Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.
B. R. Liu et al., "Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells," PLoS ONE, vol. 11, no. 3, Public Library of Science, Mar 2016.
The definitive version is available at https://doi.org/10.1371/journal.pone.0150439
Keywords and Phrases
Calcium Chloride; Enhanced Green Fluorescent Protein; L5a Cell Penetrating Peptide; Lactoferricin; Nitrogen; Phosphate; Plasmid DNA; Unclassified Drug; DNA; Fluorescein Isothiocyanate; Lactoferricin B; Lactoferrin; Cell Viability; Complex Formation; Concentration (Parameters); Controlled Study; Cytotoxicity; Dose Response; EGFP Gene; Fluorescence Microscopy; Gene Expression; Gene Targeting; Human Cell; Internalization; Particle Size; Protein Analysis; Protein DNA Interaction; Protein Expression; Protein Transport; Real Time Polymerase Chain Reaction; Static Electricity; Zeta Potential; Amino Acid Sequence; Bovine; Chemistry; Drug Effects; Endocytosis; Gene Transfer; Genetic Transfection; Metabolism; Molecular Genetics; Reporter Gene; Tumor Cell Line; Animals; Cattle; Cell Line, Tumor; Cell-Penetrating Peptides; Fluorescein-5-Isothiocyanate; Gene Transfer Techniques; Genes, Reporter; Humans; Microscopy, Fluorescence; Molecular Sequence Data; Plasmids; Transfection
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01 Mar 2016