Reconfiguring the architectures of cationic helical polypeptides to control non-viral gene delivery

Abstract

Poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) (PPABLG), a cationic helical polypeptide, has been recently developed by us as an effective non-viral gene delivery vector. In attempts to elucidate the effect of molecular architecture on the gene delivery efficiencies and thereby identify a potential addition to PPABLG with improved transfection efficiency and reduced cytotoxicity, we synthesized PEG-PPABLG copolymers with diblock, triblock, graft, and star-shaped architectures via a controlled ring-opening polymerization. The PPABLG segment in all copolymers adopted helical structure; all copolymers displayed structure-related cell penetration properties and gene transfection efficiencies. In HeLa and HepG-2 cells, diblock and triblock copolymers exhibited reduced membrane activities and cytotoxicities but uncompromised gene transfection efficiencies compared to the non-PEGylated homo-PPABLG. The graft copolymer revealed lower DNA binding affinity and membrane activity presumably due to the intramolecular entanglement between the grafted PEG segments and charged side chains that led to reduced transfection efficiency. The star copolymer, adopting a spherical architecture with high density of PPABLG, afforded the highest membrane activity and relatively low cytotoxicity, which contributed to its potent gene transfection efficiency that outperformed the non-PEGylated PPABLG and Lipofectamine™ 2000 by 3-5 and 3-134 folds, respectively. These findings provide insights into the molecular design of cationic polymers, especially helical polypeptides towards gene delivery.

Fig. 1

(A) Schematic representation of PEG-PPABLG conjugates with different architectures. (B) GPC traces of the precursors (PVBLG or PEG-PVBLG conjugates) of various polypeptides.

Fig. 2

CD spectra of polypeptides in aqueous solution at pH = 7 (0.1 mg/mL) (A). Helicity of polypeptides at different concentrations (B), pH (C), and NaCl concentrations (D) as demonstrated by the molar ellipticity at 222 nm.

Fig. 3

DNA condensation by polypeptides at different N/P ratios as evaluated by the gel retardation assay (A) and EB exclusion assay (B). N represents naked DNA.

Fig. 4

Particle size (A) and zeta potential (B) of polyplexes at different N/P ratios as determined by DLS measurement.

Fig. 5

Transfection efficiencies of polyplexes in HeLa (A) and HepG-2 (B) cells at various N/P ratios. Results were indicated as mean ± SD (n=3).

Fig. 6

Uptake levels of YOYO-1 labeled pDNA in HeLa (A) and HepG-2 (B) cells when complexed with polypeptides at different N/P ratios. (C) CLSM images of HeLa cells following treatment with 8-arm PEG-PPABLG/YOYO-1-DNA complexes (N/P ratio of 15) at 37 °C for 1 h and 4 h. Bar represented 20 µm. (D) Cell uptake level of FITC in HeLa cells following co-incubation with polymers for 2 h at 37 °C. (E) Mechanistic probes of the intracellular kinetics of 8-arm PEG-PPABLG/YOYO-1-DNA complexes (N/P ratio of 15) in HeLa and HepG-2 cells by monitoring the cell uptake level in the presence of various endocytic inhibitors. Results were indicated as mean ± SD (n=3).

Fig. 7

Cytotoxicity of polymer/DNA complexes (N/P ratio of 20) towards HeLa (A) and HepG-2 (B) cells as determined by the MTT assay. Results were indicated as mean ± SD (n=3).