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The aim of the study was to formulate solid lipid nanoparticulate gel of Econazole nitrate by cold homogenization process. The solid lipid nanoparticles comprises of stearic acid and palmitic acid; poloxamer 188 as stabilizer and they were evaluated for FTIR, DSC, particle size, zeta potential, percentage drug entrapment efficiency and in vitro drug diffusion studies. FTIR and DSC studies confirmed that there was no incompatibility between drug and lipids. The formulation code F2 was selected as optimized formulation based on the higher composite desirability value obtained by Minitab Qsutra 18 software. The scanning electron micrographs revealed that particles were non-spherical, agglomerated and were of nanometre range. Entrapment efficiency was in the range of 22.19% and 78.58%. Further optimized solid lipid nanoparticles were formulated as topical gel using HPMC K4M and were evaluated for spreadability, viscosity, pH, drug content, in vitro drug diffusion studies and skin irritation study. The primary skin irritation index was found to be 0.00 which inferred no skin irritation in rats. Three months of stability studies revealed F2 formulation and solid lipid nanoparticulate gel of Econazole nitrate were stable at 40±2° C and 75±5% RH.
Key words: Econazole nitrate, solid lipid nanoparticles, entrapment efficiency, cold homogenization.

Fungal infections are the most commonly experienced diseases affecting the skin, hair and nails. The treatment approaches for these infections includes both topical and oral antifungal agents. In topical administration of antifungals, the drug should pass through the stratum corneum that is the outermost layer of the skin to reach the lower layers of the skin especially viable epidermis.
The administration of these antifungals in the form of creams, gels, powder, and solution has the major drawbacks such as allergic and severe skin reaction when they are designed into these dosage forms directly. In order to reduce these drawbacks, novel drug delivery approaches are being projected and to maximize their activity.

Solid lipid nanoparticles (SLNs) are colloidal lipid nanoparticles with the sub-micron size range of 50-1000 nm and composed of biodegradable solid lipophilic matrix in which drug molecules can be loaded. SLNs combine the superiorities of all other nanoparticles such as liposomes, fat emulsion and polymeric nanoparticles.
Econazole nitrate (ECN) is an imidazole antifungal drug that inhibits the conversion of lanosterol to ergosterol by interacting with 14-? demethylase enzyme by which it prevents fungal organisms to produce vital substances required for their growth and function. ECN has the topical half-life of 35 min and very slightly soluble in water. ECN possess adverse effects such as irritation, burning sensation and contact dermatitis when it is applied topically.
SLNs ensures the close contact of antifungal drugs with the stratum corneum which results in the increase of the amount of encapsulated drugs penetrating into viable skin and their application on the damaged or inflamed skin as they are based on non-irritant and non-toxic lipids of Generally Recognized as Safe (GRAS) listed approved by FDA. An adhesive effect can be seen by solid lipid nanoparticles as they form a film on the skin after application, the adhesion increases with decreasing particle size. Occlusion can enhance the penetration of drugs through the stratum corneum by increased hydration. Moreover, these lipids are likely to minimize the danger of allergic contact dermatitis that may be induced by the drug.
The aim of this work was to formulate solid lipid nanoparticulate gel of Econazole nitrate and evaluated the dosage form for various parameters. In this work, an attempt was made to retain the solid lipid nanoparticulate gel of Econazole nitrate in the absorption site more than its half-life, reduction in dose frequency and toxicity and to control the drug release from the dosage form.

MATERIALS AND METHODS
Lipids such as stearic acid, palmitic acid and preservatives such as methyl parabhen and propyl parabhen were obtained from Karnataka Fine Chem, Bengaluru. Econazole nitrate, Poloxamer 188 and HPMC K4M were procured from Yarrow Chem Products, Mumbai. HPLC graded water was procured from SDFCL, Mumbai. All solvents and reagents used were of analytical grade.

