INTRODUCTION:

In recent years, the scientists have focused a significant portion of their research efforts on the development of innovative drug delivery systems. These systems target the delivery of the drug to a particular area of the body in order to enhance the drug's therapeutic efficacy while simultaneously mitigating the drug's negative side effects. Research in the field of therapeutics has focused on controlling various issues by targeting medications through carrier systems. The utilisation of a carrier, such as albumin conjugates, antibodies, lectins, glycoprotein DNA, dextran, polysaccharides, nanoparticles, or liposomes, is typically required in order to successfully target the medicine. Liposomes are microscopic vesicles that are composed of one or more concentric phospholipid bilayers surrounding an aqueous membrane. Liposomes can be found in a variety of different organisms. Since hydrophilic medications are captured in the aqueous sections and hydrophobic items are located in the hydrocarbon zone, this makes it possible to combine a diverse assortment of compounds. In comparison to the standard dosing system, the lipidomal delivery method for a greater amount of medicine to be accumulated in the dermis while simultaneously requiring a lower penetration flux. There is no doubt that this will result in the continued use of an older medicine that has a superior and well-established therapeutic index and fewer adverse effects. A wide variety of bacterial illnesses can be remedied with the use of the antibiotic drug known as azithromycin. Illnesses of the middle ear, strep throat, pneumonia, traveler's diarrhoea, and particular other intestinal infections are included in this category. In combination with other antimalarial drugs, it is sometimes employed in the treatment of malaria. It can be administered intravenously or orally, with daily doses being given only once^1-8.

The leaves and buds of the Camellia sinensis plant are used to manufacture green tea. Unlike the leaves and buds used to make oolong and black teas, the Camellia sinensis leaves and buds used to make green tea have not been withered or oxidised. It is believed that China was the first country in East Asia to produce green tea, but since that time, its production and manufacture have expanded to other countries in the region. There is more than one type of green tea, and these teas are distinguished from one another in significant ways by the type of C. sinensisutilised, the growing conditions, the horticulture practises, the production processes, and the time of harvest. Although a significant amount of research has been conducted on the topic of the possible health consequences of drinking green tea on a regular basis, there is little evidence to suggest that drinking green tea has any effects on health^9-13.

Formulation of Liposomes:

Preparation of liposomes containing Azithromycin: Using the thin film hydration approach, azithromycin, soya lecithin, and cholesterol were used into the preparation of five different liposomal formulations. According to table 4, the soya lecithin, cholesterol, and azithromycin that were used in the experiment were combined in the following proportions: 50:50:20 (F1), 60:40:20 (F2), 70:30:20 (F3), 80:20:20 (F4), and 90:10:20 (F5). Each preparation consisted of the utilisation of azithromycin. Because azithromycin is water soluble, it was combined with the aqueous phase throughout the manufacturing process. After dissolving cholesterol and soy lecithin in 10 millilitres of chloroform solution, the solution was allowed to settle for evaporation until a thin coating of the solid admixture was deposited on the walls of the flask. The flask was then discarded. In order to create poly dispersed liposomes from the dried lipid film, it was first rehydrated with 10 millilitres of distilled water that included azithromycin. This was done by stirring the solution in a vibrator for approximately one hour^14-18.

Preparation of liposomes containing Green tea:

The green tea, soya lecithin, and cholesterol that were used in the preparation of the five different liposomal formulations were subjected to the thin film hydration method. According to the information presented in table 4, the soya lecithin, cholesterol, and green tea that was utilised came in the following ratios: 50:50:20 (F6), 60:40:20 (F7), 70:30:20 (F8), 80:20:20 (F9), and 90:10:20 (F10). In each of the preparations, green tea was utilised. Due to the fact that green tea dissolves well in water, it was added to the aqueous phase. After dissolving cholesterol and soy lecithin in 10 millilitres of chloroform solution, the solution was allowed to settle for evaporation until a thin coating of the solid admixture was deposited on the walls of the flask. The flask was then discarded. In order to create poly dispersed liposomes from the dried lipid film, it was first rehydrated with 10 millilitres of distilled water that contained the medicine. This was followed by shaking the mixture in a vibrator for approximately one hour^14-18.

