INTRODUCTION:

The creation of a new drug delivery system (NDDS) has concentrated significant attention over the previous few decades. Ideally, the NDDS should fulfill two preconditions. First, it should supply the drug throughout the therapy at a pace that is guided by the body's requirements. Second, the active entity should be channeled to the action site. None of these can be met by conventional dosage forms, including prolonged-release dosage forms. At current, no drug delivery scheme is ideal, but genuine efforts have been made to accomplish this through numerous new methods in the delivery of drugs.^1-3

The aim of the novel drug delivery system is to provide some control of the release of drugs in the body, whether temporal or spatial in nature or both. In recent years, vesicles have become the vehicle of choice in shipping medicines.

In immunology, membrane biology, diagnostic methods, and most lately genetic engineering⁴, lipid vesicles have been discovered to be of importance. Vesicles can play an important role in modeling biological membranes as well as in transporting and targeting active agents. It can be anticipated that a drug's encapsulation in vesicular structures will prolong the drug's presence in systemic circulation and may reduce toxicity if selective uptake is possible.⁵ The phagocytic uptake of the systemic transmission of the drug-loaded vesicular delivery system offers an effective technique for delivering the drug straight to the infection site, resulting in reduced drug toxicity without adverse effects.

Yohimbine is an alpha-2 adrenergic antagonist. Yohimbine is generally used as a nutritional supplement for athletic performance, erectile dysfunction, weight loss, high blood pressure, diabetic neuropathy, chest pain, and more. Yohimbine HCL has a half life of up to 5 hours and has a bioavailability of 7-87% with a mean value of 33%. Yohimbine HCl usually undergoes hepatic first pass metabolism^6-7.

Table 1: Formulation Design:

Ingredients	F1	F2	F3	F4	F5	F6	F7	F8	F9
Yohimbine HCl (mg)	10	10	10	10	10	10	10	10	10
Phosphatidyl Choline (mg)	100	100	150	150	50	150	100	50	50
Cholesterol	150	100	50	100	100	150	50	50	150
Mannitol (g)	1	1	1	1	1	1	1	1	1
Chloroform (ml)	6	6	6	6	6	6	6	6	6
Methanol (ml)	6	6	6	6	6	6	6	6	6

MATERIALS AND METHODS:

Divis Laboratories, Hyderabad, gathered Yohimbine HCl as a donation sample. Phosphatidyl Choline, Mannitol, Cholesterol, Mannitol, Carbopol 934 was purchased from SD Fine Chemicals, Mumbai. All other chemicals and reagents used were analytical grade.

Preparation of Yohimbine HCl Loaded Proliposomes:

The proliposomes containing Yohimbine HCl was prepared by film deposition on carrier method using vacuum rotary evaporator. The Yohimbine HCl proliposomes was done by preparing the different formulations by varying the concentration of phosphatidylcholine and cholesterol. The required quantity of mannitol was placed in 100ml round bottom flask which was held at 60-70⁰C temperature, and the flask rotated at 80-90 rpm for 30 min under vacuum. After complete drying, the temperature of the water bath was lowered to 20-30⁰C. Yohimbine HCl, Phosphatidyl Choline and Cholesterol were dissolved in a mixture of organic solvents (chloroform: methanol, 6:4, v/v) and 5ml of an aliquot of the organic solution was slowly introduced into the flask via the solvent inlet tube. After complete drying second aliquot (5ml) was introduced. After complete drying, the vacuum was released, and Proliposomes were put overnight in a desiccator and then 100 mesh-sieved. The gathered powder was transported to a glass bottle and stored at the temperature of freezing.^8-10(table-1).

Preparation of Carbopol Gel Base:

2gm of carbopol 934 was weighed and dispersed in distilled water. Then propylene glycol was added, neutralizing the blend by adding 1 percent of triethanolamine drop-wise. Mixing continued until the transparent gel was acquired and permitted for 24 hours to swell. Similarly, 2% and 3% of carbopol gels have beenready¹¹.

Preparation of Proliposomal Gels:

Proliposomes containing Yohimbine HCL (separated from the unentrapped drug) were mixed into the 2% carbopol gel by using mortar and pestle, the concentration of proliposomes in the gel being 1%. All optimized formulations were incorporated into different carbopol gels (1% and3%)^12-14.

