ABSTRACT
Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.
Subject(s)
Beclin-1/pharmacology , Cell-Penetrating Peptides/metabolism , Drug Carriers/metabolism , Peptide Fragments/pharmacology , Peptide Nucleic Acids/metabolism , Autophagy/drug effects , Beclin-1/metabolism , Beclin-1/toxicity , Cell-Penetrating Peptides/toxicity , Drug Carriers/toxicity , Drug Liberation , HeLa Cells , Humans , Leucine Zippers , Oligopeptides/metabolism , Oligopeptides/toxicity , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Peptide Nucleic Acids/toxicity , Protein BindingABSTRACT
Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.
Subject(s)
Cells/metabolism , Lipids/chemistry , Oligonucleotides, Antisense/metabolism , Peptide Nucleic Acids/metabolism , Peptides/metabolism , Amino Acid Sequence , Arginine/metabolism , Cations/chemistry , Cells/cytology , Endosomes/metabolism , Fatty Acids/metabolism , Gene Products, tat/metabolism , HeLa Cells , Humans , Luciferases/metabolism , Molecular Sequence Data , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/toxicity , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/toxicity , Peptides/chemistry , Protein Structure, TertiaryABSTRACT
Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.
Subject(s)
Burkitt Lymphoma/genetics , Enhancer Elements, Genetic , Immunoglobulin mu-Chains/toxicity , Mutagens/toxicity , Nuclear Localization Signals/toxicity , Peptide Nucleic Acids/toxicity , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Animals , CHO Cells , Cricetinae , Cricetulus , Escherichia coli , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Lymphocytes/drug effects , Micronucleus Tests , Protein Transport , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Salmonella typhimuriumABSTRACT
Establishing a general and effective method for regulating gene expression in mammalian systems is important for many aspects of biological and biomedical research. Herein we report the antisense activities of a cell-permeable, guanidine-based peptide nucleic acid (PNA) called GPNA. We show that a GPNA oligomer designed to bind to the transcriptional start-site of human E-cadherin gene induces potent and sequence-specific antisense effects and is less toxic to the cells than the corresponding PNA-polyarginine conjugate. GPNA confers its silencing effect by blocking protein translation. The findings reported in this study provide a molecular framework for designing the next generation cell-permeable nucleic acid mimics for regulating gene expression in live cells and intact organisms.
Subject(s)
Cadherins/genetics , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/pharmacology , Transcription, Genetic/genetics , Base Sequence , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Survival/drug effects , Humans , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/toxicity , Peptide Nucleic Acids/genetics , Peptide Nucleic Acids/toxicity , Sensitivity and SpecificityABSTRACT
Peptide nucleic acids (PNA) are very promising antisense agents, but their in vivo application is often hampered by their low bioavailability, mainly due to their limited uptake through cellular and nuclear membranes. However, PNA chemical synthesis easily allows modification with functional structures able to improve the intrinsically low permeability and great interest is arising in finding specific and efficient delivery protocols. Polymeric core-shell microspheres with anionic functional groups on the surface were tested for their ability to reversibly bind lysine modified PNA sequences, whose antisense activity against COX-2 mRNA was already demonstrated in murine macrophages.
