# Comparison of Drying Techniques to Produce Stable and Bioavailable Encapsulated ACE-2 Nanoparticles

## TL;DR

This study compares drying methods to create stable ACE-2 nanoparticles that can help reduce SARS-CoV-2 infection by blocking viral binding to human cells.

## Contribution

The study identifies spray-freeze drying as the optimal method for dehydrating ACE-2 nanoparticles with minimal cryoprotectant use.

## Key findings

- Spray-freeze drying produced higher yield rates and better particle properties with less cryoprotectant.
- The best formulation used pH 5.5 and a 2:1 chitosan–ACE-2 ratio, achieving high encapsulation efficiency and loading content.
- ACE-2 NPs showed effective release and uptake in Caco-2 cells, indicating potential for dry powder applications.

## Abstract

Background/Objectives: COVID-19 infection continues globally, with frequent emergence of unfamiliar SARS-CoV-2 variants acting to impair immunity. The competitive binding of SARS-CoV-2 spike proteins and angiotensin-converting enzyme 2 (ACE-2) can decrease the binding of the virus on native ACE-2 receptors on healthy human cells. It remains a practical approach to lessen viral spread. In this study, a method to encapsulate ACE-2 in the form of chitosan/tripolyphosphate cross-linked nanoparticles (NPs) was developed with emphasis placed on the best dehydration method to secure functional ACE-2 nanoparticles. Methods: Methods: Preparation conditions were assessed by varying pH (4.0–6.5) and the ratio between chitosan and ACE-2 mixing ratios (1:1, 1.5:1, 2:1, 2.5:1, and 3:1). The formulated NPs were then dehydrated using different approaches that included spray-drying (SD), freeze-drying (FD), and spray-freeze drying (SFD) and used varying mannitol concentrations (0, 1:1, and 5:1 of total weight). The mannitol was served as a cryoprotectant in this study. Results: The best formulation achieved used a pH 5.5 with a mixing chitosan–ACE-2 ratio of 2:1, where ACE-2-loaded NPs had an average particle size of 303.7 nm, polydispersity index (PDI) of 0.21, encapsulation efficiency (EE) of 98.4%, and ACE-2 loading content (LC) of 28.4%. After reconstitution, all SD samples had a relatively low yield rate, but the ACE-2 NPs dehydrated specifically using SFD required a lower amount of added mannitol (1:1 of its total weight) and produced a higher yield rate (p < 0.05) and similar PDI and EE values, along with relatively good particle size and LC. This formulation also produced a high ACE-2 release and uptake in differentiated Caco-2 cells, thus representing an effective ACE-2 encapsulation procedure for use with dry powders. Conclusions: This work showed that spray-freeze drying was the best method to dehydrate ACE-2 NPs, using less cryoprotectant to create a significant advantage in terms of greater loading capacity with lower additive requirements.