Characterization of Proliposomes Vesicle Size and Count:

Average size and size distribution proliposomes were determined using an optical microscope. On a glass slide without a cover slip, a drop of distilled water was added to proliposome granules and the method of liposome formulation was observed using a 100X optical microscope. Size of liposomal vesicles was measured at different locations on the slide. From the obtained results, size distribution and the average size of liposome vesicles was determined.¹⁵

Surface Morphology:

The surface morphology of proliposomes and plain mannitol particles were examined by scanning electron microscopy (SEM) after coating with gold. After the gold coating of proliposome and plain mannitol particles, their surface morphology was viewed and photographed.^16-17

Drug Content:

Yohimbine HCl content in proliposomes was assayed by an UV-visible spectrophotometer. Proliposomes (100mg) were dissolved in 10ml methanol by shaking the mixture for 5 mins. One ml of the resulting solution was drawn and methanol was diluted to 10 ml. Then, aliquots were withdrawn, and absorbance was recorded at 323 nm using UV-visible spectrophotometer (Lab India3200)^.18

Entrapment Efficiency:

Separation of the unentrapped drug from the liposomal suspension was done by centrifugation method. The entrapment efficiency of proliposomes was determined after hydration with distilled water. 10ml of phosphate buffer (pH 7.4) was added to proliposomes granules and then subjected to sonicate for 10 mins using ultra sonicator (Citizen, India). For the removal of the unentrapped drug, the liposomal suspension was subjected to centrifugation on a cooling centrifuge (REMI TR-01) for 30 minutes at 15000rpm.¹⁹ The clear supernatant (1ml) was taken and diluted to 10ml with buffer and absorbance was recorded at 323 nm using UV-visible spectrophotometer (Lab India 3200). Then calculate the percentage drug in each formulation.

Ct – concentration of total drug Cf – concentration of free drug

The Yield of Proliposomes:

After complete drying, the proliposome powders were collected and weighed accurately. The yield of proliposomes was calculated using the formula.

Total weight of proliposmes

Percentage yield = -------------------------------------- ×100

Total weight of drug + weight of added materials

Characterization of Gel:

Gel base was evaluated for the following parameters for both plain gel and gel loaded withproliposomes.²⁰

Physical Appearance:

All prepared proliposomal gel formulations have been observed for their visual appearances, such as transparency, color, texture, grittiness, greasiness, stickiness, smoothness, stiffness, tackiness and clarity was determined by using clarity chamber with black and white background.

The pH of Formulation:

The pH of the gel was evaluated using a digital pH meter (Lab India SAB 5000) and the glass electrode was fully immersed in the gel structure. The observed pH values were recorded for all formulations (F1-F9) in triplicates.

Rheological Properties:

The rheological properties of prepared gels were estimated using a Brookfield viscometer. The sample holder of the Brookfield viscometer was filled with the gel sample, and then spindle was inserted into the sample holder. The spindle was rotated at 100 rpm. All the rheological studies were carried out at room temperature. A viscosity measurement was done in triplicate.^[32] The viscosity of 1, 2 and 3% carbopol gel were determined and selected the optimized formulation.

Drug Content:

For determination of drug content, accurately weighed quantity (1gm) of gel equivalent to 10 mg of Yohimbine HCL was dissolved in phosphate buffer (PH 7.4) and analyzed by UV-Vis Spectrophotometer at 323 nm and the drug content was calculated^.21

In-vitro Studies:

For the research of drug release in vitro, Franz diffusion cell was used. The semi-permeable membrane was put between the diffusion cell's donor and receptor chamber. Freshly prepared 30ml 7.4 PH phosphate buffer filled the receiver chamber. On a semi-permeable membrane, proliposomal gel equivalent to 1gm was put. The Franz diffusion cell was placed at 500rpm over the magnetic stirrer and maintained at 37±1⁰C temperature. Periodically 5ml of samples were removed and replaced by fresh buffer. The withdrawn samples were periodically diluted and analyzed for drug content using UV visible spectrophotometer (Lab India 3200) at 323 nm^21-24.

RESULTS AND DISCUSSION:

In a preformulation study, the optimum concentrations of mannitol, phospholipid and cholesterol were determined to obtain stable liposomes devoid of aggregation, fusion and sedimentation. Yohimbine HCl proliposomes was prepared using thin-film hydration technique and method was found to be well suited for the production of liposomes without aggregation. Amount of mannitol, phospholipid and cholesterol was found to be very critical in the preparation and stabilization of proliposomes. The most important parameter, which needs to monitor during proliposome preparation for its best performance, is the vesicle size and size distribution of liposomes. Several reports showed the effect of liposome size on the drug release as well as drug deposition in the skin.

A positive correlation was observed for both variables phospholipid and cholesterol in case of liposome vesicle size. Thus, the vesicle size was discovered to be enhanced with a rise in phospholipid and cholesterol concentration. Proliposomal sample was placed under a digital microscope (Metzer, India) and hydrated with water. Then the formation of a vesicle was observed within the liposomal dispersion. Results of average vesicle size and distribution were calculated for count and distribution. Entrapment efficiency is an important parameter in the case of liposomes as it majorly affects the drug release and skin deposition. Results indicate that the effectiveness of phospholipid and cholesterol trapping has been improved with an rise in concentration. In the present study, the observed entrapment efficiency for all batches of Yohimbine HCL proliposome formulation was in the range of 85.12 to 96.5% were tabulated in table no.2. Among all Yohimbine HCL proliposomal formulations, F1-F9 had maximum vesicle size and entrapment efficiency, which were selected for further study.

