(M1430-03-17) Optimizing Microfluidic Process Parameters and Lipid-Cholesterol Composition for Lysozyme-Loaded Liposome Preparation

Purpose: The conventional methods used for producing protein-loaded liposomes face stability issues, as protein drugs are prone to denaturation, aggregation, agglomeration, and adsorption. In contrast, microfluidics technology offers diverse advantages, including ease of particle size manipulation, reproducibility, minimal reagent usage, and enhanced stability compared to conventional methods like thin film hydration. Therefore, the purpose of this study is to apply microfluidics technology to protein-loaded liposomes and optimize parameters such as the lipid (DSPC: Cholesterol) molar ratio, total flow ratio (TFR), and flow rate ratio (FRR). Additionally, lysozyme, a 14 Kda protein, was utilized as a protein model drug. The aim is to determine the optimal combination of these parameters that results in the smallest particle size and maximum encapsulation efficiency of the protein drug.

Methods: Liposome Preparation and Characterization: Lysozyme, a protein model drug, was encapsulated in liposomes composed of DSPC and cholesterol. The lysozyme concentration in the aqueous phase was 0.1 mg/ml, and the lipid concentration in the organic phase was 5 mg/ml in methanol. Liposomes were prepared using a Microfluidic Chip (5 input chip, 3D 150um Dolomite Microfluidics, Royston, UK) with varying lipid ratios (DSPC:Cholesterol) - F1(2:1), F2(3:2), F3(7:3), and F4(4:1), total flow ratios (TFRs) of 1.5 to 5 ml/min, and flow rate ratios (FRRs) of the aqueous phase to organic phase of 2:1 to 4:1. TFRs and FRRs were controlled using two single-syringe pumps (New Era Pump Systems, Inc., Farmingdale, NY, USA). The Microfluidic chip had three inlet channels (two for the aqueous phase, one for the lipid phase) and one outlet channel. The aqueous and organic phases, along with lysozyme, were injected into the chip using the single-syringe pumps to form self-assembled liposomes. Methanol was removed from the liposomes using a rotary evaporator (EYELA N-1110 rotary evaporator, Tokyo, Japan) at 45°C. After methanol evaporation, the liposomes were placed in PBS at 4°C for 4 hours. Subsequently, dialysis was performed for 10 hours with regular changes of fresh PBS to evaluate encapsulation efficiency (EE %) using a dialysis device (Spectrapor Float-A-Lyzer G2, Sigma Aldrich, St. Louis, MO, USA) and quantify with a detergent-compatible Bradford assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Particle size (PS nm) and polydispersity index (PI %) were evaluated using DLS (Dynamic light scattering).

Results: F2, with a higher concentration of Cholesterol compared to F1, exhibited smaller particle sizes at TFR 3 mL/min with FRRs of 2:1, 3:1, and 4:1 (148.75±10.57 nm, 146.19±1.92 nm, and 187.59±13.82 nm, respectively). F1 showed larger particle sizes under the same FRR ratios (179.07±5.28 nm, 192.53±15.06 nm, and 357.5±104.41 nm) (Figure 2A and Figure 3). Increasing TFR to 5 ml/min resulted in both F1 and F2 achieving particle sizes and polydispersity index (PI%) below 200 nm and 25%, respectively. F1 exhibited a PI% of 23.1%±1.1% at FRR 2:1, while F2 showed a polydispersity index of 19.6%±5.0% at FRR 2:1, 21.1%±1.5% at FRR 3:1, and 24.9%±0.7% at FRR 4:1 (Figure 3). Comparing zeta potentials, F1 displayed more negatively charged values (-11.2 mV at TFR 5 ml/min, FRR 4:1, to -21.8 mV at TFR 3, FRR 2:1), indicating stronger repulsion forces among liposomal particles and improved stability (Figure 3). Encapsulation efficiency (EE%) evaluation, based on minimum particle size < 200 nm and PI% < 25% after methanol evaporation at 45°C and dialysis, showed that at TFR 5 ml/min and FRR 2:1 (Aqueous: Organic), the mean EE% was 69.87%±1.18% for F1 and 34.96%±0.44% for F2. At FRR 3:1, F1 exhibited an EE% of 70.0%±1.05%, while F2 demonstrated an improved EE% of 43.07%±6.94% (Figure 2B and Figure 3). Based on these results, the suitable formulation for microfluidics technology, achieving the highest EE% with particle size < 250 nm and PI% < 25%, was determined to be F1 at TFR 5 ml/min and FRR 3:1. This formulation is selected as the optimal lysozyme-loaded liposome formulation.

Conclusion: This study optimized the production of lysozyme-loaded liposomes using microfluidics technology. Parameters such as Cholesterol concentration, total flow ratio (TFR), and flow rate ratio (FRR) were investigated. Higher Cholesterol concentration, TFR, and FRR resulted in smaller liposome size, improved stability, and higher encapsulation efficiency. These findings provide valuable insights for the production of hydrophilic drug-loaded liposomes using microfluidics technology, facilitating further research in liposomal drug delivery systems.

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).

Figure 1. General setup of microfluidics system applied to the study

Figure 2. A) The effect of different DSPC: Cholesterol ratios and microfluidics parameters FRRs (Aqueous phase: Organic phase) and TFRs on the mean particle size of lysozyme loaded liposomes±SD (n=3) B) Encapsulation efficiency (%) for F1 and F2 at microfluidics parameters which produced lysozyme loaded liposomes with particle size < 25.0 nm

Figure 3. Summary of Physicochemical properties of lysozyme loaded liposome depending on parameters of microfluidics system parameters