Design of the Zinc Ion and Plasmid DNA Co-Delivery System by Poly(1-Vinylimidazole) Derivatives for Myoblast Differentiation

The co-delivery system of zinc ions (Zn2+) and plasmid DNA (pDNA) has been designed by the use of poly(1-vinylimidazole) (PVIm) derivatives for myoblast differentiation. Six PVIm derivatives were synthesized, followed by the optimization of the chemical structure. As a result, methylated and carboxymethylated PVIm (CM-PVIm-Me) delivered the highest amount of Zn2+ ions inside C2C12 myoblast cells. The CM-PVIm-Me also delivered pDNA inside the myoblast cells to exhibit pDNA gene expression which was upregulated by the co-delivered Zn2+ ions. The co-delivered Zn2+ ions were localized in the cell nucleus presumably to affect cellular functions. Actually, the myoblast cells treated with the Zn2+/CM-PVIm-Me/pDNA complexes differentiated to myotubes. These results suggest that the Zn2+ ions delivered by the CM-PVIm-Me inside the cells differentiated myoblasts to myotubes. Our co-delivery system of Zn2+ ion and pDNA without zinc transporter can be a unique tool of regenerative medicine for muscular injury

Zinc-Chelated Poly(1-vinylimidazole) and a Carbohydrate Ligand Polycation Form DNA Ternary Complexes for Gene Delivery

Zinc-chelated poly(1-vinylimidazole) (PVIm-Zn) and a carbohydrate ligand polycation, a poly(l-lysine) conjugated with lactose molecules (PLL-Lac), have formed DNA ternary complexes for gene delivery. The particle size of the PVIm-Zn/DNA complexes with negative zeta potential was decreased by the addition of the PLL-Lac. The resulting PLL-Lac/PVIm-Zn/DNA ternary complexes, which exhibited the pH-dependent dissociation of the PLL-Lac, mediated more gene expression than the PVIm/DNA binary complexes. The PLL-Lac/PVIm-Zn/DNA complexes with the specific recognition of cell surface receptors mediated the highest gene expression without cytotoxicity at a relatively lower charge ratio (positive/negative = 2.5). These results suggest that the pH-dependent dissociation of the carbohydrate ligands after the recognition of cell surface receptors, including the physicochemical and biochemical function of PVIm-Zn, played an important role in gene expression

Synthesis and Characterization of Methylated Poly(l-histidine) To Control the Stability of Its siRNA Polyion Complexes for RNAi

Poly­(l-histidine) (PLH) with dimethylimidazole groups has been synthesized as a pH-sensitive polypeptide to control the stability of its small interfering RNA (siRNA) polyion complexes for RNA interference (RNAi). The resulting methylated PLH (PLH-Me) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid–base titration and solution turbidity measurement. Agarose gel retardation assay proved that the quaternary dimethylimidazole groups worked as cationic groups to retain siRNA. The stability of the PLH-Me/siRNA complexes has depended on the content of hydrophobic groups, that is, τ/π-methylimidazole groups as well as deprotonated imidazole groups. PLH-Me exhibited no significant cytotoxicity despite the existence of cationic dimethylimidazole groups. By use of PLH-Me as a pH-sensitive siRNA carrier, the PLH-Me/siRNA complexes mediated efficient siRNA delivery attributed to the dimethylimidazole groups, and the gene silencing depended on the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups. These results suggest that PLH-Me controls the stability of siRNA polyion complexes by enhancing noncytotoxic siRNA delivery by optimizing the content balance of dimethyl, τ/π-methyl, and unmodified imidazole groups

Synthesis and Characterization of Alkylated Poly(1-vinylimidazole) to Control the Stability of its DNA Polyion Complexes for Gene Delivery

Poly(1-vinylimidazole) (PVIm) with alkylated imidazole groups has been synthesized as a pH-sensitive polycation to control the stability of its DNA polyion complexes for gene delivery. The resulting alkylated PVIm (PVIm-R) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid−base titration and solution turbidity measurements. Agarose gel retardation assay proved that the alkylated imidazole groups worked as anchor groups to retain DNA. Pyrene fluorescence measurement showed that the hydrophobic domain of the DNA complex with butylated PVIm (PVIm-Bu) increased after the protonation of imidazole groups of the PVIm-Bu to enhance the membrane disruptive activity. The PVIm-Bu exhibited no significant cytotoxicity in spite of the existence of cationic groups. The resulting PVIm-Bu/DNA complexes easily released DNA, as compared with the octylated PVIm, which was examined by competitive exchange with dextran sulfate. As a result, the PVIm-R/DNA complexes mediated efficient gene delivery, and the gene expression depended on the length and density of the alkyl chains. These results suggest that pH-sensitive PVIm-R’s control of the stability of DNA polyion complexes enhanced noncytotoxic gene delivery by the optimized alkylated imidazole groups

Plasmid DNA Mono-Ion Complex Stabilized by Hydrogen Bond for In Vivo Diffusive Gene Delivery

Our original concept of the mono-ion complex (MIC) between plasmid DNA (pDNA) and a monocationic biocompatible polymer has been stabilized by hydrogen bond formation. To form the hydrogen bond with pDNA, ω-amide-pentylimidazolium end-modified poly­(ethylene glycol), that is, APe-Im-PEG, has been synthesized. Agarose gel retardation assay and circular dichroism measurement have revealed that the MIC between pDNA and APe-Im-PEG has been stabilized by the hydrogen bond between pDNA and the ω-amide group and that the stable MIC has surprisingly further migrated into gel, as compared with naked pDNA. The rise of melting temperature suggests that the specific hydrogen bond forms between an adenine-thymine base pair and the ω-amide group. The resulting pDNA MIC with APe-Im-PEG has enhanced gene expression by intramuscular administration in mice, as compared with a poly­(ethylenimine) polyion complex (PIC). These results suggest that the pDNA MIC is diffusive in vivo administration site, as compared with pDNA PICs. Our methodology for MIC stabilization by a ω-amide group is expected to offer superior supramolecular systems to those by ubiquitous PICs for in vivo diffusive gene delivery

