Advantages as drug delivery systems which are as fallows.

• Entirely biodegradable, biocompatible, flexible, nonimmunogenic, non-toxic, drug delivery system for non-systemic and systemic administrations
• Solubility enhancement of amphiphilic and lipophilic drugs
• Increased effectiveness and potency
• To attain active targeting flexibility to pair with site-specific ligands
• For labile drugs superior stability
• Decreased toxicity due to drug targeting properties
• Site avoidance effect resulting in reduced toxicity
• Improved pharmacokinetic effects

Skin chronic illness is atopic dermatitis (AD) which is described by itchy, scaly, and reddish lesions along with incessant inflammatory patterns. Usually in a familial context AD happens with an occurrence of atopic conditions like bronchial asthma, allergic rhinitis, and food allergies. The major therapeutic approach for AD is still topical drug administration. Numerous disadvantages may be mentioned as low efficiency, decreased patient fulfillment (a result of unfavorable effects as allergy or irritation of the skin), and the specificity of these systems in delivering active substance. The improved drug delivery must demonstrate the stratum corneum penetration capacity, to a range of substances that is naturally impermeable, so as to boost drug targeting and decrease adverse effects. To inverse the pathological effects of AD, there is still no medication good enough. Because of this, for topical drug administration, the preparations based on nanoparticles (NPs) have been used and are predicted to overcome the afore mentioned limitations. Atopic eczema is another name of AD, which is the most general disease of the skin, concerned with skin deterioration, a pattern of pruritus, and chronic inflammation. In the early-mid- or late-stages of lives, this disease affects people and there is no identified cure to date. The levels of immunoglobulin E (IgE) are enhanced significantly over the course of the disease, producing cutaneous signals that come into view at an early age and remain till late stages of life, after they start to fade away. Due to this, AD is also called an “allergic march”. There is variety of factors for AD and it is caused by the response of the immune system, impairments against the skin natural barrier, and the environmental damages. Due to this the activity of T-lymphocytes and mast cells gets accelerated, along with inflammatory signals. Increased IgE levels in the patient’s serum, and pruritus. Serous exudate, skin rashes, pruritus, xerosis, papules related to erythematous pruritic lesions are predominant features in AD patients. Two theories are specifically advanced to explain the appearance of skin rash/cutaneous lesions and skin disruption. The first theory is called “in-out” and on an imperfect skin, it originates from the idea of the burden related to adaptive immune system, the second theoryis named “inside-out” and was introduced subsequently. The first theory is more up-to-date, and in the immune system activation its foundation explains, the vital role of skin impairment. The two theories are complementary and are not self-excluded.

Materials

Table 1: List of Materials

b. Instruments and Equipments

Table 2: List of Equipment’s

Preformulation Studies

UV Spectroscopic Estimation of Hydroquinone: Determination of lambdamax of Hydroquinone in phosphate buffer pH 7.4. Stock solution of 100 mg/ 100ml hydroquinone solution was prepared by dissolving 100 mg of pure drug in 10 ml of ethanol and made up to 100 ml with phosphate buffer pH 7.4. This is designated as stock solution A (1 mg/ml). From the stock solution A, 5 ml was taken and diluted to 100 ml with phosphate buffer pH 7.4 to give the concentration 50 ?g/ml (stock solution B). The above solution was scanned between 200-400 nm. The sample showed a ?max of 293 nm.

 Standard plot of Hydroquinone: From the above stock solution B?, aliquots of 1, 2, 3, 4, and 5 ml were transferred to 10 ml volumetric flasks and made up to the mark with methanol to get concentrations of 5, 10, 15, 20, and 25 ?g/ml. The absorbance of these solutions was measured at 228 nm and a graph of concentration versus absorbance was plotted. The calibration curve data are shown in Table 2 and calibration curve is shown in the Figure 7.

COMPATIBILITY STUDIES:

Done by Fourier transform infrared spectroscopy. FT-IR analysis was carried out for pure drug and drug with polymers, using KBr pellet method on FTIR spectrophotometer (FTIR-8400S, Shimadzu, Japan). The pure drug was mixed with KBr in the ratio of 1:3 and punched in a hydraulic press at 5-6 ton load. The prepared pellets were scanned from 4000 to 400 cm-1 using FT-IR spectrophotometer. The FT-IR spectra of the physical mixture were compared with the spectra of pure drug.

PREPARATION OF HYDROQUINONE LIPOSOMES:

A mixture of different ratios of phospholipids (P-90H) and cholesterol in chloroform were taken in round bottom flask. A thin film was formed on the inner side of round bottom flask by evaporating chloroform under vacuum in rotary flash evaporator at 40°C. The required amount of phosphate buffer saline (PBS), pH 7.4 containing hydroquinone (2%) was added at 40°C and the same temperature was maintained for 1 hour to anneal liposome structures. Further the flasks were shaken for 5 h with intermittent sonication using a bath sonicator99,100.

PREPARATION OF HYDROGEL:

To create a 0.5% gel, 0.5 g of carbopol 934 was weighed, distributed in 100 ml of distilled water, and gently stirred. This was done for 24 hours [Table 4]. To keep the gel consistent, 2 milliliters of glycerin were added later. Two types of preservatives were added to the gel: methyl and propylparaben. In the same way, Carbopol gels at 1% and 2% were made.

