Search

In this work, we synthesized a series of poly-L-lysine (PLL)-based polymers for gene delivery, by modifying the PLL with both cationic peptide and histidine. The peptide moieties serve as cationic centers for polyplex formation, and also as substrates for protein kinase Cα (PKCα), which is specifically activated in many types of cancer cells, to achieve cancer-specific gene expression. The histidine groups serve as buffering moieties to increase the ability of the plasmid DNA (pDNA)-polymer complex (polyplex) to escape the endosome and thus to promote expression of the pDNA in the transfected cells. The facile synthesis of the polymers proceeded by modifying the PLL with side-group-protected peptide and protected histidine, followed by deprotection of the functional groups. The synthesized polymers showed significant buffering capacity over the neutral to acidic pH range and showed less cytotoxicity in vitro compared with histidine-unmodified polymers. The polyplexes successfully showed PKCα-responsive gene expression immediately after their introduction into cancer cells and the gene expression continued for at least 24 h. These PLL-based carriers thus show promise for cancer-targeted gene therapy.

Here, we developed a new gene carrier, comprising a linear polyethylenimine (LPEI) grafted with a hydrophobically modified cationic peptide containing a long alkyl chain, for use in cancer-specific gene delivery. The cationic peptide is a substrate of protein kinase Cα (PKCα), which is known to be activated specifically in cancer cells. The hydrophobically modified LPEI-peptide conjugate (LPEI-C10-peptide) could form a polyplex with DNA through electrostatic and hydrophobic interactions between the anionic DNA strands and the cationic peptide substrate. The hydrophobic modification of the peptide did not affect the reactivity of the peptide toward PKCα, while the polyplex showed improved intracellular uptake. Because of the efficient endosomal escape and enhanced stability, the polyplex significantly improved the transgene regulation responding to intracellular PKCα activity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We examined in vitro performance of the branched polyethylenimine (bPEI)-based gene carriers which respond to cancer-specific activation of protein kinase Cα (PKCα) to express plasmid DNA. The carriers were synthesized straightforward by using amide bond formation between a peptide terminal carboxyl and a primary amine group of bPEI. To examine the effect of the peptide contents in the carrier, we prepared several carriers with various peptide contents. The obtained polymers form polyplexes with tighter condensation of plasmid DNA than our previous gene carriers. After internalization of the polyplexes via endocytosis, the polyplexes effectively escaped from the endosome into cytosol. Then, the polyplexes showed a clear-cut response to PKCα to release plasmid DNA for gene expression. We determined the optimum contents of the peptides in carriers as 5 mol% to achieve the clear-cut response to PKCα.

A novel intracellular delivery method both for genes and proteins is one of the most coveted systems in the drug delivery field. In the present study, we developed a double-coating carrier loaded with gene and protein produced by solid-in-oil and solid-in-water nanodispersion technology. The double-coating carriers did not require electrostatic interactions during the preparation so were able to encapsulate plasmid DNA, ovalbumin (pI 4.5), horseradish peroxidase (pI 7.2), and cytochrome-c (pI 10.5) in a consistent manner. The carriers had practical encapsulation efficiencies and release profiles for genes and proteins. Furthermore, effective gene expression and cellular uptakes of both anionic and cationic proteins were achieved by modification of carriers with functional molecules. These findings indicate that the double-coating carrier has high potential for cellular delivery of various drugs and is a novel, superior method for both gene and protein delivery into cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Recently, our group has proposed a novel gene-regulation system responding to cAMP-dependent protein kinase (PKA) that has been applied to living cells. In this study, human liver-specific bionanocapsules (BNCs) are used as a gene-delivery system to increase transfection efficiency and to target specific cell types. BNCs can efficiently deliver a target gene to human hepatocytes and hepatoma cells in vitro or in vivo. The combination of a signal-responsive gene-delivery system with BNCs led to an increase in the transfection efficiency and selectivity for hepatoma cells. Expression from the delivered gene was identified from PKA-activated hepatoma cells (HepG2), but not from colon tumor cells (WiDr). These results show that the combination of a gene-regulation system responding to an intracellular signal with BNC can be used for the selective treatment of human hepatoma cells., (Copyright 2010 Elsevier B.V. All rights reserved.)

