(628g) Polypeptide Micelles for Stimuli-Responsive and Targeted Nonviral Gene Delivery

Gene delivery using polypeptide-based nonviral vectors has been of great interest. Polypeptides are biodegradable and can be easily functionalized for versatile applications simply by tailoring their amino acid sequences. Although poly-L-lysine (PLL or poly[Lys]) efficiently condenses nucleic acids and has been popularly employed in clinical nonviral gene therapy, its low buffering capacity affords only modest transfection. Consequently, poly(Lys) is frequently co-administered with additional endosomolytic agents (e.g., chloroquine and fusogenic peptides) in order to achieve acceptable gene transfection. Moreover, the cytotoxicity of poly(Lys) is not negligible and poly(Lys)/DNA polyplexes are unstable in serum, which poses significant limitations in nonviral gene therapy.

In this study, poly(Lys)-mimicking but acid-transforming polypeptides were developed. The hydroxyl group of serine residue was conjugated with an acid-cleavable amine-bearing branch. The resulting poly(ketalized serine) (or poly[kSer]) is designed to 1) hydrolyze in the mildly acidic endosome, 2) enhance endosomal escape, and 3) release the payload (nucleic acids) in the cytoplasm, upon transformation from cationic poly(kSer) to neutral poly(Ser). Finally, the poly(kSer) was further conjugated with polyethylene glycol (PEG) and used to complex DNA in a form of micelles.

The PEG-poly(kSer)/DNA micelles showed significantly improved transfection efficiency (2-3 folds) than PEG-poly(Lys)/DNA micelles and poly(Lys)/DNA polyplexes, via enhanced cytosolic release and DNA unpackaging. In addition, cross-linked PEG-poly(kSer)/DNA micelles resulted in much higher transfection than non-cross-linked ones in the presence of serum. PEG-poly(kSer) was further functionalized with folic acid (FA), a popular molecule used for cancer cell-targeted delivery, and selective transfection of HeLa human cervical cancer cells over NIH 3T3 murine fibrobalst cells was obtained by FA-PEG-poly(kSer)/DNA micelles. The acid-transforming PEG-poly(kSer)/DNA micelles, which were rationally designed to overcome key extra- and intracellular barriers, are promising nonviral vectors for efficient, safe, serum-resistant, and cancer-targeted gene delivery.