Preformulation study:
Melting point of Econazole nitrate was determined by thiele’s tube method. Drug excipients compatibility studies were carried out by Fourier-transform infrared spectroscopy (FTIR spectrophotometer, Tensor 27, Bruker optics, Mumbai). In FTIR study, drug and lipids were individually analysed initially and later physical mixture of drug and lipid were analysed. The FTIR spectrums of drug with lipids were compared with the standard FTIR spectrum of the drug. DSC studies were carried out on DSC Q60, Shimadzu, Japan. Sealed and perforated aluminium pans were used for all samples in the experiments. Temperature calibrations were performed using indium as standard. An empty pan sealed in the same way as for the sample was used as a reference. The entire samples were run in nitrogen atmosphere at a scanning rate at 10° C/ min from 50 – 300° C. By comparing the DSC curves of a pure drug sample with that of formulation, the presence of an impurity can be detected in a formulation.

Standard calibration of Econazole nitrate was done using UV-Visible spectrophotometer (UV-1700, Shimadzu, Japan) with phosphate buffer pH 5.5 and the absorbance was measured at 271 nm.
Preparation of Econazole nitrate loaded solid lipid nanoparticles:
Econazole nitrate loaded solid lipid nanoparticles were prepared by using cold homogenization technique. The lipid was melted to approximately 10° C above its melting point; econazole nitrate was dispersed in this lipid melt and kept the same on ice bath to get drug lipid melt. This melt had to be grounded to obtain a fine powder. Drug lipid melt was dispersed in the cold aqueous phase of poloxamer 188 under magnetic stirring. The dispersion was homogenized at 15,000 rpm for 45 min by maintaining temperature below 10° C using IKA Ultra-turax homogenizer, Germany. The obtained emulsion was lyophilized to get the final product (Table 1).

Particle size analysis:
The particle sizes of solid lipid nanoparticles (SLNs) were measured by using Malvern Zeta sizer Nano ZS-90 which works on the principle of Dynamic Light Scattering that measures the diffusion of particles moving under Brownian motion and converts this to size and a size distribution using the Stokes-Einstein relationship. The nanosuspension was sonicated for 30 min followed by further dilution with HPLC graded water. The average particle size was determined from the particle size distribution data.

Zeta potential:
The zeta potential of SLNs were measured by using Malvern Zeta sizer Nano ZS-90 which works on the principle of Lasser Doppler Micro-electrophoresis which is used to measure zeta potential. An electric field is applied to a solution of molecules or a dispersion of particles, which then move with a velocity related to their zeta potential. This velocity is measured using patented laser interferometric technique called M3-PALS (Phase analysis Light Scattering).
Scanning electron microscopy:
SEM photographs were taken for the prepared nanoparticles using a scanning electron microscope (Carl Zeisus FESEM model number: Ultra 55 USA) at different required magnifications at room temperature. The photographs were analysed for morphological characteristics.
Drug entrapment efficiency:
The prepared SLN dispersion was centrifuged at 7500 rpm for 20 min at 4° C using REMI cooling centrifuge. The supernatant was analysed for the free drug content. The entrapment efficiency (%) of drug was calculated by the following equation:
%Entrapment efficiency = total drug content – free drug content / total drug content *100
Loading capacity:
Loading capacity is the amount of drug loaded per unit weight of the nanoparticles, indicating the percentage of mass of the nanoparticles that is due to the encapsulated drug. Loading capacity can be calculated by the amount of total entrapped drug divided by volume of water that is required to re suspend the nanoparticles.

Loading capacity = total drug content – free drug content / volume of water required to re suspend SLNs.

