Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.     

Gene transfection into HeLa cells by vesicles containing cationic peptide lipid

Lipid vesicles are potentially useful as microcapsules for drug and/or gene delivery. We developed cationic lipid vesicles consisting mainly of sorbitan monooleate (Span 80) and cationic peptide lipid (CPL), and evaluated the CPL vesicles as gene transfection vectors. The optimum CPL concentration for gene transfection into HeLa cells was found to be 20 wt % of total lipid, and such CPL vesicles did not exhibit significant cytotoxicity. Co-culture of Poly-L-lysine and plasmids prior to making CPL vesicle-plasmid complexes was effective. Lipofection using LipofectAMINE was suppressed in 10% serum-supplemented medium. The transfection efficiency of 20 wt % CPL vesicles, however, was not affected by serum in the medium when plasmids were treated with poly-L-lysine.     

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.     

Entry of a cationic lytic-type peptide into the cytoplasm via endocytosis-dependent and -independent pathways in human glioma U251 cells

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.     

Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes.

Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes.     

A peptide carrier for the delivery of biologically active proteins into mammalian cells.

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.     

Cell surface oligonucleotide-binding proteins of human squamous carcinoma A431 cells

Affinity modified with Flu-DAP-p(N)16degU oligonucleotide-binding proteins were isolated by affinity chromatography using Ultrogel A2-anti fluorescein antibodies. After separation by SDS-PAGE the proteins with molecular masses about 68 kDa were MS/MS sequenced and identified as keratin K1, keratin K10, keratin K2e and albumin.     

A second activation peptide from bovine cationic trypsinogen.

1. Although only one activation peptide of bovine cationic trypsinogen has been reported previously, the peptide fraction obtained from activation mixtures shows several bands on paper electrophoresis at pH 6.5. 2. The major band was the peptide previously described. The band second in intensity of staining with ninhydrin (10-20% of that of the main band, as judged by eye) had an electrophoretic mobility consistent with its being related to the main peptide. It appeared on activation both of bulk commercial samples of trypsinogen and, as the Appendix shows, of samples prepared from pancreases obtained at the local abattoir. 3. The second peptide proved to be Phe-Pro-Val-Asp-Asp-Asp-Asp-Lys, and we conclude that it is another activation peptide. We discuss briefly the genetic and phylogenetic implications of our findings.     

Interaction of cationic alpha-helical peptide with phosphorothioate DNA hybrid.

Synthetic amphiphilic alpha-helix peptides were found to bind to stabilize double or triple stranded DNA. The stabilization effect was significant for cationic alpha-helix peptides which indicated the importance of electrostatic interaction of positive charge of peptide and negative charge of DNA. It should be also pointed out that hybrid double or triple helical complexes containing phosphorothioate oligonucleotide were stabilized to a larger extent respect to phosphodiester oligonucleotides. Since it was shown that cationic amphiphilic alpha-helix peptide accelerate membrane permeability of DNA, the present study can provide a solution for the problems of antisense or triplex oligonucleotide in their practical application.     

New cationic liposomes for gene transfer into mammalian cells with high efficiency and low toxicity

Novel cationic amphiphiles, based on hydrophobic cholesterol linked to L-lysinamide or L-ornithinamide, were designed and tested as nonviral gene transfer vectors. Each amide form of amino acid was conjugated to cholesterol by a carbamate ester bond to facilitate efficient degradation in animal cells. Cytotoxicity tests were performed for some cell lines. The transfection efficiency of the amphiphiles on different cell lines was evaluated as a liposomal solution in the presence of the fusogenic helper lipid, dioleoyl phosphatidylethanolamine (DOPE). The efficiency was also compared with other generally used gene carriers, such as lipofectin, 3 beta[N-(N'N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) liposome, and polyethylenimine (PEI).     

Evaluation of cationic liposome suitable for gene transfer into pregnant animals.

Cationic liposome-mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the beta-galactosidase gene through intravenous injection into pregnant animals. Transduction efficiency was assessed by Southern blot analysis and expression of the transferred gene was evaluated by enzymatic demonstration of beta-galactosidase activity. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE-C reagent, a liposome formulation of the cationic lipid DMRIE (1, 2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol in membrane-filtered water met our requirements. When the plasmid DNA/DMRIE-C complexes were administered intravenously into pregnant mice at day 11.5 post coitus (p.c.), transferred genes were observed in several organs in dams and were expressed. Furthermore, although the copy numbers transferred into embryos were low, we observed reporter gene expression in the progeny.     

Aggregation and membrane permeabilizing properties of designed histidine-containing cationic linear peptide antibiotics.

