Abstract
We designed a series of peptide vectors that contain functional fragments with the goal of enhancing cellular internalization and gene transfection efficiency. The functional fragments included a cell-penetrating peptide (R9), a cationic amphiphilic α-helical peptide [(LLKK)3-H6 or (LLHH)3], a stearyl moiety, and cysteine residues. Vectors were also synthesized with D-type amino acids to improve their proteolytic stability. The conformations, particle sizes, and zeta potentials for complexes of these peptides with pGL3 plasmid DNA were characterized by circular dichroism and dynamic light scattering. In addition, cellular uptake of the peptide/DNA complexes and gene transfection efficiency were investigated with fluorescence-activated cell sorting and confocal laser-scanning microscopy. Greater transfection efficiency was achieved with the vectors containing the R9 segment, and the efficiency was greater than Lipo2000. In addition, the D-type C18-c(llkk)3ch6-r9 had about 7 times and 5.5 times the transfection efficiency of Lipo2000 in 293T cells and NIH-3T3 cells at the N/P ratio of 6, respectively. Overall, the multifunctional peptide gene vectors containing the R9 segment exhibited enhanced cellular internalization, a high gene transfection efficiency, and low cytotoxicity.
Highlights
Gene therapy has been investigated for its potential to treat genetic diseases such as cancer, severe combined immunode ciency, cystic brosis, and various monogenic diseases
The subsequent increase in concern regarding patient safety led to increased consideration of non-viral vectors, including cationic liposomes,[7] polymers,[8] nanoparticles,[9] peptides,[10] and multifunctional envelope-type nano devices (MEND),[11] for the delivery of gene molecules
The peptide vectors generated for this study contained four functional segments: a cell penetrating peptides (CPPs) segment (R9 or TAT), an endolysosomal membrane disrupting (ELMD) segment [(LLHH)[3] or (LLKK)3-H6], a stearyl segment, and cysteine residues
Summary
Gene therapy has been investigated for its potential to treat genetic diseases such as cancer, severe combined immunode ciency, cystic brosis, and various monogenic diseases. Hydrophobic modi cations of cationic peptide vectors can enhance the cellular uptake and endosomal escape of complexes by facilitating interactions with membranes via hydrophobic moieties.[34,35] For example, a stearyl moiety at the N-terminus of cationic peptide vectors has been shown to increase a-helicity and enhance gene delivery.[36] In an aqueous environment, cationic amino acids of amphiphilic peptide vectors extend into the aqueous phase, while stearyl moieties will self-assemble inside vector structures.[37] As a result, the vectors will form a secondary amphiphilic a-helical structure which enhances the binding ability of plasmid DNA and cellular uptake efficiency.[38] The incorporation of cysteine residues into peptides can increase a-helical content, potentially according to the sequence of the peptide molecule. The stabilities of these peptide vectors were examined in the presence of proteinase K
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