In vitro drug release studies:
The in vitro drug release profile of econazole nitrate loaded SLNs were studied using vertical diffusion cell. The dialysis membrane was soaked overnight in the pH 5.5 phosphate buffer. The calculated amount of nanoemulsion of econazole nitrate was kept in the donor compartment above the dialysis membrane. In the receptor compartment, 250 ml of phosphate buffer pH 5.5 was taken and placed on magnetic stirrer with the temperature of the assembly and rpm was maintained at 37 ± 0.5° C and 100 rpm throughout the study. Samples of 1ml were withdrawn at predetermined time intervals (1, 2, 3, 4,5,6,7 and 8 h) and replaced simultaneously with equal amounts of fresh buffer. After suitable dilution, the samples were analysed for drug concentration by UV spectrophotometer at 271 nm.
The aim of the study was to formulate solid lipid nanoparticulate gel of Econazole nitrate by cold homogenization process. The solid lipid nanoparticles comprises of stearic acid and palmitic acid; poloxamer 188 as stabilizer and they were evaluated for FTIR, DSC, particle size, zeta potential, percentage drug entrapment efficiency and in vitro drug diffusion studies. FTIR and DSC studies confirmed that there was no incompatibility between drug and lipids. The formulation code F2 was selected as optimized formulation based on the higher composite desirability value obtained by Minitab Qsutra 18 software. The scanning electron micrographs revealed that particles were non-spherical, agglomerated and were of nanometre range. Entrapment efficiency was in the range of 22.19% and 78.58%. Further optimized solid lipid nanoparticles were formulated as topical gel using HPMC K4M and were evaluated for spreadability, viscosity, pH, drug content, in vitro drug diffusion studies and skin irritation study. The primary skin irritation index was found to be 0.00 which inferred no skin irritation in rats. Three months of stability studies revealed F2 formulation and solid lipid nanoparticulate gel of Econazole nitrate were stable at 40±2° C and 75±5% RH.
Key words: Econazole nitrate, solid lipid nanoparticles, entrapment efficiency, cold homogenization.

Fungal infections are the most commonly experienced diseases affecting the skin, hair and nails. The treatment approaches for these infections includes both topical and oral antifungal agents. In topical administration of antifungals, the drug should pass through the stratum corneum that is the outermost layer of the skin to reach the lower layers of the skin especially viable epidermis.
The administration of these antifungals in the form of creams, gels, powder, and solution has the major drawbacks such as allergic and severe skin reaction when they are designed into these dosage forms directly. In order to reduce these drawbacks, novel drug delivery approaches are being projected and to maximize their activity.

Solid lipid nanoparticles (SLNs) are colloidal lipid nanoparticles with the sub-micron size range of 50-1000 nm and composed of biodegradable solid lipophilic matrix in which drug molecules can be loaded. SLNs combine the superiorities of all other nanoparticles such as liposomes, fat emulsion and polymeric nanoparticles.
Econazole nitrate (ECN) is an imidazole antifungal drug that inhibits the conversion of lanosterol to ergosterol by interacting with 14-? demethylase enzyme by which it prevents fungal organisms to produce vital substances required for their growth and function. ECN has the topical half-life of 35 min and very slightly soluble in water. ECN possess adverse effects such as irritation, burning sensation and contact dermatitis when it is applied topically.
SLNs ensures the close contact of antifungal drugs with the stratum corneum which results in the increase of the amount of encapsulated drugs penetrating into viable skin and their application on the damaged or inflamed skin as they are based on non-irritant and non-toxic lipids of Generally Recognized as Safe (GRAS) listed approved by FDA. An adhesive effect can be seen by solid lipid nanoparticles as they form a film on the skin after application, the adhesion increases with decreasing particle size. Occlusion can enhance the penetration of drugs through the stratum corneum by increased hydration. Moreover, these lipids are likely to minimize the danger of allergic contact dermatitis that may be induced by the drug.
The aim of this work was to formulate solid lipid nanoparticulate gel of Econazole nitrate and evaluated the dosage form for various parameters. In this work, an attempt was made to retain the solid lipid nanoparticulate gel of Econazole nitrate in the absorption site more than its half-life, reduction in dose frequency and toxicity and to control the drug release from the dosage form.

MATERIALS AND METHODS
Lipids such as stearic acid, palmitic acid and preservatives such as methyl parabhen and propyl parabhen were obtained from Karnataka Fine Chem, Bengaluru. Econazole nitrate, Poloxamer 188 and HPMC K4M were procured from Yarrow Chem Products, Mumbai. HPLC graded water was procured from SDFCL, Mumbai. All solvents and reagents used were of analytical grade.

