The development of efficient delivery systems for nucleic acid-based cancer therapies is an on-going challenge. Lipid-based nanocarriers are widely used; however, they present limitations, in-cluding poor specificity, toxicity, and instability under physiological conditions. Peptide-based systems are emerging as promising alternatives due to their tunable properties. Nevertheless, their clinical application is hindered by susceptibility to proteases and limited stability. In this study, we investigate the potential of D-peptides, protease-resistant synthetic peptides composed exclusively of D-amino acids (MIPs, Mirror-Image Peptides)1, as nanovectors for nu-cleic acid delivery. Due to their inverted chirality, D-peptides demonstrate remarkable resistance to protease activity while retaining biological functionality. Among various constructs, we select-ed a promising sequence, RALA2, characterized by the presence of charged amino acids, which enable self-assembling and the complexation of negatively charged nucleic acids (NAs). Using both manual and automated microfluidic-based synthesis we produce nanoparticles complexed with either RNA or double-stranded DNA. Our results demonstrate that microfluidics synthesis ensures superior size control compared to manual methods and, importantly, offers a scalable and reproducible platform suitable for future clinical translation. Obtained nanoparticles were evaluated for their stability and protease-resistance through gel electrophoresis, which revealed significantly enhanced retention of NAs cargo in D-form nanoparticles relative to their natural L-form counterparts. Moreover, D-form nanoparticles were tested for their ability to silence gene expression in MDA-MB-231 luciferase-expressing cells, showing comparable siRNA-mediated knockdown efficiency. The ultimate goal of this approach is to activate the cGAS-STING innate immune pathway3 by delivering dsDNA (cGAS agonists) to the tumor microenvironment. A key advantage of this sys-tem lies in the modularity of the peptide scaffold, which can be rationally engineered to include targeting moieties, enabling selective delivery to desired cellular subpopulations These findings provide a foundation for the development of next-generation D-peptide-based na-noparticles as effective, customizable, and scalable vectors for targeted nucleic acid delivery and immunomodulation in cancer therapy.
Banfi et al. (Wed,) studied this question.