Figure 1: Average Particle Size Distribution for All formulations of Proliposomes.

Table 2: Average Particle Size distribution of Proliposomes.

Sr. No.	Formulation Code	Avg particle Size (nm)	Drug Content (%)	Percentage Yield (%)	Entrapment Efficiency (%)
1	F1	5.34±0.023	95.03±0.543	93.4±0.324	94.9±0.244
2	F2	4.43±0.123	86.4±0.734	90.7±0.534	85.12±1.48
3	F3	2.65±0.076	93.7±0.664	89.5±0.654	91.02±0.613
4	F4	6.06±0.012	96.8±0.249	95.4±0.123	96.5±0.205
5	F5	4.34±0.231	94.7±0.984	94.3±0.221	92.7±0.249
6	F6	5.12±0.167	94.8±0.860	94.8±0.212	94.1±0.509
7	F7	3.21±0.221	92.4±1.70	88.7±0.321	88.1±2.19
8	F8	2.69±0.148	90.6±0.748	89.2±0.817	89.2±0.817
9	F9	2.34±0.321	87.5±0.953	86.5±0.265	86.02±2.90

Figure 2: Percent Drug Entrapment Efficiency for All formulations of Proliposomes.

The Yohimbine HCl content in the proliposomes was observed in the range of 86.4% to 96.8% at the various drug to phospholipid ratios. It was found from the above-mentioned outcomes that formulations F4, F1, F5 and F6 showed peak drug content relative to other formulations. The per cent yield of formulations was found to increase with the increase in phospholipid concentration. The percentage yield outcomes of different formulations were discovered to be in the range of 86.5 ± 0.265 to 95.4±0.221 percent as the drug-to-phospholipid ratio was altered in proliposomes.

Viscosity Measurement:

Rheological studies revealed that 2% carbopol gel showing better rheological properties when compared to 1% and 3% carbopol gels. So 2% of carbopol was used for the preparation of proliposomal gel. The viscosity of the gel was measured by Brookfield viscometer. The viscosity of proliposomal gel showed 1156cps at 100rpm.

pH Measurement:

The P^H of the developed formulation was in accordance with human skin pH rendering them more acceptable. Therefore formulated Proliposomal gel was suitable for topical application. The P^H values of prepared Proliposomal gels were within the limits of 5.5 to 5.8.

Drug Content (%):

The prepared Yohimbine HCl proliposomal gel was subjected to drug content uniformity, and it was found to 98.55 %, which indicated the drug uniformly dispersed throughout the formulation.

In-vitro Drug Release:

The result of in vitro release of Yohimbine HCl from the gel formulation clearly shows that the gels have the ability to retain the drug for prolonged periods. The % CDR of proliposomal gel formulation F4 was found to be 80.5 %, as shown in Fig. And that follows the model of Higuchi. For all the formulation, the ' n ' values were discovered to exceed 0.5. This shows that the release approximates the mechanism of non-Fickian diffusion for a longer period of time, which shows the sustained release behaviour of formulations. Phospholipid-rich vesicle domains may have helped to improve the percentage of lipophilic drug molecule trapping such as Yohimbine HCL in a lipid bilayer, indicating that Yohimbine HCL trapping in reconstituted liposome was found to be predominantly lipid-dependent. It was also possible to regulate the release of the active ingredient by increasing lipid concentration in proliposome

CONCLUSION:

In conclusion, sustained delivery of Yohimbine HCl can be achieved by a proliposomal drug delivery system. As the significant element of the liposomal system, phospholipids can be readily incorporated with the skin lipids and retain the required hydration conditions to enhance drug permeation. The fusion of lipid vesicles with skin contributed to the permeation enhancement effect. It was discovered that the phospholipid has an important impact on the stratum corneum's lipid matrix, indicating a disturbance of the intercellular lamellar lipid structure and acting as a penetration enhancer. Hence as the phospholipid concentration was increased, it would increase the permeation of drug following application on the skin. The free-flowing properties of the proliposomes granules will be beneficial in formulating the proliposomes as a solid dosage form. In-vitro studies concluded that enhanced skin permeation and retention of Yohimbine HCl was observed and were due to lipo-solubilized state of drugs within proliposomes, which helped to produce the depot effect. The data show that.

ACKNOWLEDGEMENT:

The author was thankful to the principal and management for providing chemicals and premises for the carrying out of the research work.

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Received on 20.12.2021 Modified on 26.02.2022

Asian J. Pharm. Res. 2022; 12(4):302-306.

DOI: 10.52711/2231-5691.2022.00049