(W1130-03-19) Optimization of Process Parameters for Apixaban Long-Acting Parental PLGA Microspheres

Purpose: In this study, apixaban-loaded PLGA microsphere as a controlled release long-acting parental process parameters were optimized by a novel microfluidic technique. These studies will contribute to developing the controlled production of microspheres for pharmaceutical application.

Methods: Experimental design: Statistical analysis was conducted by using JMP® software (version 14.2.0; SAS Institute Inc., Cary, North Carolina, USA) to determine the optimal process parameters of microfluidic equipment with three factors: X1(Frequency, Hz), X2(Core flow, μL/min), and X3(Core nozzle, μm). The D-optimal design was used to optimize and evaluate main effects, interaction effects and quadratic effects of the process permeates on the responses, Y1 (drug entrapment efficiency, EE %), Y2 (mean particle size, μm), Y3 (SPAN), Y4 (10 % particles below this size range, D10, μm), Y5 (50 % particles below this size range, D50, μm), and Y6 (90 % particles below this size range, D90, μm). Fifteen experimental Runs are in Figure 1. Microsphere preparation by microfluidic technique: Shell solution was dissolved in 10 g of PVA in 1 L (1% w/v) of distilled water at 90 ± 1 ℃ for 2h. After cooling down to room temperature 5 mL of Tween 80 (0.5% v/v) was added to the prepared solution and mixed. Core solution was dissolved in 20 mg of Apixaban (1% w/v) and RESOMER® RG 502H 80 mg (4% w/v) and caprylic acid (10% w/v) of 1 mL DCM. After 5 min vortexing proper dissolution was observed visually and the core solution was colledcted in a 10 mL syringe (NORM-JECT, D-78532 Tuttlingen, Germany). The core solution containing the syringe was connected to the microfluid dispensing head in the core side and the syringe was set to the equipment. The core solution flow was observed form the microfluid dispensing head after core flow started. The vibration unit was also set to the predefined frequency. Uniform laminar flow of core entrapped shell was observed, and the dispensed particles were collected in the 1000 mL collecting glass beaker. Characterization study: The mean particle size and distribution of microspheres were determined by laser diffraction technique. The morphology of the prepared microspheres was observed by an optical microscope (Axio scope. A1, Carl Zeiss, Oberkochen, Germany). Drug loading and entrapment efficiency was calculated by using freeze-dried 5 mg of apixaban-loaded microspheres dissolved in 1 mL of ACN.

Results: The mean particle size, SPAN, D10, D50, and D90 was determined by the microfluidic technique. The particle size increased with the increased diameter of core nozzle size from 120 μm to 150 μm. Microscopic images were collected with 20x magnification. The morphology of the prepared microspheres shows that frequency is the major influential factor for particle size. None of the formulations reached a theoretical drug loading of 18.18%. Drug loading increased with increased core flow and core nozzle size. Entrapment efficiency was observed for 92.74, 93.53, and 95.48%. Effect of factors: By the D-optimal Design of this study, different factors and their influence on the response were observed (Figure 1.). The most influential factors were core nozzle size and frequency. When core nozzle size increase, particle's diameter, and distribution also increased. The optimal process parameters from the design of experiments showed that the optimal frequency for higher desirability is 573 Hz, which is the optimized frequency used for the formulation preparation of optimal formulations. The next and last factor as the core flow rate (μL/min) was observed quite opposite to the frequency and core nozzle. When the core flow volume increase from 50 μL/min to 75 μL/min particle size increase, however, with a further increase to 100 μL/min, particle size start to decrease (Figure 1.). Optimal formulation and design space: After statistical analysis, the prediction profiler function was used to obtain the optimal value that satisfies the QTPP. Through Frequency 573 Hz, Core flow rate 54.3 μl/min, and Core nozzle size 120 μm, entrapment efficiency, mean particle size, SPAN, D10, D50, D90 were estimated to be 97.29 %, 34.05 μm, 1.50, 14.20 μm, 29.77 μm, and 54.62 μm, respectively. At the same time, the Desirability value was observed, 83.07 % (Figure 2-a.). The Design space (white area in Figure 2-b.) was derived by setting the optimum value within the JMP® software. Optimal formulation preparation: The optimum process parameters were obtained from the D-optimal design by JMP® software and optimized formulations were prepared through the process parameters. Fixing the core nozzle to 120 μm, frequency, and the core flow rate was set to 573 Hz and 54 μL/min, respectively (Figure 3.).

Conclusion: From the pre-screening study of solubility, stability, and pre-formulation screening of apixaban in DCM, formulations were prepared by a novel microfluidic technique. Different crucial characteristics of microspheres as entrapment efficiency, drug loading, mean particle size, SPAN, D10, D50, and D90 of particles were selected as responses. The novel microfluidic technique was proved quite effective in the production of uniformed size microsphere.

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

Factors and responses from design of experiments.

a. Predicition profiler and desirability b. Design space of optimal formulation with 120 μm core nozzle.

Optimized process parameters for microfluidic technique.