Formulation and Evaluation of Voriconazole Solid Lipid Nanoparticles as a Shampoo

Introduction to Solid Lipid Nanoparticles (SLNs)

Introduction of solid lipid nanoparticles (SLN) in 1991 provided an alternative to the traditional carrier systems such as emulsions, liposomes and polymeric micro and macro-particles. Solid lipid nanoparticles are composed of a high melting point lipid as a solid core coated by aqueous surfactant and the drugs used are generally of BCS Class II and IV with solubility related problems. Solid lipid particulate system is gaining more consideration as novel colloidal drug transporter for intravenous applications. This particulate system is sub-micron colloidal transporters (50–1000 nm), which are self-possessed of physiological lipid, distributed in water in aqueous surfactant solution. It has various advantages over traditional colloidal carrier system such as less size, higher surface area, effective drug loading and capacities.

Solid Lipid Nanoparticles for Topical Delivery

Solid lipid nanoparticles are composed of a high melting point lipid as a solid core coated with an aqueous surfactant and the drugs used are generally of BCS Class II and IV with solubility related problems. In recent years, solid lipid nanoparticles have been studied as a potential carrier for oral, intravenous, ocular, dermal, nasal, buccal, and vaginal drug delivery system. Solid lipid nanoparticles employ use of liquid lipids which safe, stable, and biodegradable in nature. Solid lipid nanoparticles have several advantages not only oral but for the topical delivery as well inducing drug penetration into the skin with reduced systemic exposure and less irritation. The drug delivery through the transdermal route has many advantages for administration of the drug in local therapy. Where the drugs are applied topically, the stratum corneum is the main barrier for the percutaneous absorption. The authorization of site-specific delivery to the skin is due to the small size and relatively narrow size distribution. Solid lipid nanoparticles offer a high affinity toward the stratum corneum and consequently an improved bioavailability of the encapsulated material to the skin is reached.

Advantages of Solid Lipid Nanoparticles

• These offers better bio compatibility due to the lipid content.
• Does not require special solvents for formulation.

Disadvantages of Solid Lipid Nanoparticles

• Particle growth which may lead to stability problems.
• Sometime burst release is offered.

Applications

• Can offer controlled drug release and drug targeting.
• Offers increase drug stability.

Methods of Preparation of Solid Lipid Nanoparticles

1. 1. High pressure homogenization.
      1. Hot homogenization
      2. Cold homogenization
   2. Ultrasonication / high speed homogenization.
      1. Probe ultrasonication
      2. Bath ultrasonication
   3. Solvent emulsification-diffusion method.
   4. Supercritical fluid method.
   5. Micro emulsion-based method.
   6. Double emulsion method.
   7. Spray drying method.
   8. Precipitation technique.
   9. Film-ultra sound dispersion.
   10. Solvent injection technique.

Principles of Drug Release

• In general, the principles of drug release from the SLNs formulations are as follows:
• There is an inverse relationship between the release of the drug and its partition coefficient.
• Drug release from the nanometer size range is higher due to higher surface area.
• Sustain drug release can be obtained by mixing the drug homogenously in the lipid matrix. Drug release depends on the drug entrapment model and type of lipid used in the preparation of SLNs.
• Fast drug release depends on the crystallinity behaviour of the lipid and high mobility of the drug in lipid. Crystallization degree and drug release are inversely proportional to each other.

Storage Stability of SLNs: The physical properties of the SLNs formulation in storage for a long interval of time can be estimated by monitoring changes in the value of zeta potential, drug content, particle size, its appearance and viscosity of the formulation with the function of time. Other parameters such as temperature and light appear to be of essential partner for long term stability. The zeta potential should remain higher than 60mv for a stable dispersion.

• • 4^oC-Most favourable storage temperature.
  • 20^oC - Long term storage did not result in drug loaded SLN aggregation or loss of drug.
  • 50^oC–at this temperature instant growth of particle.

