particles were maintained at room temperature for 30 days, which were characterized for particle size, poly dispersity index and zeta potential. About 1ml of prepared solid lipid nano particles were diluted appropriately using distilled water, which was then taken individually in a zeta cell and measured the average particle size, poly dispersity index and zeta potential using Zetasizer. The experiments were performed in triplicate.

Drug content, Encapsulation efficiency and Drug loading estimation:

Drug content of prepared solid lipid nano particles was evaluated by performing assay using the established HPLC methods for Felbamate.

HPLC Method to estimate samples of Felbamate:

The chromatographic separation was performed with Shimadzu HPLC system with the best chromatographic conditions equipped with C18 column (ODS 250 mm X 4.6 mm with 5 micron pore size, Phenomenax) using a mobile phase combination of potassium dihydrogen phosphate buffer (50mM, pH4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) in an isocratic mode elution with the flow rate was set at 0.8 mL/min. The samples were analyzed by PDA detector.

Estimation of encapsulation efficiency and drug loading:

Prepared solid lipid nano formulations were centrifuged using a refrigerated centrifuge (Remi) for 45 minutes at 19,000 rpm at -20°C and supernatant was separated and stored individually for further analysis. About 1 ml of supernatant was mixed with 1 ml of methanol, which was vortexed for 5 minutes and filtered through 0.22 µm membrane. Estimated amount of free drugs were expressed as W_free. The experiments were performed in triplicate.

Encapsulation efficiency (EE) and drug loading (DL) were estimated as follows

Drug Content (W_total)] – [Drug in the supernatant (W_free)

EE (%) = –––––––––––––––––––––––––––––––––––––––––––– × 100

Drug Content (W_total)

Drug Content (W_total) – Drug in the supernatant (W_free)

D(%) = –––––––––––––––––––––––––––––––––––––––––––– × 100

Weight of the polymer used in the formulation (W_polymer)

In-vitro drug release study:

In-vitro release study of Felbamate loaded nano scale solid lipid particles was determined by using dialysis bag method. Accurately weighed quantities of Nano particle were placed in the dialysis bag (cut-off 5 kDa, Himedia, India) and both ends were tightly sealed. The dialysis bag was positioned in a Beaker containing 100 ml phosphate buffer at pH 7.4, which was magnetically stirred at 100rpm (Remi, India) and maintained at 37°C ±0.5°C. At schedule time intervals, the 1ml of the release medium was withdrawn using micropipette and replaced with the same volume of fresh PBS. The samples were immediately filtered through a 0.45μm membrane filter (Elix, Mill-Q) and the content of Felbamate was estimated after suitable dilution with a Thermo scientific HPLC (Spectra system P-4000, USA) with UV detector (Kromosil 100) and C18 column (Particle size 5μm, 250mm×4mm) at 265nm.

Stability studies:

The stability study provides the evidence of fabricated nano scale solid lipid particles quality varies with time under different environmental factors includes temperature, humidity and light. The study was carried out according to the international conference on harmonisation (ICH-QIA (R2) guidelines, 2003), with Felbamate loaded nano scale solid lipid particles. The samples were studies were performed over a period of 6 months at 40°C±2°C/75% RH±5% RH (Accelerated condition). The samples were evaluated at 0, 3 and 6 months for their particle size, poly dispersity index, zeta potential and drug content. In addition, samples were visually examined for any physical instability (separation and aggregation).

RESULTS AND DISCUSSION:

Development of solid lipid Nano particulate drug delivery system:

Felbamate solid lipid nanoparticles were prepared using hot homogenization followed by ultrasonication method.

Prepared nanoparticles were characterized for distribution width, mean particle size, surface area, span, and uniformity using laser particle size analyser. However, these characterization parameters depends on process parameters such as organic solvent, lipid concentration, surfactant percentage, percentage of organic solvent, volume of aqueous phase, temperature generated during sonication process, sonication duration and drug concentration. Hence, a step-by-step optimization was carried out to evaluate the effect of these process parameters on prepared solid lipid nanoparticles (F1 to F6). The experiments were performed in triplicate and characterization results were expressed as mean ± standard deviation.

