Characterization of Interpolyelectrolyte Complexes between Double-Stranded DNA and Polylysine Comb-Type Copolymers Having Hydrophilic Side Chains

Maruyama, Atsushi; Watanabe, Haruo; Ferdous, Anwarul; Katoh, Maiko; Ishihara, Tsutomu; Akaike, Toshihiro

doi:10.1021/bc9701510

Cited by 120 publications

(127 citation statements)

References 30 publications

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“…Aminated dextran was prepared as described by Maruyama et al 15 with minor modifications. Dextran 180 mg was dissolved in dimethyl sulfoxide, and sodium cyanoborohydride (189 mg, 3 mM) was added.…”

Section: Synthesis Of Dexblg Block Copolymermentioning

confidence: 99%

Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer

Jeong

Kim²,

Chung³

et al. 2011

IJN

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Background Polymeric micelles using amphiphilic macromolecules are promising vehicles for antitumor targeting. In this study, we prepared anticancer agent-incorporated polymeric micelles using novel block copolymer. Methods We synthesized a block copolymer composed of dextran and poly (DL-lactide-co-glycolide) (DexbLG) for antitumor drug delivery. Doxorubicin was selected as the anticancer drug, and was incorporated into polymeric micelles by dialysis. Polymeric micelles were observed by transmission electron microscopy to be spherical and smaller than 100 nm, with a narrow size distribution. The particle size of doxorubicin-incorporated polymeric micelles increased with increasing drug content. Higher initial drug feeding also increased the drug content. Results During the drug-release study, an initial burst release of doxorubicin was observed for 10 hours, and doxorubicin was continuously released over 4 days. To investigate the in vitro anticancer effects of the polymeric micelles, doxorubicin-resistant HuCC-T1 cells were treated with a very high concentration of doxorubicin. In an antiproliferation study, the polymeric micelles showed higher cytotoxicity to doxorubicin-resistant HuCC-T1 cells than free doxorubicin, indicating that the polymeric micelles were effectively engulfed by tumor cells, while free doxorubicin hardly penetrated the tumor cell membrane. On confocal laser scanning microscopy, free doxorubicin expressed very weak fluorescence intensity, while the polymeric micelles expressed strong red fluorescence. Furthermore, in flow cytometric analysis, fluorescence intensity of polymeric micelles was almost twice as high than with free doxorubicin. Conclusion DexbLG polymeric micelles incorporating doxorubicin are promising vehicles for antitumor drug targeting.

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Section: Synthesis Of Dexblg Block Copolymermentioning

confidence: 99%

Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer

Jeong

Kim²,

Chung³

et al. 2011

IJN

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“…We have been interested in soluble complex formation observed with cationic graft copolymers containing poly(Llysine) and dextran, PLL-g-Dex ( Figure 1); PLL-g-Dex has a polycation backbone and hydrophilic side chains. 2 The copolymer with a high degree of grafting forms totally soluble DNA complexes without inducing collapse. 3 We have shown that PLL-g-Dex increases stability of DNA hybrids, 2 accelerates DNA hybridization, 4 and activates DNA strand-exchange reactions.…”

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“…2 The copolymer with a high degree of grafting forms totally soluble DNA complexes without inducing collapse. 3 We have shown that PLL-g-Dex increases stability of DNA hybrids, 2 accelerates DNA hybridization, 4 and activates DNA strand-exchange reactions. 5 However, interaction of the copolymer with DNA is not thoroughly understood and the influence of the copolymer grafting degree on conformation of DNA has not been assessed.…”

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confidence: 99%

“…2 The nomenclature used for the copolymers reflects the degree of grafting: Polymers with grafts of 38, 61, and 91 wt % are referred to as 40K38D, 40K61D, and 40K91D, respectively. First, we observed the effect of these copolymers on conformation of bacteriophage -DNA (48.5 kbp) in aqueous buffer solution using a method described by Yoshikawa et al 7 We observed images of cationic polymers in the same mixtures by employing a second fluorescent label.…”

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Cationic Comb-type Copolymers Do Not Cause Collapse but Shrinkage of DNA Molecules

