(T0930-12-71) Preparation and Characterization of Apixaban with Anti-crystallizer PLGA Microsphere Using Prilling by Vibrating Microfluidic Device

Purpose: The purpose of this study is to present and evaluate a new concept of microsphere fabrication technology to inhibit crystal formation of apixaban in Dichloromethane (DCM) because crystal formation can break the microsphere structure and to produce microspheres with uniform particle size distribution using a prilling by vibrating microfluidic device.

Methods: 1. Screening of solubilizer and anti-crystallizer for apixaban in DCM: To improve the solubility and stability of apixaban in DCM, solubility enhancers or solubilizers were screened. Various fatty acids including Caprylic acid (C₈; CH₃(CH₂)₆COOH), Capric Acid (C₁₀; CH₃(CH₂)₈COOH), Lauric acid (C₁₂; CH₃(CH₂)₁₀COOH) and Stearic acid (C₁₈; CH₃(CH₂)₁₆COOH) were screened as solubility enhancers. 20 mg of apixaban was dissolved in 1 mL of DCM. Various fatty acids with various ratios were added. Solubility or precipitation were visually observed. Figure 2 a) showed the ratio of apixaban and fatty acid. 2. Equipment setup and process parameters: Formulation of apixaban-loaded microspheres was prepared by using microfluidic technique. The core solution was prepared by dissolving PLGA, apixaban and fatty acids in DCM. The shell solution contains 1% of PVA (poly (vinyl alcohol)) and 0.5% Tween 80 in distilled water. Figure 1 showed the composition of formulation and process parameters. The Encapsulator-B 390 equipment comprises two flow nozzle (core and shell) and a vibrating unit. Microsphere size can be altered or influenced by parameters like nozzle size (a major influence); core flow rate (moderate influence); vibration frequency (moderate influence). The effect of vibration frequency was analyzed. The shell nozzle flows stabilizing solution (water phase) by pressure drive force at a constant flow rate of 4 mL/min. The Fusion 100 equipment flows the core solution by a 15.40 mm internal diameter 10 mL syringe connected to the main equipment head. 3. Characterization: The morphology of the prepared microspheres was observed by an optical microscope (Axio scope. A1, Carl Zeiss Oberkochen, Germany). Microphotographs were captured at 20X magnification. For the determination of drug loading and entrapment efficiency, freeze-dried 5 mg of apixaban-loaded microspheres were dissolved in 1 mL of acetonitrile. After 5 min of vortexing, the tube was swung in an incubator at 37 ± 0.5 ◦C for 1 hour. The solution was centrifuged at 10,000xg for 15 min and the supernatant was collected. The collected supernatant was analyzed by HPLC for apixaban content after necessary dilution with the mobile phase. Drug loading (DL %) and entrapment efficiency (EE %) was calculated using the following Equation. DL % = (mg of apixaban microsphere evaluated / mg of microsphere) * 100
EE % = (actual mg of apixaban found/ theoretical apixaban should be) * 100

Results: For caprylic acid formulations were stable and small precipitation of apixaban was observed at the 6^th hour. According to Figure 2 b), the optimum ratio of apixaban to fatty acids was observed 1: 0.25 and above. It was observed that lower carbon chain fatty acids were more effective to solubilize apixaban in DCM. Figure 3 showed the characterization of microspheres. The microspheres were observed non-uniform distribution with distinguishable particle size. The surface of these microspheres was smooth, however aggregated particles occurred. Both formulation 01 and formulation 02 were prepared with a theoretical drug loading of 18.18% and the actual drug loading was observed at 15.33 ± 1.57 and 15.85 ± 0.86 for formulation 01 and 02. The entrapment efficiency for each formulation was observed as more than 80%.

Conclusion: In this study, it was observed that caprylic acid acts as inhibitor of crystal formation of apixaban. After a successful screening study of fatty acid, formulations were prepared by using the microfluidic technique. Particle size and uniformity of prepared microspheres were quite non-uniform. However, drug loading and entrapment efficiency were observed high for the microfluidic technique. Therefore, further study is needed to optimize the process parameters of this microfluidic technique.

Acknowledgements:

Funding: This work was supported by the Mid-Career Researcher Program (No. NRF-2021R1A2C2008834) and Basic Research Infrastructure Support Program (University-Centered Labs-2018R1A6A1A03023718) through the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT)


a) Formulation composition for microfluidic technique
b) Process parameters for the microfluidic technique used for the preparation of formulations
c) Schematic diagram of microfluidic technique


a) Composition of apixaban and fatty acids in screening study for the improvement of solubility and stability of apixaban in DCM.
b) Solubility and re-crystallization of apixaban in DCM with various composition (w/w) of apixaban: caprylic acid (C8), lauric acid (C12)


a) Particle size and morphology of apixaban-loaded microsphere. Left, formulation-01; Right, formulation-02. b) Encapsulation efficiency and drug loading for formulation 1 and 2