The amphiphilic alpha-helical peptide, Td3717, is a bi-functional synthetic peptide that acts as both a polycation for DNA binding and a ligand for targeted delivery to tumor cells. Td3717 forms a stable complex with plasmid DNA, and the complex maintained high transfection efficiency after storage at 4 degrees C for six months and after four freeze/thaw cycles. During the storage and freeze/thaw cycling, the particle size of the DNA/Td3717 complex remained less than 100nm. The size of the complex is an important factor for its internalization into cells via the endocytosis pathway; therefore, the stability of the particles will strongly contribute to high transfection efficiencies after storage and repeated freezing/thawing.

We recently proposed a novel gene regulation system responding to specifically and abnormally activated intracellular enzymes in diseased cells. In the present study, we focused on protein kinase C (PKC)alpha, which is hyper-activated in most tumor cells, as a trigger for transgene regulation. We prepared cationic copolymers comprising hydrophilic and neutral polymers in main chains and cationic peptide substrates with different contents in side chains. Our copolymer with high peptide content (>3 mol%) condensed with pDNA more weakly than with poly(L-lysine) (pLL) having a similar molecular weight, but gene suppression was nearly identical to that of pLL, probably due to the steric hindrance of the main chains in our copolymer. Steric hindrance of the main chains barely affected the phosphorylation reaction of the pendant peptide. In cell and mouse experiments, higher gene expression was observed in complexes of pDNA with copolymers pended PKC alpha-specific substrate peptide than that in complexes with negative copolymers pended peptide substituted phosphorylation site of serine residues with alanine. These results indicate that our system can recognize intracellular PKC alpha as a trigger to regulate transgene expression, and may be useful for tumor gene therapy.

We newly prepared lipopolyplexes containing N-lauroylsarcosine (LS) as a hybrid vector for pulmonary gene delivery via the systemic route. Lipopolyplexes were composed of polyethylenimine (PEI), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA), LS, and plasmid DNA (pDNA). The particle size of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was not largely changed by the addition of LS, although the addition of LS decreased the high zeta potential. Lipopolyplexes containing LS with low zeta potential showed little aggregation with erythrocytes and low cytotoxicity. In HepG2 cells, lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D. After intravenous injection of the complexes into mice, lipopolyplexes containing LS showed high transgene efficiency, comparable to lipopolyplex 2P-2D. In particular, lipopolyplexes containing LS showed extremely high transgene efficiency in the lung. As a result of the analysis to identify optimum formulations, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index. These results suggest that lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity.

Cationic gold nanoparticles, prepared by reducing HAuCl(4) with NaBH(4) in the presence of 2-aminoethanethiol, formed a complex with plasmid DNA, and could be transfected into cultivated cells.

Novel carboxylic acid pendant-containing poly(ethylene glycol) (PEG) derivatives (PEG-Cs) were synthesized by the chemical modification of the carbon-carbon double-bond side chain of copoly(allyl glycidyl ether/ethylene oxide) (copoly(AGE/EO)). PEG-C showed a protecting ability for the DNA/polycation complex from the albumin-induced aggregation. It also expressed the enhanced efficiency on the polycation-mediated gene transfection on the cultured cells (up to 3-4-times higher), probably due to its disperse-stabilizing property and also the proton-sponge effect.

The success of gene therapy is largely dependent on the development of the gene delivery vector. Recently, gene transfection into target cells using naked DNA, which is a simple and safe approach, has been improved by combining several physical techniques, for example, electroporation, gene gun, ultrasound and hydrodynamic pressure. Chemical approaches have been utilized to improve the efficiency and cell specificity of gene transfer. Novel gene carrier molecules, which facilitate DNA escape from the endosome into the cytosol, have been developed. Several functional polymers, which enable controlled release of DNA in response to an environmental change, have also been reported. Plasmids with reduced number of CpG motifs, the use of PCR fragments and the sequential injection method have been established for the reduction of immune response triggered by plasmid DNA. Construction of a long-lasting gene expression system is also an important theme for nonviral gene therapy. To date, tissue-specific expression, self-replicating and integrating plasmid systems have been reported. Improvement of delivery methods together with intelligent design of the DNA itself has brought about large degrees of enhancement in the efficiency, specificity and temporal control of nonviral vectors.