Characterization of SLNs

Characterization of SLNs is very important due to colloidal particles of the formulation. The important parameters which can be evaluated for SLNs are particle size, zeta potential, and degree of crystallinity (polymorphism), time scale of distribution processes, drug content, surface morphology and in-vitro drug release. These parameters are evaluated by using different techniques like Photon correlation spectroscopy (PCS), laser diffraction (LD), Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Atomic force microscopy (AFM) and freeze fracture electron microscopy (FFEM).

Sterilization of SLN

SLNs must be sterile for intravenous and ocular route of administration. High temperature during sterilization by autoclaving causes hot o/w emulsion in autoclave as results there is modification of size of hot nano droplets. But on slow cooling SLNs are reformed but some nano droplets produce large size SLNs so to prevent this problem they are washed before sterilization and surfactant and co surfactant are used in smaller quantities in hot systems.

Fungal Infections (Ringworm of the Scalp)

Fungal infection has been divided into two major classes which are systemic and superficial categories. Severe skin diseases are caused by fungal species known as fungal infection. Classification of antifungal drugs is given according to the chemical structure such as

• • polyene anti fungals.
  • Allyl amine anti fungals.
  • Azole anti fungals.
  • Echinocandin anti fungals.
  • Miscellaneous.

Sources of Infection

Dermatophyte group of fungi cause an infection known as scalp ringworm. This group of fungi is categorized into three groups according to the area in which they usually developed. Geophilic organisms live in soil, zoophilic organisms on animals, and anthropophilic organisms on humans. In most of the cases of childhood ringworms from last twenty years have been spread by zoophilic organisms Micros Porum Canis or Trichophyton verrucose (from cattle). Recently, the predominant organism has changed to an anthropophilic one, T.Hondurans, and it spreads directly from child to child. In Britain's larger cities this organism is responsible for most of the scalp ringworm.

Treatment for Scalp Ringworm

1. Griseofulvin has been used as a best antifungal agent for the treatment of scalp ringworm from 1950?s. For the treatment of fungal infection 8-12 weeks, regular prescription of the griseofulvin is required. There is no liquid dosage form of griseofulvin is present in the market. So, in the pediatric patient is given in crushed form with a little amount of water.
2. Terbinafine is another drug which can be used in place of griseofulvin in the treatment of scalp ringworm. This drug is not licensed for the treatment of scalp ringworm but it is effection in this fungal infection. For this infection, the four-week course is required. To clear the infection completely, it is necessary to complete the course. Other drugs that can be used in the treatment Voriconazole and fluconazole.

 MATERIALS AND METHODS

List of Materials Used in the Study.

List of Equipment/ Software Used in Study.

Formulation Of Voriconazole Solid Lipid Nanoparticles

Solvent Evaporation Followed by Homogenization Method

Accurately weighed amount of Voriconazole, cholesterol and span 60 were transferred in to beaker and then transfer accurately measured quantity of methanol was added. The solubility was provided by applying sonication for 10-15 minutes. Measured quantity of tween 80 and water was added and kept for stirring on magnetic stirrer for 15 min to produce an aqueous medium. The lipid phase was then slowly added into aqueous phase under continuous stirring and the organic solution was removed by evaporation. After removal of organic solvent, the nanoparticles are formed in aqueous medium. Finally, the aqueous medium was homogenized 1000- 3000 rpm for 15-30 min.

Formulation Development Trials

Preparation of Optimized Formulation by Doe Technique

A Central composite design (CCD) was selected for two factors at three levels (X1and X2) to optimize the response variables Y1 and Y2 respectively i.e. entrapment efficiency, drug permeation at one hour (Q1). Design expert software was used for employing this design. It is summarizing an account of twenty-eight experimental runs studied. Formulation at central point (0, 0) was studied in quintuplicate. Three levels - 1, 0 and +1 were decided. Based on the preformulation studies, formulations were designed. CCD for two factors at three levels, each was selected to optimize the varied response. Design expert software was used for employing this design. The variables used were amount of Voriconazole, span 60 and methanol. The translation of coded factors levels and amount of ingredient is listed.