Table 1: Formulation table of liposomes

Drug Used	Formulations	Formulation Ratios (Lipids: Cholesterol: Drug)
Azithromycin	F1	50:50:20
	F2	60:40:20
	F3	70:30:20
	F4	80:20:20
	F5	90:10:20
Green Tea	F6	50:50:20
	F7	60:40:20
	F8	70:30:20
	F9	80:20:20
	F10	90:10:20

Characterization of Liposomes^19-25:

1. Determination of the average size and size distribution in liposomes:

2. Determination of shape of the liposome

3. Drug content

4. Encapsulation efficiency

5. Stability of liposomal suspension

Stability of the selected suspension formulation were carried out at 25⁰C±2⁰C / 60% RH±5% RH for 3 months. Effects of temperature and RH on the vesicle size and drug content were studied.

6. In-vitro drug release study

Preparation of Liposomal Gels²⁶:

Formula:

Carbopol 940: 2% w/w

Triethanolamine: q.s.

Methyl hydroxy benzoate: 0.15 % w/w

Distilled water: 95.8 % w/w

Procedure:

1. Preparation of Carbopol 940 Gel Base:

In order to obtain a carbopol 940 dispersion that was uniform across the medium, the carbopol was slowly dusted into 5 ml of water while the water was continually agitated. After first being dissolved in a separate volume of water (five millilitres), the methyl hydroxy benzoates were then added to the carbopol dispersion. Water was used to make the necessary adjustments to the final quantities, and triethanolamine was applied to bring the pH back to a neutral state. This preparation was left out at room temperature overnight in order to ensure that the carbopol would have a homogeneous texture and appearance and that any air bubbles would be eliminated.

2. Incorporation of Liposomes into Gel Base:

The drug-loaded liposomes were integrated into the gel base (carbopol 940) in such a way that the final formulation contained 1% w/w of the drug. This was accomplished by levigating 0.2 g/10 ml of liposomal suspension with 10 g of gel base.

Physical Evaluations:

Preliminary evaluations of formulations were carried out as follows:

pH, Viscosity, Spreadability, In-vitro drug release, Stability study

RESULTS:

Characterization of Liposomes:

Determination of Average Particle Size of Liposomal Formulation: The average particle size of liposome was carried out by using the microscopic method. The results are given in tables 2 and 3 and figures 1 and 2.

Tab 2: Size Analysis of Liposomal Formulations (F1)

Size Range		Mid value (d)	Particles (n)*	Nd
Eye Piece Division	µm	Mid value (d)	Particles (n)*	Nd
0-2	0 - 6.25	3.125	109	340.6
2-4	6.25-12.50	9.375	39	365.6
4-6	12.5-18.75	15.600	2	31.2
6-8	18.75-25	21.870	0	0

Average particle size = Σ nd = 737.445 =4.91 μm.

Σn 150

In the similar way average particle size of other formulations was calculated.

Table 3: Data of Average Particle Size Determinations

S. No	Formulations	Average Particle Size* (μm±SD)
1	F1	4.91±0.015
2	F2	5.20±0.016
3	F3	5.75±0.0137
4	F4	5.79±0.0205
5	F5	6.25±0.024
6	F6	4.26±0.0127
7	F7	4.57±0.0214
8	F8	5.29±0.0205
9	F9	5.87±0.0169
10	F10	6.58±0.025

Fig. 1: Size distribution of azithromycin liposomes

Fig. 2: Size distribution of green tea liposomes

Drug content:

The process was followed to determine the amount of drug contained within the liposomal formulation. After being centrifuged and rinsed three times with distilled water, the liposomal suspension was lysed with a 1% v/v solution of triton X100. After being centrifuged for one hour at 2000 rpm, the 0.5 ml supernatant was separated and examined.

Encapsulation efficiency:

Complete vesicle disruption was followed by extraction using a solution of triton X-100 that was 1% volume-to-volume (v/v), which allowed for the determination of the quantity of medication that was enclosed inside the liposomes. Table 4 presents the percentage of the medicine that is encapsulated inside the liposomal formulations.

Table 4: Table showing the drug content and Encapsulation Efficiency

Formulation	*Drug Content (%)**	*Encapsulation Efficiency (%)**
F1	71.2±0.71	69.5±0.98
F2	64.2±0.23	56.4±0.37
F3	57.8±0.65	52.6±0.58
F4	53.5±0.77	48.2±0.41
F5	49.3±0.47	45.7±0.95
F6	69.9±0.39	66.2±0.34
F7	56.3±0.87	54.3±0.46
F8	51.5±0.58	47.6±0.74
F9	48.3±0.44	44.4±0.65
F10	43.7±0.37	40.1±0.54

In-vitro drug release study:

The egg membrane was used to conduct the in-vitro drug release investigation. After conducting the release trials for an uninterrupted period of eight hours, the final sample was collected after twenty-four hours in order to ascertain whether or not the drug release was still ongoing. The findings are depicted in figures 3 and 4 which are presented below.

