Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

Abstract

A safe and efficient quaternary gene delivery system (named Q-complexes) was constructed based on self-assembly of molecules through noncovalent bonds. This system was formulated through the cooperation and competing interactions of cationic liposomes, multifunctional peptides, and DNA, followed by coating hyaluronic acid on the surface of the ternary complexes. The multifunctional peptide was composed of two functional domains: penetrating hepatic tumor-targeted cell moiety (KRPTMRFRYTWNPMK) and a wrapping gene sequence (polyarginine 16). The effect of spacer insertion between the two domains of multifunctional peptide on the intracellular transfection of Q-complexes was further studied. Experimental results showed that the formulations assembled with various peptides in the spacer elements possessed different intercellular pathways and transfection efficiencies. The Q-complexes containing peptide in the absence of spacer element (P_a) showed the highest gene expression among all samples. The Q-complexes containing peptides with a noncleavable spacer GA (P_c) had no ability of intracellular nucleic acid delivery, whereas those with a cleavable spacer RVRR (P_d) showed moderate transfection activity. These results demonstrated that the different spacers inserted in the multifunctional peptide played an important role in in vitro DNA transfection efficiency. Atomic force microscopy images showed that the morphologies of ternary complexes (LP_cD) and Q-complexes (HL_cPD) were crystal lamellas, whereas those of other nanocomplexes were spheres. Circular dichroism showed the changed configuration of peptide with spacer GA in nanocomplexes compared with that of its free state, whereas the P_a configuration without spacer in nanocomplexes was consistent with that of its free state. The present study contributed to the structural understanding of Q-complexes, and further effective modification is in progress.

Keywords:

• Q-complexes
• spacer elements
• multifunctional peptides
• lipid component
• intracellular transfection

Supplementary materials

Figure S1 (A–D) The results of hydrodynamic size and zeta potential measurements of ternary complexes and Q-complexes wrapping pGl3 plasmid.

Notes: The data is the weight ratio of each component in Q-complexes. For example, “H:L:P_a:D-14:1:8:1” refers to hyaluronic acid, cationic liposome, P_a and DNA at 14:1:8:1 weight ratios. LPD: a cationic liposome, multifunctional peptide, and DNA at optimized ratios; HLPD: H represents hyaluronic acid, L represents cationic liposome that was composed of DOTAP/DOPE at a 1:1 weight ratio, P represents peptide (P_a–P_d refers to the different peptide used), and D represents DNA.

Abbreviation: PDI, polydispersity index.

Figure S2 Electrophoretic mobility in 1% agarose gel for ternary complexes and Q-complexes wrapping pGL3 plasmid.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No 30901881 and 31470587) and Engineering and Medical Cooperation Projects of Shanghai Jiao Tong University (No YG2013MS42 and YG2014QN04).

Disclosure

The authors report no conflicts of interest in this work.