In-vitro Drug Release

Release of drug from the prepared nano suspension formulations was studied in-vitro using the semi permeable membrane dialysis bag. The dialysis bag was soaked in glycerin for whole night i.e. for 12 h before performing the permeation study. The soaking helps to soften the membrane and opening of pores. One end of the tube was closed by tying a tight knot with a short piece of string about 1 cm from the end of the tubing. The tube was then filled the tube with 5 ml of prepared nano suspension through the open end. 100 ml of pH 5.5 phosphate buffer solution containing 30 % methanol was transferred into a clean 250-ml beaker. The dialysis bag was placed in a beaker containing the buffer solution and provided continues stirring with the help of magnetic stirrer. The samples were collected from the beaker at different time intervals, by replacing the same amount of fresh sample to maintain the sink condition. The collected samples were analyzed by using the UV Visible spectrophotometer at 260 nm for determining the concentration of drug.

Incorporation of prepared nanoparticles into shampoo

The shampoo prepared with variable ratio of components were as shown by using the appropriate number of prepared nanoparticles, peppermint oil, lemon oil, carboxy methyl cellulose (CMC) (2% gel) and sodium lauryl sulphate (SLS) the ingredients were transferred into motor pestle and mixed uniformly then it is transferred in clean and dried glass container. Small amount of EDTA was mixed in little amount of water and added in to the glass container. Then, few drops of saturated NaCl solution were added to modify or increase the viscosity of the shampoo. Finally, the volume was made up to the 100 ml by using the distilled water.

Preparation Of Voriconazole Shampoo

• Voriconazole (active pharmaceutical ingredient).
• Lemon Oil (for fragrance and potential soothing properties).
• Peppermint Oil (for fragrance and cooling effect).
• Sodium Lauryl Sulfate (SLS) (surfactant).
• Carboxymethyl Cellulose (CMC) (thickening agent).
• Ethylenediaminetetraacetic Acid (EDTA) (chelating agent).
• Saturated Sodium Chloride (NaCl) (osmotic agent).

The Voriconazole solid lipid nanoparticle shampoo was formulated by using F4 optimized formulations.

Method

• Select Lipids: Choose an appropriate solid lipid such as stearic acid. The lipid phase will form the matrix for the SLNs.
• Melting the Lipid: Weigh the solid lipid and melt it at around 70°C using a water bath or heating plate. Ensure the lipid is completely melted and homogenized.
• Add Voriconazole: Weigh the appropriate amount of Voriconazole and dissolve it in the molten lipid phase. Ensure complete dissolution by stirring gently.
• Incorporate Oils: Add Lemon oil and Peppermint oil to the lipid phase at this stage. These       essential oils will add fragrance and may enhance the user experience.
• Dissolve Surfactants: In a separate container, dissolve Sodium Lauryl Sulfate (SLS) in distilled water. SLS will act as an emulsifier, helping to stabilize the SLNs in the shampoo formulation.
• Add CMC: Dissolve Carboxymethyl Cellulose (CMC) in the aqueous phase to act as a thickening agent, giving the shampoo the desired viscosity. Stir until fully dissolved and homogeneous.
• Incorporate EDTA: Add EDTA (typically in a concentration of 0.05–0.1%) to the aqueous phase as a chelating agent. It will help reduce metal ion interference that might destabilize the formulation.
• Prepare Saturated NaCl Solution: Prepare a saturated sodium chloride solution in water. This will help with adjusting the osmotic pressure of the formulation and help in the stability of the SLNs.
• High-Shear Homogenization: Slowly add the lipid phase (containing Voriconazole, Lemon Oil, Peppermint Oil, and melted lipid) into the aqueous phase (containing SLS, CMC, EDTA, and Saturated NaCl) under continuous stirring. This step is crucial for forming the SLNs. Use a high-shear homogenizer or sonicator to reduce the particle size of the SLNs and ensure a stable dispersion.
• Cooling: Once the SLNs are formed, allow the mixture to cool down to room temperature. During cooling, the solid lipid will form nanoparticles encapsulating Voriconazole.
• Adjust pH: After the SLN dispersion has been cooled, check the pH of the formulation. The ideal pH for shampoos is typically in the range of 4.5 to 5.5. Adjust, if necessary, by adding small amounts of citric acid or sodium hydroxide.
• Final Viscosity Adjustment: If the shampoo is too thin, adjust the viscosity further by adding more CMC or other thickeners. If it is too thick, add more water or a small amount of saline.

