Novel Concentration-Based Freezing Method for Efficient Protein Delivery

We developed a novel, well-controlled slow-freezing concentration-based strategy for effective protein delivery using polyampholyte NPs in the absence of a cryoprotectant. Here, we demonstrate the feasibility of this freeze concentration method, which is exceptionally favorable for cytoplasmic protein delivery, and focus on a simple slow freeze-thaw method using slow freezing program. We used confocal laser scanning microscopy to investigate the adsorption and internalization of lysozyme proteins at variable freezing temperatures. Quantification of fluorescence intensity revealed that proteins were more easily internalized by gradually decreasing the temperature. We believe that application of this concentration-based freezing technique could be extended to gene therapy and cancer immunotherapy