Optimization Of Peptide-Tagged Cationic Lipid Nanoparticles For Targeted Gene Delivery

A limitation in the use of cationic lipids as gene delivery vectors is lack of in vivo targeting. PEGylation (PEG; polyethylene glycol) sterically stabilizes cationic lipid-nucleic acid (CL-NA) nanoparticles but reduces their therapeutic efficacy through inhibition of cell-nanoparticle interactions [1]. To successfully deliver genetic material, nanoparticles must overcome barriers, including cellular binding and uptake, endosomal escape, and release of cargo. Both binding and endosomal escape require attractive interactions between the nanoparticle and cellular membranes. To recover cell binding and internalization, targeting ligands (e.g. peptides) must be attached to the distal end of PEG chains [2]. Peptide sequence and structure determine the receptor-ligand interaction strength, while mole fraction of ligand-PEG-lipid modulates efficacy due to competing factors of adequate cell binding and decreased stealth properties. We have also seen cell detachment due to blocking of receptors. Further, the addition of acid-labile PEG-lipids (HPEG-lipid) increases membrane interactions in late endosomes via shedding of PEG molecules [1]. A reduction in ligand-PEG-lipid allows for replacement with HPEG, promoting endosomal escape.We used CL-NA nanoparticles displaying linear and cyclic variations on RGD as well as C-end Rule motifs (e.g. iRGD and RPARPAR), which target integrin and neuropilin-1 receptors, respectively [3]. We determined the efficacy of CL-NA nanoparticles as a function of ligand type and ligand-tagging densities using flow cytometry, fluorescent colocalization with Rab proteins (GFP-Rab-GTPases; markers of endocytic organelles), and gene expression measurements. Our goal is to determine the relationship between different nanoparticle formulations and their ability to overcome the barriers in gene delivery and to identify regions in the nanoparticle formulation phase diagram that give optimized vectors.[1] Chan, C.L.et al.; Biomaterials, 33, 4928-4935, 2012[2] Majzoub, R. N. et al.; Biomaterials, 35, 4996-5005, 2014[3] Teesalu, T. et al.; Front Oncol, 3, 216, 2013