Lipophilic Peptides for Gene Delivery

Abstract

DNA transfections are widely performed in research laboratories and in vivo gene delivery holds the promise for curing many diseases. The synthetic carriers or vectors for DNA are typically cationic lipids. However, in biology, the recognition of nucleic acids by proteins involves both electrostatic and stacking contributions. As such we have prepared a series of new lipophilic peptide vectors that possess lysine and tryptophan amino acids for evaluation. These lipophilic peptides show minimal cytotoxicity and enhanced in vitro gene transfection activity.The complexation of cationic amphiphiles with DNA or RNA and the resulting supramolecular structures are of interest for the delivery of nucleic acids to cells. As such, a means to correct a defective gene, introduce a new gene, or knock-down a gene is provided for a specific in vitro or in vivo application (1-7). This complexation between cationic amphiphiles and nucleic acids is governed by electrostatic interactions between the positively charged amphiphiles and the negatively charged phosphate backbone of the nucleic acid and the hydrophobic chainchain packing forces between the individual assembled amphiphiles. In biology, the recognition of nucleic acids by proteins involves more than electrostatic interactions (8-10). Examination of these protein nucleic acid recognition motifs typically reveals structures rich in basic and aromatic amino acids that provide important electrostatic and stacking contributions to binding. With this inspiration in mind, we are designing peptide-based amphiphiles for gene delivery. Herein we describe a series of lipophilic peptides including the synthesis, physiochemical properties, cytotoxicity, and in vitro gene transfection activity.To mimic structural characteristics present in nucleic acid binding proteins, we selected the tripeptide Lys-Trp-Lys, KWK, to be the headgroup for the amphiphile because it possesses cationic charges and an aromatic side chain and is relatively small in size. Moreover, this peptide is known to bind DNA with a binding constant on the order of 10 4 M and is a model peptide used to study protein-nucleic acid interactions, and lysine and tryptophan provide important interactions in a number of structurally characterized DNA binding domains of proteins (11)(12)(13)(14). In addition, lysine-based small molecule amphiphiles and macromolecules (e.g., polylysine) are being explored and used for gene transfection (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In order to perform a systematic study to identify the key molecular components responsible for transfection © 2008 American Chemical Society * Correspondence should be addressed to M.W.G.; author E-mail: mgrin@bu.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript activity with these new lipophilic peptides, we prepared a series of amphiphiles where the headgroup (charge, aliphatic content, aromatic content) and fatty acid chain length (C12:0, C14:0, C16:0, and C18:0) were varied. As shown in Figure ...

In Vitro Reporter Gene Transfection via Plasmid DNA Delivered by Metered Dose Inhaler

“…8,54 The absence of effect of washed, aerosolised pEGFP-N1 formulation (without DOTAP) on cell viability is promising. The significant reduction in cell viability in the presence of DOTAP concurs with previously published reports 55,56 and it may be appropriate to explore the use of alternative transfection agents 10,56,57 and 'helper' lipids. 58,59 Indeed, in vivo gene expression has been achieved in the past without the use of a transfection agent, although transfection efficiency was found to be relatively low.…”

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates

“…Recent reports have demonstrated delivery of cholesterol-modified siRNA via lipoprotein-based materials (15,18). Amino acids and peptides are natural building blocks of apolipoproteins, and previous studies have examined the potential of amino acid derivatives for gene delivery or siRNA delivery (19)(20)(21)(22)(23)(24). However, to our knowledge, none of these systems have shown potencies and/or specificities that rival traditional lipids or lipidoids.…”

Synthetic Nucleic Acid Delivery Systems: Present and Perspectives

“…A similar trend was observed in CHO cells, where lipopeptide KGG-C14 75f was the most effective lipopeptide, at a +/-charge ratio of 16. 161 Building on these conclusions, the same group investigated C14-lipopeptides 75j-75m having di-and tripeptide headgroups with different (pro)cationic amino acids separated by a mono/di-glycine spacer. Biological testing in CHO and NIH 3T3 cell lines revealed that lipopeptides having a (pro)cationic amino acid separated by a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 47 bis-glycine spacer 75f-75i displayed much higher DNA transfection activity that their congeners 75j-75m having single glycine spacers.…”