Background/Objective: Surface enrichment of hydrophobic excipients via spray drying has been demonstrated as an efficient way to protect the dry powder inhaler formulations against moisture-induced deterioration in aerosol performance. However, the impact of such surface enrichment on dissolution and cellular uptake is less investigated, which can affect the safety and efficacy of dry powder inhalers (DPIs). Methods: In the present work, hygroscopic colistin was coated with leucine or trileucine, at different weight ratios during spray drying. All the powders were exposed to 75% relative humidity for one week. The aerosol performance was compared before and after the moisture exposure. Various solid-state characterizations, including particle size, particle morphology, crystallinity, water sorption/desorption, and surface composition, were conducted to evaluate the properties of spray-dried colistin with/without leucine or trileucine. Results: The results indicated that leucine or trileucine could protect the aerosol performance of spray-dried colistin against moisture deterioration. Leucine crystallized after spray drying with colistin, and such crystal leucine could further hinder water uptake when leucine was at a 20% or higher weight ratio. Trileucine did not crystallize after spray drying with colistin nor reduce the water uptake. Interestingly, trileucine showed a superior moisture protective effect to that of leucine, which could be attributed to its better surface enrichment efficiency than that of leucine due to its lower water solubility. Conclusions: Importantly, our results showed that the surface enrichment with leucine and trileucine did not significantly affect in vitro dissolution of colistin in the Franz cell test and cellular uptake of colistin in the H441 lung epithelium cell model, which could be attributed to small particle size and incomplete surface coverage by leucine or trileucine.

Subcutaneous administration has become an increasingly important route for delivering protein therapeutics, driven by patient convenience and the growing use of self-administration devices. However, conventional subcutaneous injection systems are typically limited to injection volumes of approximately 1–2 mL, posing significant formulation challenges for protein drugs requiring high therapeutic doses. Monoclonal antibodies (mAbs), for example, often require concentrations exceeding 100 mg/mL to enable subcutaneous delivery, which introduces challenges related to limited solubility, elevated viscosity, and an increased risk of physical and chemical instability. Therefore, high-concentration protein suspensions have emerged as a promising formulation strategy to overcome these limitations and enable subcutaneous administration of high-dose proteins. In such systems, therapeutic protein solid particles are suspended in vehicles in which they are insoluble, giving rise to unique considerations related to particle properties, protein stability, and suspension behaviors such as viscosity, injectability, and sedimentation. Accordingly, multiple particle production approaches have been explored to enable the development of ultra-high-concentration protein suspensions (>200 mg/mL). This review article aims to provide a comprehensive overview of particle formation techniques and the relationships between key particle properties and suspension performance attributes relevant to the development of high-concentration protein suspensions for injectable applications, as well as future directions in this field.