Preformulation study:
Melting point of Econazole nitrate was determined by thiele’s tube method. Drug excipients compatibility studies were carried out by Fourier-transform infrared spectroscopy (FTIR spectrophotometer, Tensor 27, Bruker optics, Mumbai). In FTIR study, drug and lipids were individually analysed initially and later physical mixture of drug and lipid were analysed. The FTIR spectrums of drug with lipids were compared with the standard FTIR spectrum of the drug. DSC studies were carried out on DSC Q60, Shimadzu, Japan. Sealed and perforated aluminium pans were used for all samples in the experiments. Temperature calibrations were performed using indium as standard. An empty pan sealed in the same way as for the sample was used as a reference. The entire samples were run in nitrogen atmosphere at a scanning rate at 10° C/ min from 50 – 300° C. By comparing the DSC curves of a pure drug sample with that of formulation, the presence of an impurity can be detected in a formulation.

Standard calibration of Econazole nitrate was done using UV-Visible spectrophotometer (UV-1700, Shimadzu, Japan) with phosphate buffer pH 5.5 and the absorbance was measured at 271 nm.
Preparation of Econazole nitrate loaded solid lipid nanoparticles:
Econazole nitrate loaded solid lipid nanoparticles were prepared by using cold homogenization technique. The lipid was melted to approximately 10° C above its melting point; econazole nitrate was dispersed in this lipid melt and kept the same on ice bath to get drug lipid melt. This melt had to be grounded to obtain a fine powder. Drug lipid melt was dispersed in the cold aqueous phase of poloxamer 188 under magnetic stirring. The dispersion was homogenized at 15,000 rpm for 45 min by maintaining temperature below 10° C using IKA Ultra-turax homogenizer, Germany. The obtained emulsion was lyophilized to get the final product (Table 1).

Particle size analysis:
The particle sizes of solid lipid nanoparticles (SLNs) were measured by using Malvern Zeta sizer Nano ZS-90 which works on the principle of Dynamic Light Scattering that measures the diffusion of particles moving under Brownian motion and converts this to size and a size distribution using the Stokes-Einstein relationship. The nanosuspension was sonicated for 30 min followed by further dilution with HPLC graded water. The average particle size was determined from the particle size distribution data.

Zeta potential:
The zeta potential of SLNs were measured by using Malvern Zeta sizer Nano ZS-90 which works on the principle of Lasser Doppler Micro-electrophoresis which is used to measure zeta potential. An electric field is applied to a solution of molecules or a dispersion of particles, which then move with a velocity related to their zeta potential. This velocity is measured using patented laser interferometric technique called M3-PALS (Phase analysis Light Scattering).
Scanning electron microscopy:
SEM photographs were taken for the prepared nanoparticles using a scanning electron microscope (Carl Zeisus FESEM model number: Ultra 55 USA) at different required magnifications at room temperature. The photographs were analysed for morphological characteristics.
Drug entrapment efficiency:
The prepared SLN dispersion was centrifuged at 7500 rpm for 20 min at 4° C using REMI cooling centrifuge. The supernatant was analysed for the free drug content. The entrapment efficiency (%) of drug was calculated by the following equation:
%Entrapment efficiency = total drug content – free drug content / total drug content *100
Loading capacity:
Loading capacity is the amount of drug loaded per unit weight of the nanoparticles, indicating the percentage of mass of the nanoparticles that is due to the encapsulated drug. Loading capacity can be calculated by the amount of total entrapped drug divided by volume of water that is required to re suspend the nanoparticles.

Loading capacity = total drug content – free drug content / volume of water required to re suspend SLNs.

In vitro drug release studies:
The in vitro drug release profile of econazole nitrate loaded SLNs were studied using vertical diffusion cell. The dialysis membrane was soaked overnight in the pH 5.5 phosphate buffer. The calculated amount of nanoemulsion of econazole nitrate was kept in the donor compartment above the dialysis membrane. In the receptor compartment, 250 ml of phosphate buffer pH 5.5 was taken and placed on magnetic stirrer with the temperature of the assembly and rpm was maintained at 37 ± 0.5° C and 100 rpm throughout the study. Samples of 1ml were withdrawn at predetermined time intervals (1, 2, 3, 4,5,6,7 and 8 h) and replaced simultaneously with equal amounts of fresh buffer. After suitable dilution, the samples were analysed for drug concentration by UV spectrophotometer at 271 nm.

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