Stability Testing

Ensure the formulation remains stable over time (e.g., after 24-48 hours) to check if the SLNs are uniformly dispersed and not settling out. You may want to perform a particle size distribution analysis to ensure consistent SLN size.

Evaluation Of Voriconazole Shampoo

pH Determination

The pH of the shampoos (S1-S6) was determined by using digital pH meter.

Rheology   

Viscosity of the shampoo preparations was determined by using Rheodyne Rheometer at 37 °C temperature with a shear rate (1/S) for 4 minutes. The plot of shear stress Vs shear strain was obtained. Similarly, a plot of shear rate Vs viscosity was plotted. The equation was applied to power law Kt n. Where K represents the consistency, n helps to represents flow of the system.

Drug Content and Content Uniformity

To ensure the drug content and uniform distribution of Voriconazole,10 ml of prepared shampoo was taken and was shaken with sufficient quantity of methanol to extract the drug. The drug content was then determined by using UV spectrophotometer at λmax of 260 nm. The procedure was repeated in triplicate to ensure the uniform drug content. For analysis of uniform drug distribution, the samples of shampoo were taken from 3 to 4 separate points and were determined spectrophotometrically.

In-vitro Permeation Study

The in-vitro permeation studies were carried by using the diffusion membrane, which was activated by keeping it in glycerin for overnight. The fresh diffusion membranes were taken each time for carrying in vitro permeation studies employing Franz diffusion cell. The different batches of shampoo were observed for their permeability to find the optimized batch. The diffusion membrane was mounted on the receptor chamber with cross sectional area of 3.91cm². The receptor compartment was filled with 25 ml of pH. 5.5 phosphate buffer. The cell was jacketed to maintain the temperature similar to skin i.e.32 ± 0.5°C at 50 rpm. Each batch of shampoo was taken on the membrane and 5 ml aliquot of sample was withdrawn at different time intervals. Same amount of buffer was replenished to the compartment to maintain the phase equilibrium. The samples withdrawn were quantified spectro photometrically at λmax of 260 nm. Release study of blanks i.e. formulation without drug was also employed for each formulation.

Stability Study of Shampoo

The stability study of shampoo was carried at different temperatures i.e. 4±3°C (Refrigerator; RF) and under stress conditions 50±2°C for a period of 15 days. The samples were taken periodically to analyze drug content for Voriconazole shampoo.

Analysis of Release Mechanism     

In-vitro release kinetics of Voriconazole from shampoo was analyzed by mathematical modeling. The in vitro drug release data obtained were fitted to various release kinetics models (Higuchi 1963; Korsemeyer, Gurnyetal. 1983; Peppas and Sahlin 1989) viz., zero-order, first-order, Higuchi, Hixson-Crowell cube root and Korsemeyer- Peppas mathematical models. Selection of a suitable release model was based on values of r² (correlation coefficient), k (release constant) and n (diffusion exponent) obtained from curve fitting of release data. The data for optimized shampoo was compared with the marketed antifungal shampoo.

In-vitro Anti-Fungal Study

The prepared optimized shampoo formulation was tested in a triplicate manner using agar cup method against Candida albic and strain. Cup of 10 mm in diameter were made aseptically in Sabouraud dextrose agar after being inoculated with tested fungal suspension strain by spreading on the agar surface. The cups were filled with formulation and control (Iraz® shampoo) by micro pipette. Then, the zone of inhibition of each cup was observed; the radius of the zone of inhibition was calculated and was compared with the control formulation.

RESULTS AND DISCUSSIONS.

