Method Development and Characterization of Liposomal Formulation of Isotretinoin

This study aims to develop a liposomal drug delivery system of isotretinoin, an acne drug-using spray drying, as a cost-effective and time-effective technique. The liposomal formulation was prepared by using spray drying; three different strategies were adopted: suspension spray drying (SSD), thin-film hydration and spray drying (TFHSD), and emulsion spray drying (ESD). Isotretinoin was 99% bound with lipid, so lipids hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol were selected for the formulation development. The HSPC, DSPG, cholesterol, and isotretinoin were taken in the ratio 4 : 1 : 0.16 : 3.1 mmol. In vitro drug release studies, microscopy, drug content, and related substance characterizations were done to formulate each strategy of spray drying prepared dry liposomes of isotretinoin. Results were compared with the USP monograph of isotretinoin. It was revealed that isotretinoin's liposomal formulation using ESD was having drug release according to the USP limits. Drug content was also according to the USP requirement; no free drug crystals were found in microscopy, multivesicular vesicles were found in shape, a particle size of up 60 μ was found. The ESD technique was a successful, time-effective, and cost-effective technique for preparing a liposomal drug delivery system for isotretinoin.


Abstract
This study aims to develop a liposomal drug delivery system of isotretinoin, an acne drug-using spray drying, as a cost-effective and time-effective technique. The liposomal formulation was prepared by using spray drying; three different strategies were adopted: suspension spray drying (SSD), thin-film hydration and spray drying (TFHSD), and emulsion spray drying (ESD). Isotretinoin was 99% bound with lipid, so lipids hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylglycerol (DSPG), and cholesterol were selected for the formulation development. The HSPC, DSPG, cholesterol, and isotretinoin were taken in the ratio 4 : 1 : 0.16 : 3.1 mmol. In vitro drug release studies, microscopy, drug content, and related substance characterizations were done to formulate each strategy of spray drying prepared dry liposomes of isotretinoin. Results were compared with the USP monograph of isotretinoin. It was revealed that isotretinoin's liposomal formulation using ESD was having drug release according to the USP limits. Drug content was also according to the USP requirement; no free drug crystals were found in microscopy, multivesicular vesicles were found in shape, a particle size of up 60 µ was found. The ESD technique was a successful, time-effective, and cost-effective technique for preparing a liposomal drug delivery system for isotretinoin.   were done with the exact quantities of lipids but with the increasing quantity of isotretinoin 20 , as shown in Table II. pump rate at 5-10%; and nitrogen pressure 2 kg/cm 2 . The dried powder so retained from the spray drier was characterized further. The batch was named SSD 1.

Thin-film hydration and spray drying
The TFHSD technique's objective was to overcome the hygroscopicity problem that was coming in the previous SSD 23 . The TFHSD was conducted in several steps.  Spray drying -The prepared emulsion was then spray dried using the following parameters: inlet temperature 70°C; outlet temperature 42°C; aspirator rate 70%; pump rate 5%; nitrogen pressure 2 kg/cm 2 ; and humidity of room 55-60%. The spray-dried powder was collected and filled in the hard gelatin capsules, and analyzed further.

Preparation of solution -
The batch was named ESD 1.

Physical characterization of isotretinoin liposomal formulation
Drug content -As much as 10 mg of spray-dried powder was dissolved in 100 mL of solvent, i.e., chloroform : methanol (2 : 1) in a 100 mL volumetric flask. As much as 2 mL was taken from this solution and diluted up to 10 mL with the solvent in a 10 mL volumetric flask. The absorbance of the resulting solution was measured at the maximum at 346 nm using a UV spectrophotometer 27 .  Selection of lipids -Excipient for isotretinoin liposomal formulation was selected based on maximum solubility, in which isotretinoin was found to be a lipophilic drug.

RESULTS AND DISCUSSION
Batches were prepared and observed under the microscope 50X lens for free drug crystals, and the shape and size of vesicles formed to determine the excipient ratios used. Batch no. 1 had shown the presence of free drug crystals while vesicles were irregular in shape.
Batch no. 2 had also shown the presence of free drug crystals, and vesicles were also irregular in shape and size. Batch no. 3 had not shown any free drug crystals, but multivesicular structures were regular in round shapes.
As observed, batch 3 excipient ratios were selected for the preparation of the liposomal formulation 31 . Clear vesicular structures with no free drug crystals found with HSPC : cholesterol 4 : 1. Therefore, the 4 : 1 ratio was found to be sufficient.  Table III. Microscopy -Microscopy of batches was done to observe particle size, shape, and free drug crystals. The particle size multivesicular vesicles of sizes 2 to 60µ were observed. Figures 3a and 3b of SSD 1 microscopy showed that multivesicular vesicles of drug solubilized lipids were found. Free drug crystals were not found. The drug was solubilized entirely in lipids. In Figure 3c of TFHSD 1, microscopy-free drug crystals were found, and the size and shape of the vesicles were also irregular.
Thus, the strategy was dropped, but beneficial points were taken into considerations from this strategy, such as this strategy was successfully able to remove hygroscopicity. In Figure 3d of ESD 1, microscopy large lipid vesicles in the aqueous medium were found. No free drug crystals were found.
In SSD 1, no free drug crystals were found, and the vesicles were also in regular shape and size, which states that the drug was utterly solubilized in lipids 34 . In ESD 1, large lipid vesicles were found, and no free drug crystals were found. Thus, both these strategies pass the microscopy test. In TFHSD 1, free drug crystals were found, and the vesicles were irregular in shape; this strategy fails the microscopy test.   Table IV. The analysis revealed that the impurities of batch SSD 1 and ESD 1 were according to the USP limits.
The impurities of batch THFSD 1 (using nitrogen; 1N and using air; 1A) were not found out of the limits of USP.