Fig. 3: Graph showing in-vitro drug release of azithromycin liposomal formulations

Fig. 4: Graph showing in-vitro drug release of green tea liposomal formulations

Stability study of liposomal suspension:

The effect on vesicle size and drug content during stability studies was done at 25⁰C±2⁰C / 60%RH±5%RH and results are given in the table7.

Table 7: Effect on vesicle size and drug content during stability

Parameter	Azithromycin suspension [F1]		Green tea suspension [F6]
Parameter	Initial	Final	Initial	Final
Vesicle size	4.91±0.015	5.06±0.018	4.26±0.012	4.75±0.016
Drug content (%)	71.2±0.71	70.2±0.97	69.9±0.39	68.2±0.29

Table 8: Table showing results of physical evaluations

Formulations	pH *	*Viscosity (cps)**	*Spreadability (g.cm/sec)**
F1	6.6±0.25	9750±23	2.93±0.012
F2	6.8±0.27	8752±24	3.19±0.017
F3	6.8±0.31	7450±41	3.19±0.018
F4	6.2±0.15	2510±29	3.56±0.011
F5	6.5±0.18	2452±25	3.99±0.016
F6	6.8±0.11	6770±26	2.73±0.014
F7	6.2±0.23	6016±28	3.05±0.015
F8	6.6±0.36	4433±21	3.27±0.013
F9	6.3±0.22	4520±28	3.25±0.017
F10	6.9±0.41	7752±23	3.82±0.016

Stability study of gel:

The effect on viscosity, spreadibility and in-vitro drug release during stability was done at 25⁰C±2⁰C / 60%RH±5%RH and results are given in the table 9.

Tab 9: Effect on viscosity, spreadibility and in-vitro drug release during stability

Parameters	Azithromycin liposomal gel [F1]		Green tea liposomal gel [F6]
Parameters	Initial	Final	Initial	Final
Viscosity	9750±23	9790±31	6770±26	6796±31
Spreadability	2.93±0.012	3.01±0.022	2.73±0.014	2.81±0.017
In-vitro drug release	74.8	75.3	77.5	78.1

DISCUSSION:

Characterization of Liposome:

Determination of average particle size of liposomal formulation: The microscopic approach was utilised in order to determine the typical size of liposome particles. The findings are presented in tables 2 and 3, as well as figures 1 and 2. The diameter of liposomal formulations was found to range anywhere from 4.91 micrometres to 6.75 micrometres on average, with sizes ranging anywhere from 4 micrometres to 18.75 micrometres. It would appear that the size of the liposomes has a significant impact on the results. Furthermore, the membranes of MLV are more flexible, and because of their heterogeneity, some budding of the liposome bilayer can occur. This could result in better adaptation to the surface of the skin, and it could also enable some infiltration of the bilayer into the pores that are found in the stratum corneum lamellae. Integration of phospholipid molecules with the lipids of the skin may have served an additional purpose, which was to assist in the retention of the drug molecules within the skin, which consequently led to a prolonged presence of drug molecules at the receptor site and localised drug action in the skin. Based on these findings, it was hypothesised that the produced formulations may be used as local depots for the controlled and gradual distribution of the drug that was incorporated over an extended period of time.

Shape of liposomes:

Liposomes that had been prepared were analysed under a microscope to determine their lamellarity and form. It was discovered that the majority of the vesicle had a spherical form.

Drug content:

The process was followed to determine the amount of drug contained within the liposomal formulation. Because the results indicated (Table 4) that the formulation contained between 43.7 and 71.2% drug, this indicates that there was no degradation of the drug during the process. It was discovered that the medication carrying capability of liposomes is inextricably linked to the drug-to-lipid ratio that is utilised in the formulation of the liposomal substance.