et al. 2011

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A flow-stretching assay was employed to elucidate polycation/DNA interactions at the single-molecule level. Bacteriophage -DNA (48.5 kbp) was attached at one end to a PEGmodified glass surface and stretched by buffer flow. The stretched DNA had an approximate length of 12¯m. Upon injection of polylysine homopolymer, the DNA folded into a globule structure within a few seconds. Injection of a polylysine graft copolymer having hydrophilic dextran side chains also induced collapse of the stretched DNA. In comparison to these polymers, a copolymer with higher graft content did not cause DNA collapse but rather caused 25% shrinkage of the extended DNA. These results were compared with those observed with unstretched DNA.Long DNA molecules can undergo discrete coil to globule transitions. Polycations like polylysine are effective inducers of the transition.1 Upon complex formation with polycations, the DNA molecules collapse into compact globule conformations. The complexes subsequently aggregate and precipitate from the solution. We have been interested in soluble complex formation observed with cationic graft copolymers containing poly(Llysine) and dextran, PLL-g-Dex ( Figure 1); PLL-g-Dex has a polycation backbone and hydrophilic side chains.2 The copolymer with a high degree of grafting forms totally soluble DNA complexes without inducing collapse. 3 We have shown that PLL-g-Dex increases stability of DNA hybrids, 2 accelerates DNA hybridization, 4 and activates DNA strand-exchange reactions.5 However, interaction of the copolymer with DNA is not thoroughly understood and the influence of the copolymer grafting degree on conformation of DNA has not been assessed. Here, we employed a DNA flow-stretching assay to further evaluate DNA/polycation interactions. The DNA flow-stretching assay has high spatiotemporal resolution and has been used to study DNA/protein interactions. 6 The results of the flowstretching assay were compared to that from assays with DNA and polycations in solution.PLL-g-Dex copolymers (M n of PLL: 4.0 © 10 4 , M n of Dex: 8.4 © 10 3 , Dex grafts: 38, 61, and 91 wt %) were prepared as previously reported.2 The nomenclature used for the copolymers reflects the degree of grafting: Polymers with grafts of 38, 61, and 91 wt % are referred to as 40K38D, 40K61D, and 40K91D, respectively. First, we observed the effect of these copolymers on conformation of bacteriophage -DNA (48.5 kbp) in aqueous buffer solution using a method described by Yoshikawa et al. 7 We observed images of cationic polymers in the same mixtures by employing a second fluorescent label. As shown in Figure 2, 11 DNA was visualized with SYBR Gold staining (488 nm excitation) and polymers were imaged (561 excitation) by TRITC labeling. The -DNA alone shows amorphous fluorescence images with 35¯m long axes on average. The DNA images wobbled due to Brownian motion, indicating an extended coil conformation of the DNA. When polylysine homopolymer (40K) was added to the DNA solution, bright DNA clots were observed, indicating that the 40K ...

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“…The carbohydrate lactose molecules conjugated to the polycation poly(L-lysine) can work as solubilizer 10 of the DNA polyion complexes and weaken the electrostatic interaction between the phosphate groups of DNA and the amino groups of poly(L-lysine). The protonated imidazole groups of PLH-NH 2 are considered to have taken the phosphate groups of DNA from the amino groups of PLL-Lac.…”

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pH-Dependent Dissociation of Carbohydrate Ligand Polycations from DNA Ternary Complexes

Asayama¹,

Sekine²,

Kawakami³

et al. 2005

Chemistry Letters

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Here, we have demonstrated that carbohydrate ligand polycations are pH-dependently dissociated from DNA polyion complexes. In response to pH, DNA ternary complexes have dissociated the ligand polycations, resulting in DNA binary complexes. Namely, the ternary complexes of DNA, a poly(L-histidine) modified with aminoethyl groups, PLH-NH 2 , and a poly(L-lysine) conjugated with lactose molecules, PLL-Lac, at pH 7.5 have dissociated the PLL-Lac polycations by the protonation of imidazole groups of PLH-NH 2 at pH 6.0. The pH-dependent dissociation of the ligand polycations from DNA polyion complexes at endosomal pH is expected to offer unique design for DNA delivery systems.In gene delivery systems, the formation of DNA-carrier polyion complexes is a key factor for new design of efficient delivery.1 The carrier design typically focuses on the ability of a ligand to bind tightly to its target. The ligand-receptor complexes on the cell plasma membrane are internalized into acidic endosomal vesicles where they are subjected to a significant pH change from pH 7 to 5.2 Subsequently, the targeting ligands are routed to the degradation in the acidic compartment following endosomal vesicles or are recycled to the cell surface.3 Such fate of ligands leads us to create the DNA polyion complexes dissociating ligand molecules in response to pH. In general, targeting ligands are unnecessary to make DNA complexes escape from endosomal vesicles. The endosomal escape is one of the critical factors for efficient gene delivery. If the targeting ligands are conjugated with pH-sensitive polycations, i.e., formation of binary complexes, the pH-dependent peculiar property of the polycations is considered to diminish. The formation of the ternary complexes between ligand polycations, pH-sensitive polycations, and DNA is expected to preserve the property of the pH-sensitive polycations. Here, we communicate that ligand polycations are pH-dependently dissociated from DNA polyion complexes. The concept that DNA ternary complexes dissociate ligand polycations in response to endosomal pH, resulting in DNA binary complexes, has no precedent, to the best of our knowledge. The experimental schema of the pH-dependent dissociation of carbohydrate ligand polycations from DNA polyion complexes is depicted in Figure 1. We have chosen a poly(L-lysine) conjugated with lactose (4-O--D-galactopyranosyl-D-glucose), PLL-Lac, as carbohydrate ligands for an asialoglycoprotein re-

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Characterization of Interpolyelectrolyte Complexes between Double-Stranded DNA and Polylysine Comb-Type Copolymers Having Hydrophilic Side Chains

Cited by 120 publications

References 30 publications

Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer

Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer

Cationic Comb-type Copolymers Do Not Cause Collapse but Shrinkage of DNA Molecules

pH-Dependent Dissociation of Carbohydrate Ligand Polycations from DNA Ternary Complexes

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