Mirror-Image Peptide Nanoparticles for Nucleic Acid Delivery

The development of efficient delivery systems for nucleic acid-based cancer therapies is an ongoing challenge. Lipid-based nanoparticles are widely used; however, they present limitations, including poor specificity, toxicity, and instability under physiological conditions. Peptide-based systems are emerging as promising alternatives due to their tunable physiological properties. Nevertheless, their clinical translation is hindered by limited stability and susceptibility to proteolytic degradation. Here, we present the synthesis of nanoparticles composed of mirror-image peptides obtained through an automated microfluidic-based system. These nanoparticles are entirely built from D-amino acids, conferring intrinsic resistance to proteolytic degradation while preserving the structural and functional properties of their L-counterparts. Among various constructs, we selected a promising 30-amino acid sequence, RALA1. This cell-penetrating2, amphipathic peptide forms nanoparticles upon complexation with negatively charged nucleic acids in a highly tunable manner. D- and L-RALA peptides were complexed with nucleic acids to form nanoparticles using an automated microfluidic platform. Physicochemical properties (size, polydispersity, and zeta potential) were assessed by Dynamic Light Scattering, while secondary structures were determined by circular dichroism spectroscopy. Encapsulation efficiency and protease resistance were evaluated by agarose gel electrophoresis after serum and trypsin exposure. In vitro transfection efficiency was tested in MDA-MB-231 luciferase-expressing cells using siRNA-complexed nanoparticles, with luciferase and MTS assays. Cellular uptake and intracellular localization were analyzed by fluorescence microscopy using labeled nanoparticles, including endocytosis inhibition studies and co-culture experiments in tumor and macrophage cell lines. Using a controlled microfluidic platform, we generated reproducible and homogeneous peptide-based nanoparticles capable of complexing negatively charged nucleic acids. The automated process ensures precise size control, improved batch-to-batch consistency, and scalability, key requirements for future clinical translation. D-peptide nanoparticles maintain structural organization comparable to L-form assemblies, while exhibiting significantly enhanced resistance to enzymatic degradation. Functional studies demonstrated their ability to efficiently deliver nucleic acids into both tumor cell lines and macrophage cell models. The combined features of protease resistance, structural stability, and effective nucleic acid delivery highlight the strong potential of mirror-image peptide-based nanoparticles as next-generation vectors for cancer gene therapy applications. 1 https://doi.org/10.1016/j.ijpharm.2021.120223 2 https://doi.org/10.1038/s41392-024-02107-5