Monodispersed dendritic poly(L-lysine)s (DPKs) of several generations were synthesized, and their characteristics as a gene transfection reagent were then investigated. The agarose gel shift and ethidium bromide titration assay proved that the DPKs of the third generation and higher could form a complex with a plasmid DNA, and the degree of compaction of the DNA was increased by the increasing number of the generation. The DPKs of the fifth and sixth generation, which have 64 and 128 amine groups on the surface of the molecule, respectively, showed efficient gene transfection ability into several cultivated cell lines without significant cytotoxity. In addition, the transfection efficiency of the DPK of the sixth generation was not seriously reduced even if serum was added at 50% of the final concentration into the transfection medium. Because we can strictly synthesize various DPK derivatives, which have several types of branch units, terminal cationic groups, and so on, they are expected to be a good object of study regarding the basic information on the detailed mechanism of gene transfection into cells. We also expect to be able to easily construct DPK-based functional gene carriers, e.g., DPKs modified by ligands such as a sugar chain, which can enable advanced gene delivery in vivo.

To develop a receptor-mediated gene delivery system into hepatoma cells using the cationic alpha-helical peptide as the gene carrier molecule, we modified an alpha-helical peptide, which is known to have transfection abilities into cells, with a multi-antennary ligand containing several galactose residues that provide efficient binding to the asialoglycoprotein receptor. The galactose-modified peptides formed complexes with a plasmid DNA and showed gene transfer abilities into HuH-7 cells, a human hepatoma cell line. The transfection efficiency of the peptide was increased by increasing the number of modified galactose residues on the peptide. Furthermore, considerable inhibition of the transfection efficiency by the addition of asialofetuin, which is a ligand for the asialoglycoprotein receptor, was observed in all galactose-modified peptides. Based on this result, we could confirm that the internalization of the galactose-modified peptides occurred by the receptor-mediated endocytosis pathway. In addition, to understand the transport route of the peptide-DNA complex in the cell, the effects on the transfection efficiencies with several endocytosis inhibitors were examined. As a result, it was suggested that the translocation of the peptide-DNA complex from the endocytic compartments to the cytosol mainly occurred during an early endosome step.

It has been showned that cationic alpha-helical peptides can be useful as nucleic acid-carrier molecules for gene transfer into cells. In order to investigate the significancemake sure of importance of the hydrophobic region in amphiphilic peptides in relation to their transfection ability, we have employed five kinds of peptides with a systematically varied hydrophobic-hydrophilic balance in the amphiphilic structures, and have evaluated the relationship between the structure and the gene transfer ability of the peptides into COS-7 cells. The peptides with a large hydrophobic region took alpha-helical structures, formed large aggregates and showed high transfection efficiency. Their high efficiency can be explained on the basis of their ability to form stable aggregates which can be internalized by endocytosis and remain resistant to digestion in lysosomal vesicles. Furthermore, it was suggested that the hydrophobic region of peptides plays an important role in the disruption of the endosomal membrane, which ca prevent the degradation of DNA in lysosomal vesicles. When peptides do not have so strong membrane-disruptive activity, but form aggregates which can be incorporated by endocytosis, the transfection efficiency can be recovered by the addition of an endosome-disruptive peptide., (Copyright 1998 Academic Press.)

A peptide consisting of 12 amino acids including 3 glutamic acids (LAEL-LAEL-LAEL; 4(3)E) underwent pH-dependent conformational change from random coil to alpha-helix when the pH was decreased from 7.4 to 5.0 in the presence of egg PC. This alpha-helical 4(3)E had higher membrane-perturbation activity at acidic conditions compared with neutral conditions. When 4(3)E was incorporated with plasmid DNA-cationic peptide complex that utilizes an endocytosis pathway for uptake into cultured cells, high transfection efficiency was observed, indicating that 4(3)E can enhance the transfection activity of cationic peptide. It is likely that 4(3)E in the multi-complex of the plasmid DNA and the cationic peptide effectively disrupts the endosomal membrane and increases the population of the complex which could transfer to cytosol. The small lysosome-disruptive peptide is very probably useful as the enhancer molecule for the gene transfer techniques mediated by the endocytosis pathway.

Abstract: For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic α‐helix peptides, mastoparan (INLK‐ALAA‐LAKK‐IL‐NH[sub 2]) obtained from wasp venom and an α‐helix model peptide (LARL‐LARL‐LARL‐NH[sub 2]). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS‐7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide. [ABSTRACT FROM AUTHOR]