Table 2: Characterization of prepared SLN’s

Run	Average Particles size (nm)	Span	Surface Area (m²g^-1)	Polydispersity index
F1	354	1.825	15.4	0.456
F2	225	1.725	22.6	0.254
F3	124	0.224	55.4	0.124
F4	364	1.756	16.5	0.546
F5	252	1.259	26.2	0.325
F6	134	0.245	52.4	0.156

Drug Content, Encapsulation Efficiency and Drug Loading Estimation:

The amount of Felbamate encapsulated in nanoparticles determines the effectiveness of prepared nano-formulations. Hence, drug content, encapsulation efficiency and drug loading estimation were performed as per procedure mentioned. Drug content was estimated by performing an assay whereas encapsulation efficiency and drug loading were calculated by measuring the free Felbamate in the nano-formulation. The results were summarised in table 3.

Table 3: Drug Content, Encapsulation Efficiency and Drug Loading Estimation of Optimized Formulation (F3)

Evaluation parameter	Nanoformulation
Process yield (%)	92.54
Mean particle size (nm)	114-134
Polydispersity Index (Pdi)	0.124
Zeta potential (mV)	- 20.2 ± 3.16
Drug loading (%)	94.93%
Encapsulation efficiency (%)	95.93 ± 0.72
Drug content (%)	99.86

Drug content in formulations was in the range of 99 to 100, which shows that there was no post-formulation degradation or drug loss.

In stirring approach, encapsulation efficiency and drug loading was found to be 94.93%. Hence, prepared Felbamate solid lipid nanoparticles is expected to display superior pharmacological activities.

In vitro drug release study:

In vitro drug release from the drug loaded nanoparticles was assessed in simulated gastrointestinal conditions. The pH condition used was pH 1.2 for a period of 2 hrs (Stomach), pH 4.5 for 2 hrs (Duodenum) followed by pH 7.4 (Distal ileum and colon) for the remaining period of the study using a USP dissolution test apparatus type 2 (Chandran et al., 2009) and in vitro drug release of optimum formulation is shown in table 4. The drug release was found to be less than 5% up to 4 hrs and the drug release increased when the pH of the medium was adjusted to 7.4.

Table4: In vitro release profile of Felbamate solid lipid nanoparticles

Time	Drug release
0 hrs	0.00 ± 0.00
1 hrs	0.00 ± 0.00
2 hrs	0.00 ± 0.00
4 hrs	1.85 ± 1.42
6 hrs	28.32 ± 1.22
8 hrs	62.54± 1.19
10 hrs	72.25 ± 0.26
12 hrs	86.34 ± 3.14
24 hrs	98.72 ± 1.71

Release kinetics:

The results of in vitro release profile obtained from the optimized formulation (F3) were plotted to know the mechanism of drug release. The data were treated according to Zero Order Release, First Order Release, Higuchi Model, Korsmeyer-Peppas Model and Hixson Crowell Cube Root Law. The release rate kinetics data of the formulation are as Zero order

r² = 0.78, First Order r² = 0.67, Higuchi model r² = 0.52, Korsmeyer-Peppas Model r² = 0.77, Korsmeyer-Peppas Model n = 0.92, Hixson Crowell Cube Root Law r² = 0.95. Where r²is determination coefficients and “n” is release exponent of kinetic data analysis of Felbamate release from nanoparticles.

Trials	Period (Months)	Average particle size (nm)	Poly dispersity index (PDI)	Zeta Potential (mV)	Drug Content (%)
Nano	0	124 ± 0.45	0.124 ± 0.04	- 20.2 ±3.16	99.86 ± 1.67
	3^rd	115 ± 0.35	0.289 ± 0.41	- 29.1 ± 1.20	97.35 ± 1.42
	6^th	116 ± 0.64	0.399 ± 0.24	- 25.2 ± 0.98	96.80 ± 1.26