(M1330-09-55) Process Optimization of Preparing Lysozyme-Loaded Liposomal Dry Powder via Design of Experiments

Purpose: Dry powder inhalation (DPI) is a promising strategy for delivering protein drugs to the lungs for both local and systemic effects. However, pulmonary enzymes, cellular uptake, and low physicochemical stability pose major challenges to protein stability. This study aims to develop a liposome-based DPI formulation that protects proteins during inhalation delivery. Using lysozyme as a model protein, a Design of Experiments (DoE) approach was used to optimize spray-drying parameters for producing a stable and inhalable liposomal powder.

Methods: Lysozyme-loaded liposomes were prepared using the most optimal formulation identified through preliminary screening. The spray-dried formulation included mannitol and L-leucine as excipients. A D-optimal Design of Experiments (DoE) with 20 runs was conducted to optimize spray drying for DPI, evaluating three factors: inlet temperature (100–140 °C), atomizing pressure (50–150 kPa), and leucine content (0–50%). Six responses were assessed: yield, particle size, moisture content, flowability (Carr’s Index), encapsulation efficiency, and lysozyme stability. Assays were performed in triplicate. Particle size was measured by laser diffraction. Flowability was assessed per USP < 616 >. Aerosol performance was evaluated using NGI at 60 L/min with 50 size 3 capsules, each filled with 42 mg of spray-dried powder, to determine emitted dose (ED) and fine particle fraction (FPF).

Results: Spray drying process optimization using DoE: A D-optimal design consisting of 20 experimental runs was conducted to evaluate the effects of inlet temperature, atomizing pressure, and leucine content on six key responses. The complete experimental matrix and corresponding results are summarized in Figure 1. The matrix includes 20 unique combinations of the three input variables and their measured responses. From the tabulated results, a general trend was observed: higher atomizing pressure led to smaller particle sizes but decreased powder yield, while increasing leucine content improved flowability across multiple runs. Among the tested parameters, atomizing pressure and leucine content were identified as statistically significant factors (p < 0.001) influencing powder yield, aerodynamic particle size, and flowability. Figure 2 illustrates predicted profiling and 3D plots from the DoE, demonstrating how each parameter influences critical outcomes. The optimized condition (100 °C, 81 kPa, 17.5% leucine) provided a desirable balance across all responses. Physicochemical characteristics of optimized formulation: The optimized spray drying condition (100 °C inlet temperature, 81 kPa atomizing pressure, 17.5% leucine) yielded a dry powder formulation with favorable characteristics for pulmonary delivery. The powder exhibited a yield of 60.97%, aerodynamic diameter of 3.55 µm, Carr’s Index of 35.71%, moisture content of 2.55%, encapsulation efficiency of 60.84%, and lysozyme stability of 72.3%. In vitro aerosol performance: Aerosol performance was evaluated using the Next Generation Impactor (NGI) at 60 L/min. As shown in Figure 3, the optimized formulation demonstrated excellent aerosolization with an emitted dose (ED) of 95.99% and fine particle fraction (FPF) of 37.28%. Run 9, which used higher atomizing pressure (100 kPa) and higher leucine content (25%), showed slightly higher FPF (37.82%) and encapsulation efficiency (76.62%). However, it required elevated inlet temperature (120 °C), which may compromise protein stability during processing. In contrast, Run 10, which lacked leucine, demonstrated poor aerosol performance with a lower ED of 46.52% and greater deposition in the throat region due to aggregation.

Conclusion: This study successfully applied a Design of Experiment (DoE) approach to optimize the spray-drying parameters for preparing protein-loaded liposomal dry powders for inhalation. Among the tested variables, L-leucine content significantly influenced the particle size, yield, and flowability of the formulations. While surface modification with leucine improved powder dispersibility and aerodynamic performance, it also reduced overall yield. The optimized formulation achieved favorable encapsulation efficiency and lysozyme stability, along with desirable aerodynamic properties such as suitable particle size and low moisture content. These findings demonstrate the feasibility of producing stable and aerosolizable protein-liposome dry powders, and suggest that surface-modified DPI formulations have strong potential for improving the efficiency of pulmonary drug delivery.

Figure 1. D-optimal design matrix and measured responses

Figure2. Predicted Profiling and 3D Plots for DoE Design Parameters

Figure 3. NGI deposition profiles of the optimized formulation, Run 9, and Run 10