Enhanced protein internalization and efficient endosomal escape using polyampholyte-modified liposomes and freeze concentration

Here we show a new strategy for efficient freeze concentration-mediated cytoplasmic delivery of proteins, obtained via the endosomal escape property of polyampholyte-modified liposomes. The freeze concentration method successfully induces the efficient internalization of proteins simply by freezing cells with protein and nanocarrier complexes. However, the mechanism of protein internalization remains unclear. Here, we designed a novel protein delivery carrier by modifying liposomes through incorporating hydrophobic polyampholytes therein. These complexes were characterized for particle size, encapsulation efficiency, and cytotoxicity. Flow cytometry and microscopic analysis showed that the adsorption and internalization of protein-loaded polyampholyte-modified liposomes after freezing were enhanced compared with that observed in unfrozen complexes. Inhibition studies demonstrated that the internalization mechanism differs between unmodified and polyampholyte-modified liposomes. Furthermore, polyampholyte-modified liposomes exhibited high efficacy in facilitating endosomal escape to enhance protein delivery to the cytoplasm with low toxicity. These results strongly suggest that the freeze concentration-based strategy could be widely utilised for efficient cargo delivery into the cytoplasm in vitro not only in cancer treatment but also for gene therapy as well

Interaction of fengycin with stratum corneum mimicking model membranes: a calorimetry study

CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY–siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. We calculated and compared several complexes with peptide/siRNA ratios of up to 40. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)), CADYs can adopt multiple positions at the siRNA surface. Nevertheless, several complex properties are common: an average of 14 ± 1 CADYs is required to saturate a siRNA. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA and the peptide cage is stabilized by hydrophobic CADY–CADY contacts. The analysis demonstrates that the hydrophobicity, the positive charges and the polymorphism of CADY are mandatory to make stable the CADY–siRNA complexes.IAP/Belspo P7/44 project : Integrative Protein Science (iPROS