Calibration Data of Voriconazole.

The calibration plot of Voriconazole was prepared by taking 4, 8, 12, 16, 20 μg/ml concentrations of Voriconazole in methanol as shown in table 6.3. The experiments were performed in triplicate to find the standard deviation and percentage relative standard deviation. Absorbance range was found to be 0.171- 0.850. The regression coefficient (R² value) was 0.9993 which showed linearity between 4-20 μg/ml concentrations. The Lambert Beer law was obeyed within the linearity range. The standard regression equation was found to be y = 0.0422 x + 0.0042.

FTIR Spectra

Solubility Studies:

Solubility of Voriconazole in Various Organic Solvents and Buffers IP, 2010. The solubility data was obtained for Voriconazole at 25°C using an ultraviolet absorption assay method to determine the concentration of drug present in the saturated solutions (IP, 2010). The solubility profile of drug with the organic solvents and buffers was helpful to determine that whether the drug was dispersed or solubilized in the organic solvents and buffers systems. The solubility profile in the decreasing order of solubility was found to be as follows: pH 5.5 phosphate buffer > pH 4.6 acetate buffer > water. The pH solubility profile of Voriconazole was generated and was reported and shown in table 6. The solubility profile signifies that the drug get sparingly solubilized in the methanol so it can be dispersed in the lipid system during the formulation. The dispersibility can help in enhancing the pay load of drug in the SLNs. Thus, the solubility profile helped to generate the supportive information regarding the final formulation.

Particle Size Analysis

The mean particle size and Poly dispersity Index (PI) of nanoparticles are presented in table. The differences in the particle size of SLNs formulations prepared with variable ratios of drug, span 60, methanol, tween 80 and homogenization were utilized to find the optimized formulation. The particle sizes were falling in the range of 10 -100 nm as shown. In general, nano and micro carriers with Poly dispersity Index (PI) value higher than 0.3 shows large size distributions and have the tendency to aggregate. Smaller value of PI (PI<0.3) indicates a homogeneous population of nano particles. The optimized formulation showed average particle size of 94.5 nm with PI of 0.261

Optical Microscopy

Transmission Electron Microscopy

Entrapment Efficiency of Nanoparticles of Prepared Formulations.

Drug Permeated at 90 min for Prepared Formulations.

The comparatively depicts entrapment efficiency of all the 28 formulations. From the entrapment data it was observed that the ratio of the five components within an optimum range offered good entrapment efficiency. The effect of the quantity of methanol was also determined from the study. When the formulation with absence of methanol was prepared, it appeared as a turbid form and was not able to form the nano suspension dispersion. The entrapment efficiency could not be determined because of the turbidity. Thus, it was observed that the methanol plays an important role in the formation of the nanoparticles by promoting particle dispensability. Thus, it may be inferred that as the nano suspension shows better drug entrapment, they also have effect on the drug loading. The enhanced entrapment efficiency is because of the structure of the composition of nano suspension which offers more space for the drug particles to get entrapped. The maximum entrapment efficiency was absorbed for F4 with 91.48% and minimum was for F10 with 14.45 %, although formulation F12 and F28 offered no entrapment due to absence of methanol and drug in the formula respectively.

In vitro drug permeation of formulation was performed for the prepared 28 formulations were shown in table 6. The drug permeation was absorbed for 90 minutes which also serves as one of the responses in the optimization study. The results depicted the drug permeation for the formulation with the minimum release of 24.52 % for F23 and maximum permeation was 89.90% for F4 formulation. Although in formulation F12, there was no permeation was observed for the drug due to absence of methanol in the formula. Additionally in case of F28 there was no permeation due to absence of drug in the formula. Therefore, the observed range of permeation include formulation F12 and F28 was within 24.52-89.90%.

Validation of Optimized Batch of SLNs Dispersion.

