Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

Abstract

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.

Polyhistidine facilitates direct membrane translocation of cell-penetrating peptides into cells

“…HL6 (CHHHHHRRWQWRHHHHHC), N-terminal fluorescein isothiocyanate (FITC) labeled HL6 (FITC-HL6), FITC-L6 (RRWQWR) 17 , FITC-HR9 (CHHHHHRRRRRRRRRHHHHHC) 12 , and FITC-nonCPP (RPPGFSPFR, bradykinin) 19 peptides were purchased from Genomics (Taipei, Taiwan). Carboxyl-functionalized and water-soluble QDs (CdSe/ZnS-PEG-COOH-520) with a maximal emission wavelength of 523 nm were purchased from Mesolight (Zhejiang, China).…”

“…In our previous study, a bovine lactoferricin L6 CPP 17 has been demonstrated to noncovalently interact with carboxyl-functionalized quantum dots (QDs) and form stable L6/QD complexes that can enter cells by endocytosis 18 . The aim of the present study was to assess whether modification of L6 with additional penta-histidine and mono-cysteine residues on both sides would influence its 1) ability to form noncovalent complexes with QDs, 2) transduction efficiency, and 3) mechanism of cellular uptake.…”

Lactoferrin, a multi-functional glycoprotein: Active therapeutic, drug nanocarrier & targeting ligand

“…In addition, a cell penetrating peptide derived from lactoferricin termed as L5a CPP (RRWQW) was shown to efficiently deliver DNA into the nucleus of human A549 lung cancer cells without induction of cytotoxic effect [ 204 ]. Compared to Tf, Lf was shown to be apically internalized into Caco-2 intestinal epithelial cells and then localized to the nuclei while Tf was internalized from the basolateral side and localized in the cytoplasm [ 205 ].…”

Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple Organisms: From Prokaryotes to Mammalians Cells

“…Entry of molecules via these four pathways is trapped in lysosomes and their bioactive and therapeutic characteristics would be lost in the low pH environment [63]. CPP-mediated direct membrane location is an option for delivering drugs and other bioactive molecules [52,53,64].…”

“…CPPs can interact with cargoes in a covalent, noncovalent, or covalent and noncovalent protein transduction (CNPT) manner. Cargoes are various including proteins, DNA, siRNA, and semiconductors QDs [44,[64][65][66][67][68]. CPP-mediated cellular uptake can be found from prokaryotic to eukaryotic organisms including mammalian cells, insect cells, aquatic microorganism, yeasts, plant tissue, mice dermis, Gram-negative and Gram-positive bacteria, and archaea [53,65,[69][70][71][72][73].…”