Double-Stranded RNA Homopolymer Poly(rC)·Poly(rG) for a New pH-Sensitive Drug Carrier

Double-stranded RNA homopolymer poly(rC)·poly(rG) has been used as a new pH-sensitive drug carrier. The poly(rC)·poly(rG) had proton buffering capacity around pH 6, owing to protonation of cytosine, as determined by acid–base titration. By circular dichroism measurement, the protonation caused conformational change of the RNA. The poly(rC)·poly(rG) and doxorubicin (Dox), as an anticancer drug, formed the complexes which released the drugs at endosomal pH. The resulting complex exhibited higher anticancer activity than the Dox alone. These results result suggest that the poly(rC)·poly(rG) is a promising biopolymer for a new class of pH-sensitive drug carriers

Carboxymethyl Poly(l-histidine) as a New pH-Sensitive Polypeptide To Enhance Polyplex Gene Delivery

Carboxymethyl poly(l-histidine) (CM-PLH) as a new pH-sensitive polypeptide has enhanced polyplex gene delivery. Agarose gel retardation assay and zeta potential measurement proved that the anionic CM-PLH at physiological pH coated the PEI/DNA binary complexes. The resulting CM-PLH/PEI/DNA ternary complexes showed the gene expression value 300 times higher than that of the PEI/DNA binary complexes. These results suggest that the synergistic effect of the pH-sensitive imidazole groups at endosomal pH and the anionic carboxymethyl groups at physiological pH in the CM-PLH enhanced polyplex gene delivery

Alkylimidazolium End-Modified Poly(ethylene glycol) To Form the Mono-ion Complex with Plasmid DNA for <i>in Vivo</i> Gene Delivery

In this study, we consider that the decrease in the transfection activity of polycations in vivo, compared with that in vitro, results from their polyion complex formation. Namely, owing to cross-linking between polycations and plasmid DNAs (pDNAs), the disadvantage of in vivo gene delivery mainly stems from the difficulty in controlling the properties of the resulting polyion complex at the nanoscale size. To avoid the cross-linking by polycations, we have establish the concept of “mono-ion complex” formation between pDNA and a monocationic biocompatible polymer. Here we have synthesized alkylimidazolium end-modified poly­(ethylene glycol), that is, R-Im-PEG, and have tuned the electrostatic interaction between the resulting alkylimidazolium group and the phosphate group of pDNA by the length of the alkyl chain to achieve “mono-ion complex” formation with pDNA for in vivo gene delivery. Instead of a polyion complex, our original concept of the “mono-ion complex” consisting of the Bu-Im-PEG and pDNA is expected to offer unique tools to break through the barriers of in vivo gene delivery. As well as the field of gene delivery, this study is considered to have exploded the common sense that it is impossible to form not a polyion complex but a “mono-ion complex” under aqueous conditions for all fields of the modification of biomacromolecules

Design of Aminated Poly(1-vinylimidazole) for a New pH-Sensitive Polycation To Enhance Cell-Specific Gene Delivery

Poly(1-vinylimidazole) (PVIm) with aminoethyl groups has been synthesized as a new pH-sensitive polycation to enhance cell-specific gene delivery. The resulting aminated PVIm (PVIm-NH2) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid−base titration and solution turbidity measurement. Hemolysis assay showed that PVIm-NH2 enhanced membrane disruptive ability at endosomal pH, owing to the protonation of the imidazole groups with a pKa value around 6.0. Agarose gel retardation assay proved that the introduced aminoethyl groups worked as anchor groups to retain DNA. Furthermore, the ternary complex of DNA, PVIm-NH2, and a poly(l-lysine) conjugated with lactose molecules, PLL-Lac, at pH 7.4 dissociated the PLL-Lac polycation by protonation of the imidazole groups of PVIm-NH2 at pH 6.0. The resulting PVIm-NH2/DNA binary complexes easily released DNA, as compared with the PLL-Lac/PVIm-NH2/DNA ternary complex, which was examined by competitive exchange with dextran sulfate. By using PVIm-NH2 as a pH-sensitive DNA carrier, as well as PLL-Lac as a cell-targeting DNA carrier, the resulting ternary complex specifically mediated the gene expression, which depended on the protonation of the imidazole groups, on human hepatoma HepG2 cells with asialoglycoprotein receptors. These results suggest that the cell-specific gene delivery mediated by PLL-Lac was enhanced by PVIm-NH2 as a new pH-sensitive polycation

Pharmaceutical Effect of Manganese Porphyrins on Manganese Superoxide Dismutase Deficient Mice

Mice lacking manganese-superoxide dismutase (Mn-SOD) activity exhibit typical pathology of dilated cardiomyopathy (DCM). In the present study, the structure–activity relationship between the water-soluble manganese (Mn) porphyrin with SOD activity and the <i>in vivo</i> pharmaceutical effect on DCM is reported. The Mn-SOD-deficient mice were treated with Mn-porphyrins for 3 weeks. The treatment of a Mn-porphyrin, MnM2Py₂P, suppressed the progression of cardiac dilation. These results suggest that the Mn-porphyrin MnM2Py₂P treatment is proposed as a potential therapy for DCM