Members of the LAH4 family of cationic linear peptide antibiotics have been designed to form amphipathic helical structures in membrane environments and switch from alignments parallel to the bilayer surface to transmembrane orientations in a pH-dependent manner. Here the aggregation in aqueous buffer of two members of the family has been investigated by DLS. The peptides form monomers or small oligomers at pH = 5 but associate into nano-sized aggregates at physiological pH. The diameter of these latter complexes can be considerably reduced by sonication. Furthermore, the membrane interactions of the various supramolecular aggregates with POPC or mixed POPC/POPS vesicles have been investigated in calcein-release assays. In all the cases tested, the large preformed oligomeric peptide aggregates of 20-40 nm in size were more active than the structures with the smallest hydrodynamic radii in releasing the fluorescent dye from LUV. In contrast, the relative activity after sonication depends on the specific environment tested. The data suggest that these amphiphiles form micellar structures and support the notion that they can act in a manner comparable to detergents.     

Penetration of the peptide lactaptin into human cancer cells

Lactaptin, a human milk protein with a molecular weight of 8.6 kDa, is a fragment of human ?casein, which has cytotoxic activity toward mammalian cancer cells in vitro. RL2 is a recombinant analogue of lactaptin, which induces the apoptosis of human cancer cells in culture and suppresses the tumor growth in vivo. It has been shown earlier that RL2 penetrates into both human cancer and nonmalignized cells and binds to cytoskeletal structures. In this process, it induces the apoptosis of cancer cells and does not diminish the viability of normal cells. The mechanism of the penetration of RL2 into human cancer cells has been studied by flow cytometry and fluorescence microscopy using the inhibitors of different endocytosis pathways. It has been shown that RL2 penetrates into cells partly through lipid raft-mediated dynamin-independent pinocytosis and partly through direct penetration across the plasma cell membrane. An analysis of the primary structure of RL2 and the mechanism of its penetration into the cell suggests that it can be assigned to the class of cell-penetrating peptides.     

Formate protects stationary-phase Escherichia coli and Salmonella cells from killing by a cationic antimicrobial peptide

For a sustained infection, enteric bacterial pathogens must evade, resist or tolerate a variety of antimicrobial host defence peptides and proteins. We report here that specific organic acids protect stationary-phase Escherichia coli and Salmonella cells from killing by a potent antimicrobial peptide derived from the human bactericidal/permeability-increasing protein (BPI). BPI-derived peptide P2 rapidly halted oxygen consumption by stationary-phase cells preincubated with glucose, pyruvate or malate and caused a 109-fold drop in cell viability within 90 min of addition. In marked contrast, O2 consumption and viability were not significantly affected in stationary-phase cells preincubated with formate or succinate. Experiments with fdhH, fdoG, fdnG, selC and sdhO mutants indicate that protection by formate and succinate requires their oxidation by the Fdh-N formate dehydrogenase and succinate dehydrogenase respectively. Protection was also dependent on the BipA GTPase but did not require the RpoS sigma factor. We conclude that the primary lesion caused by this cationic peptide is not gross permeabilization of the bacterial cytoplasmic membrane but may involve specific disruption of the respiratory chain. Because P2 shares sequence similarity with a range of other antimicrobial peptides, its cytotoxic mechanism has broader significance. Additionally, protective quantities of formate are secreted by E. coli and Salmonella during growth suggesting that such compounds are important determinants of bacterial survival in the host.     

Delivery of "suicide" thymidine kinase gene of herpes virus in the complex with cationic peptide into human hepatoma cells in vitro

The delivery of "suicide" herpes simplex virus type-1 thymidine kinase gene (tk) into tumor cells, followed by treatment with synthetic nucleotide analogues (gancyclovir, acyclovir), is a perspective approach to cancer therapy. Serious limitations in employment of the existing means of gene delivery into target cells constitute the main obstacle for cancer gene therapy development. In the present work a possibility to use a nonviral gene delivery system is shown based on the employment of lysine rich peptide K8 and amphipathic peptide JTS-1 for transferring tk gene into human hepatoma HepG2 cells. Cationic peptide K8 forms compact complexes with plasmid DNA, and JTS-1 acts as a pH-dependent endosomal releasing agent. Transfection of HepG2 cells by tk expression vector coupled with K8/JTS-1 peptides, followed by acyclovir administration (50-100 micrograms/ml) for 24 h leads to cell cycle arrest in the G1/S checkpoint of some cells, which eventually die through apoptosis. Treatment of HepG2 cells with higher acyclovir concentration (200 micrograms/ml) additionally results in a nonspecific toxic effect. The above results demonstrate the efficacy of K8/JTS-1 delivery system for the "suicide" cancer gene therapy, and may be regarded as a basis for further elaboration of "suicide" cancer approaches in vivo.