Based on the results of studies carried out to select suitable polymer, solvents and preparation method, different formulations were prepared. The formulations varied in terms of amount of drug, span 60, methanol, tween 80 and homogenization. A Central Composite Response Surface Rotatable Design was employed to obtain 28 different factor combinations and replicates where two independent variables were studied at two levels (Stat-Ease, 2011). Different factor combinations that were obtained and experimentally run to measure the responses Y1 (percent entrapment efficiency) and Y2 (percent permeability at 90 min) are given in table. Figure 18 shows the FDS plot of the mean standard error over the design space. A fraction of design space (FDS) graph indicates the repeatability of experiment and possibility of detecting a significant effect. The FDS curve is the percentage of the design space volume containing a given standard error. The FDS graph in figure reveals a flatter and lower curve that means the overall prediction error will be constant and small. The value of FDS was found to be 0.938 which means that fraction of design space capable of predicting the true average within 1, standard deviation was 89.2%, which is higher than the recommended 80% value.

Comparison of Experimental Results with Predicted Values with Percentage Error.

Percentage Encapsulated Drug Loss from Optimized Cubosomes at Different Temperature Depicting Stability Study.

Physical Evaluation of Formulated Shampoo Preparations.

The apperance of shampoo is one of the important aspects to be considered. The formulated shampoo preparations (S1-S6) were evaluated for physical characteristics such as color, odor, transparency, and nature.

pH Profile of Shampoo Formulations.

The pH of the shampoo is usually kept within slightly alkaline to neutral range to avoid irritation to scalp and eyes. The pH of prepared Voriconazole shampoo formulations was observed by using the pH meter (Systronic, μ pH system, India). The pH formulations were from 4.53-5.21. Therefore, by referring to literature and available reports, the shampoo with pH range for the (S6) 5.21 can be considered good which was shown. The pH profile for the formulations were compared with the marketed shampoo which was found to be 5.42

Foaming Ability of Shampoo Formulations.

The foaming index was determined to evaluate the ability of prepared shampoo to provide foam when applied. The foaming ability in shampoo is associated with the detergency ability for better cleaning. Therefore, the foaming index was calculated for the six formulations of shampoo, out of which, formulation S6 offered best related to foaming. The results were then compared with the foaming ability of marketed shampoo

Percentage Permeation of Voriconazole from Optimized Formulations and Marketed Ketoconazole Shampoo.

Percentage Permeation of Voriconazole from Optimized Formulations and Marketed Ketoconazole Shampoo.

For analysis of uniform drug distribution, the samples of Voriconazole shampoo were taken from 3 to 4 separate points and were determined spectrophotometrically. In table depicts the results for content uniformity as the drug content was found to be within the range of 97-99 % so the prepared shampoo formulation containing Voriconazole can be considered as homogeneously dispersed.

Stability Study of SLNs Shampoo at Different Temperature Conditions.

The stability study of formulated shampoo was carried at different temperatures i.e. 4- 8°C (Refrigerator; RF) and 50 ± 2°C (Room temperature; RT) for a period of 15 days as shown in table 6.27. The change in pH along with the drug content of shampoo was checked at various time interval and the results revealed that the shampoo was stable over both the conditions with small changes. The prepared shampoo thus, can be considered stable.

Various Kinetic Models of Optimized Shampoo (OPS).

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The drug release profile was also evaluated for „goodness-of-fit? into various mathematical model equations such as zero order, first order, Higuchi matrix, Korsmeyer- Peppas and Hixson-Crowell cube root equation by using DD solver® tool. These kinetic models were used to understand the release mechanism of drug from the shampoo. The r2 and k values of the model equation are shown. The model with r2 value near estto 1.000 was considered as the „best-fit? model for the formulation. The maximum r2 value was found to be for kosmeyer peppas model with n >0.5 which indicates release following fickian diffusion and secondly for Hixson Crowell model. Therefore, Korsemeyer-Peppas model depicts the release as modified due to presence of lipid and surfactants for diffusion of drug and the second model represents dissolution rate limited drug release.

The Inhibition Zones of Gels of Fluconazole.

Showing SDA Media with Cups (10mm) with Test, Standard and Control Formulation