PREPARATION OF LIPOSOMAL HYDROGEL:

To extract the not entrapped drug, 10 milligrams of liposome formulation were dissolved in 10 milliliters of ethanol and centrifuged for 20 minutes at 6000 rpm. The supernatant was decanted after the sediment was added to the gel vehicle. Using a slow mechanical stirrer (REMI type BS stirrer, Vasai, India) at 25 rpm for 10 minutes, the hydroquinone-loaded liposomes (equal to 0.1) were incorporated into the gel. The optimized formulation used three distinct gel concentrations (0.5, 1, and 2% w/w).

CHARACTERIZATION OF LIPOSOMES:

1. Vesicle shape determination:

The determination of shape and surface morphology was done by scanning electron microscope Jeol JSM-5600, Japan. SEM analysis of the samples revealed that all liposome prepared at UGC-DAE Consortium for scientific Research, Indore.

2. Entrapment efficiency:

Liposomal formulations were subjected to centrifugation using cooling centrifuge at 2,500 rpm for about 2 h. The clear supernatant was separated then carefully to separate the unentrapped nadifloxacin and sediment was then treated with 1 ml of methanol to lyse the vesicles and diluted to 10 ml with methanol and absorbance of both solutions was observed at 293 nm. The amount of nadifloxacin in supernatant and sediment gave a total amount of hydroquinone in 1 ml of dispersion. The entrapment efficiency was calculated using this formula.

% Entrapment efficiency= Entrapped drug /Total drug added ×100

3. In vitro drug diffusion studies:

The release of drug was determined by using the dialysis membrane mounted on the one end of open tube, containing 5ml of liposomal suspension (10 mg of hydroquinone). The dialysis tube was suspended in 200ml beaker containing 100 ml of PBS (pH 7.4).

EVALUATION OF LIPOSOMAL HYDROGEL

1. Percentage Drug content: The liposomal hydrogel sample (1 gm) was withdrawn and dissolved in 100ml of phosphate buffer (pH 7.4). The volumetric flask containing liposomal hydrogel solution shaken for 2hr. This solution was filtered and estimated using UV spectrophotometer at 293 nm.
2. Visual inspection: All developed liposomal hydrogel formula was checked for their homogeneity, color and presence of lumps by visual inspection after the gels have been set in the container.
3. Measurement of pH: The pH of various gel formulations was determined by using digital pH meter. one gram of liposomal hydrogel was dissolved in 100 ml distilled water and stored for two hours. The measurement of pH of each formulation was done in triplicate and average values are calculated.
4. Spreadability: A sample of 0.5 g of each formula was pressed between two slides (divided into squares of 5 mm sides) and left for about 5 minutes where no more spreading was expected. Diameters of spreaded circles were measured in cm and were taken as comparative values for spreadability. The results obtained are average of three determinations.
5. Viscosity determination: The viscosity of gel was determined by using a Brookfield DV-E viscometer model with a T-Bar spindle in combination with a helipath stand. The spindle#6 was used for determining the viscosity of the gels the factors like temperature, pressure and sample size etc. Which affect the viscosity was maintained during the process.
6. Homogeneity: After the liposomal hydrogel have been set in the container, all developed liposomal hydrogel were tested for homogeneity by visual inspection. They were tested for their appearance and presence of any aggregates.
7. Extrudability study:The liposomal hydrogel were set in the container, the formulations were filled in the collapsible tubes. The extrudability of the formulation was determined in terms of weight in grams required to extrude a 0.5 cm. ribbon of gel in 10 second.
8. Washability: Liposomal hydrogel formulation was applied on the skin and then ease and extent of washing with water were check manually.

 IN VITRO DRUG RELEASE STUDIES:

The liposomal hydrogel in release of drug was determined by using the dialyis membrane mounted on the one end of open tube, containing 1gm of liposomal hydrogel. The dialysis tube was suspended in 200ml beaker containing 100 ml of PBS (pH 7.4).

STABILITY STUDY OF LIPOSOMAL HYDROGEL FORMULATION:

The prepared liposomal hydrogel formulation was stored at room temperature and refrigeration for a period of 30 days and then visually observed for clearance of every week. No significant visual change was observed during the study period. The optimized formulation was packed in a screw capped bottle and studies were carried out for 12 months by keeping at 25±2°C and 60 ± 5% RH, 30± 2°C and 65 ± 5% RH. And for 6 months for accelerated storage condition at 40± 2°C and 75 ± 5% RH. Samples were withdrawn on 0, 3, 6 and 12 months for long term storage condition and 0, 3 and 6 months for accelerated storage condition and checked for changes in physical appearance and drug content.

PREFORMULATION STUDY

UV Spectroscopic Estimation of Hydroquinone:

Figure 6. ?max of hydroquinone in phosphate buffer pH 7.4.

Table 3. Calibration curve data of Hydroquinone in phosphate buffer pH 7.4

       
           
       
    Figure 7. Calibration curve of hydroquinone in phosphate buffer pH 7.4

By Fourier transform infrared spectroscopy. FT-IR studies are used to determine the possible interaction between the drug and excipients used. The peak obtained indicates characteristic groups and the bonds present in the compound. Hydroquinone shows the characteristic peak at 3829.67 cm-1 due to OH stretching, 3122 cm-1 due to CH stretching vibration, and a peak at 1511.88 cm C=C stretching vibration.

PREPARATION OF LIPOSOMES

Table: 5 Composition of liposomes

Table: 6. Composition of optimized liposome formulation

PREPARATION OF HYDROGEL

Table: 7. Composition of different hydrogel base

To extract the not entrapped drug, 10 milligrams of liposome formulation were dissolved in 10 milliliters of ethanol and centrifuged for 20 minutes at 6000 rpm.

CHARACTERIZATION OF LIPOSOMES