(T1330-01-07) Preparation and Evaluation of Protein Encapsulated Liposome Using Surfactant for Dry Powder Inhalation

Purpose: In this study, when encapsulating proteins in empty liposomes, surfactant was used to promote drug loading and encapsulation efficiency. Since most proteins are denatured at high temperatures, heating during hydration is difficult. Thus, empty liposomes were formulated, and physically homogenized to prepare liposomes. In addition, we would examine the changes in shape and size in the group in which the surfactant was added by constructing dry powder using the spray drying method. It was also aimed to improve the in vitro aerosol performance by increasing the emission fraction and particulate fraction values of the enlarged powder by enlarging the surfactant.

Methods: The liposomes were prepared by thin film hydration. The weight ratio of DSPC to cholesterol was 3:2. The mixture of DSPC and Cholesterol was dissolved in chloroform in a round bottom flask. Dry-down of the resultant lipidic solution from the organic solvent to form a lipid thin layer with rotary evaporator (EYELA Pte. Ltd., Singapore) (45℃, 1 h). Surfactants were dissolved in DW at weight ratios of 0.5 and 5. Hydration solution was added to empty liposomes (80℃, 1 h and sonicate 30 min). Before the extrusion process, lysozyme was added, and stirring for 30 minutes, liposome particle size was reduced using high-pressure homogenizer Nano DeBEE (BEE International Co. Ltd., South Easton, MA) at 15000 psi for 15 cycles to obtain homogeneous liposomes. The liposome was kept at room temperature before spray drying. The mixture of liposomes was spray-dried using a spray dryer SD-1000 (spray dryer SD-1000, EYELA Pte. Ltd., Singapore). The mixture was pumped into the drying chamber and atomized through a 0.7mm two-way nozzle. The spray-drying parameters were set as follows: pump speed of 300mL/h, inlet temperature of 135℃, outlet temperature 80℃ to 90℃, blower of 0.33m3/min, atomizing of 8 x 10 kPa. Drug loading (DL%), and encapsulation efficiency (EE%) were analyzed with a detergent compatible Bradford assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The surface morphology of spray-dried liposome was determined by optical microscopy using Axioscope A1 (Axioscope A1, Carl Zeiss, Oberkochen, Germany) and scanning electron microscopy (SEM) using JSM 7100F (JSM-7100F, JEOL, Tokyo, Japan).

Results: Drug loading, and encapsulation efficiency dried liposome powders were shown as below Figure 1. P188, and SE each obtained the highest drug loading results. Encapsulation efficiency was also observed like the drug loading. 30.57%, 28.32%, and 29.21% encapsulation efficiency was observed for P188(0.5), P188(5), and SE(5) respectively. P188(0.5) result presented maximum encapsulation efficiency. The drug loading, and encapsulation efficiency was highly relative with each different kind of surfactant, and ratio. When the morphology of the particles was confirmed through an optical microscope, it was confirmed that P188, and SE were Uniformly sphere-shaped liposomes. Figure 2 A) showed the results observed under an optimal microscope. For Morphology confirmed by SEM, liposomes were uniformly formed in aspherical shape with smooth surface in the group of Poloxamer 188, and Sugar ester stearate S-1670 as surfactants. Figure 2 B) showed the results observed by SEM. Aerosolization efficacy was performed by the percent of emitted fraction (EF%), fine particle fraction (FPF%) of the total dose was observed, and shown Figure 3 A), Figure 3 B), and Figure 3 C). EF% value of SE(5) in the formulation showed the most obtained result of 93.7%. In the case of the fine particle obtained inside the impactor, 28.35%, 26.33%, 26.78%, and 24.50% of the negative control P407(5), SE(0.5), and SE(5) formulations were obtained. However, among them, SE(0.5), and SE(5) formulations presented the highest percentage of 26.78%, 24.50%.

Conclusion: This study evaluated the pulmonary inhalation delivery effect using surfactants to prepare liposomes for dry powder inhalation, and the steps to establish a process of preparing liposomes in which proteins could be loaded. As a result, in the SE (5) formulation, the homogeneous spherical structure, and the surface was smooth, and the encapsulation efficiency of 29.21%, drug loading of 0.63% was presented. In vitro aerosol performance, the emitted fraction was 93%, and the fine powder fraction was 24.6%, which reached the highest stage 7. The formulation composition using sugar ester S-1670 was the most suitable for preparing an inhalation powder formulation.

References: Tagami, Tatsuaki, Mark J. Ernsting, and Shyh-Dar Li. "Optimization of a novel and improved thermosensitive liposome formulated with DPPC and a Brij surfactant using a robust in vitro system." Journal of controlled release 154.3 (2011): 290-297.
Sultana, Syeda Shabana, and A. Krishna Sailaja. "Formulation and evaluation of diclofenac sodium transferosomes using different surfactants by thin film hydration method." Der Pharmacia Lettre 7.11 (2015): 43-53.


Figure 1. Drug loading (%) and encapsulation efficiency (%) of liposomal dry powder


Figure 2. Morphology of dry powder A) optimal microscope image; N control, P188(0.5), P188(5), P407(0.5), P407(5), SE(0.5), SE(5) in order from the left B) SEM image; N control, P188(0.5), P188(5), P407(0.5), P407(5), SE(0.5), SE(5) in order from the left


Figure 3. A) Percentage of emitted dose (%) for each stage B) Fine particle fraction (FPF%) of dry powder C) Emitted fraction (EF%) of dry powder

Acknowledgements: This work was supported by the Mid-Career Researcher Program (No. NRF-2021R1A2C2008834), the Bio & Medical Technology Development Program (No. NRF-2022M3A9G8016257) and Basic Research Infrastructure Support Program (University-Centered Labs-2018R1A6A1A03023718) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science & ICT (MSIT).