Encapsulation efficiency:

Complete vesicle disruption was followed by extraction using a solution of triton X-100 that was 1% volume-to-volume (v/v), which allowed for the determination of the amount of CMP that was bound up in the liposomes. Table 4 presents the percentage of the medicine that is encapsulated inside the liposomal formulations. Comparisons are made between the encapsulation efficiencies of liposomes that are loaded with medication and those that are loaded with cholesterol and lipids in varied amounts. The amount of cholesterol present in the liposomes and the ratio of drug to lipid both had a substantial impact on the efficiency with which they encapsulated the drug. The level of encapsulation efficiency displayed by liposomes comprised of cholesterol was found to be much higher. The percentage of azithromycin that was found to be entrapped was found to be at its highest with formulation F1, and the percentage of green tea that was found to be entrapped was found to be at its highest with formulation F6. When the molar ratio of lipid to cholesterol was altered from 1:1, the entrapment efficiency of the medication dropped significantly. This may be due to the increasing concentration of cholesterol, which may increase the lipophilic properties of the lipid bilayer and consequently increase the entrapment of drugs. On the other hand, in liposomes with a lower amount of cholesterol, the lipid bilayer becomes more fragile, which leads to a decrease in entrapment efficiency due to the leaking of drug when shaking the liposomes.

In-vitro drug release study:

Egg membrane was the material that was used for the in-vitro drug release study that was conducted in distilled water. The outcomes are presented in tables 5 and 6, as well as figures 3 and 4. A comparison of the results obtained from in vitro drug release tests has been carried out for each of the ten different formulations. It was discovered that the medication release rate was lower in formulations F1 and F6 compared to other formulations. This could be the result of increased drug entrapment. Because of this, the liposomes that are made by combining phosphatidyl choline with cholesterol can be utilised for sustained release. When it comes to the formulation of liposomes, the quantity of cholesterol used is an important issue since it has an effect on the liposome's integrity, stability, and the entrapment of hydrophilic drugs. Since of the presence of many lipid bilayers, the multilamellar liposomal formulation was able to induce sustained release of the medicine. This was possible because the drug was released gradually over an extended period of time.

Stability study of liposomal suspension:

In accordance with the ICH recommendations, the stability of the liposomal suspension was evaluated over a period of three months at various temperatures. The findings are presented in the table7 below. Because of the possibility that the liposomal formulations could be damaged by the accelerated high level temperature and increased relative humidity, the stability tests were carried out at room temperature with ambient humidity. There was not a discernible shift in either the drug's concentration or its vesicle size when the conditions were held at 250C 20C and 60% RH 5% RH. An minor rise in the size of the vesicles was found, which may be attributable to a very slight fusion of the liposomes. However, this increase in size was not significant.

Stability study of gel:

In accordance with the ICH recommendations, the stability of the liposomal dispersion was evaluated over the course of three months at various temperatures. The findings are presented in the table10 below. Because of the possibility that the liposomal formulations could be damaged by the accelerated high level temperature and increased relative humidity, the stability tests were carried out at room temperature with ambient humidity. At a temperature range of 250C20C and a relative humidity range of 60%5%, there was no discernible shift in the spreadability, viscosity, or in-vitro drug release.

CONCLUSION:

Acne is typically treated topically with azithromycin, which is an antibacterial medication known as azithromycin. Azithromycin gel that is now on the market suffers from a number of drawbacks, the most notable of which are skin irritation, limited tolerability, and difficulty with patient compliance. Because of decreased efficacy or increasing adverse effects, topical azithromycin is used significantly less frequently. As a result of the difficulties associated with existing formulations, we were motivated to develop an innovative drug delivery system for azithromycin. As a result, liposomes have been chosen for this particular piece of research on the presumption that incorporating Azithromycin into liposomes will, at the very least, lessen the likelihood of the drug's adverse effects. Traditional herbal medicines have been the subject of substantial research as viable alternative treatments for a wide variety of disorders. This is done to mitigate the potential risk of bad effects and antibiotic resistance caused by prescription pharmaceuticals. Due to the antibacterial qualities of green tea, it is considered to be one of the most effective herbal medicines for the treatment of acne. In order to create a regulated and sustained release of the medicine when it is applied topically, a liposome containing azithromycin and green tea was prepared using a variable ratio of lipid and cholesterol by the thin film hydration method. This was done in order to accomplish the desired results. In conclusion, the liposomes that contain azithromycin and green tea offer significant therapeutic promise for the treatment of acne vulgaris. Additional research with animal models will shed additional information on how effective the formulation is in vivo.

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Received on 25.08.2022 Modified on 05.11.2022

Res. J. Pharma. Dosage Forms and Tech.2023; 15(2):91-96.

DOI: 10.52711/0975-4377.2023.00016