Nanoparticles Electrostatically Coated with Folic Acid for Effective Gene Therapy

Kurosaki, Tomoaki; Morishita, Tamami; Kodama, Yukinobu; Sato, Katsushige; Nakagawa, Hiroo; Higuchi, Norihide; Nakamura, Tadahiro; Hamamoto, Tomoyuki; Sasaki, Hidetada; Kitahara, Takashi

doi:10.1021/mp2001268

Cited by 43 publications

(39 citation statements)

References 31 publications

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“…Some researchers reported that anionic charged gene vector modified with anionic ligands such as hyaluronic acids (HA) and folic acid (FA) showed high transgene expression. 18,19) These vectors were taken up by HA or FA receptor-mediated endocytosis. The anionic pDNA/PEI/DOPS complex also may be specifically taken up via a DOPS-related pathway although further studies are necessary to confirm the mechanism.…”

Section: Discussionmentioning

confidence: 99%

Splenic Gene Delivery System Using Self-assembling Nano-complex with Phosphatidylserine Analog

et al. 2015

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The recognition of phosphatidylserine on the erythrocyte membrane mediates erythrophagocytosis by resident spleen macrophages. The application of phosphatidylserine to a gene vector may be a novel approach for splenic drug delivery. Therefore, we chose 1,2-dioleoyl-sn-glycero-3-phospho-L-serin (DOPS) as an analogue of phosphatidylserine for splenic gene delivery of plasmid DNA (pDNA). In the present study, we successfully prepared a stable pDNA ternary complex using DOPS and polyethyleneimine (PEI) and evaluated its efficacy and safety. The pDNA/PEI complex had a positive charge and showed high transgene efficacy, although it caused cytotoxicity and agglutination. The addition of DOPS changed the ζ-potential of the pDNA/PEI complex to negative. It is known that anionic complexes are not taken up well by cells. Surprisingly, however, the pDNA/PEI/DOPS complex showed relatively high transgene efficacy in vitro. Fluorescence microscope observation revealed that the pDNA/PEI/DOPS complex internalized the cells while maintaining the complex formation. The injection of the pDNA/PEI complex killed most mice within 24 h at high doses, although all mice in the pDNA/PEI/DOPS complex group survived. The ternary complex with DOPS showed markedly better safety compared with the pDNA/PEI complex. The pDNA/PEI/DOPS complex showed high gene expression selectively in the spleen after intravenous injection into mice. Thus the ternary complex with DOPS can be used to deliver pDNA to the spleen, in which immune cells are abundant. It appears to have an excellent safety level, although further study to determine the mechanism of action is necessary.Key words gene delivery; spleen; 1,2-dioleoyl-sn-glycero-3-phospo-L-serin; plasmid DNA; ternary complexIn gene delivery, non-viral vectors, including cationic polymers, have several advantages, e.g., they are non-immunogenic; cause few acute toxicities; and their structural and chemical properties can be tightly controlled, which allows vehicles that are suitable for mass production to be designed.1,2) Among non-viral gene vectors, polyethylenimine (PEI) is a popular cationic polymer and show high gene expression in in vitro and in vivo, because of specific mechanisms such as binding to the cell surface, being taken up by the endocytotic pathway, and release of plasmid DNA (pDNA) from endosomes via the so-called "proton sponge mechanism." On the other hand, PEI caused nonspecific gene expressions, high cytotoxicity, and aggregation with blood components because of their strong cationic charge. Recharging cationic complex with anionic compound was reported to be a promising method for overcoming these toxicities.Phosphatidylserine is a biocompatible phospholipid that has a negative charge at biological pH and is a component of the cellular membrane. In normal cells, phosphatidylserines are restricted in the inner leaflet of the cellular membrane by the function of ATP-dependent transporters. [3][4][5] In contrast, in apoptotic cells or aging erythrocytes, phosphatidylserines exposed on the...

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Section: Discussionmentioning

confidence: 99%

Splenic Gene Delivery System Using Self-assembling Nano-complex with Phosphatidylserine Analog

et al. 2015

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“…These transfection efficiencies were as great as the commercially available transfection reagent, lipofectin (1.49×10 8 RLU/mg protein). 23) This high gene expression of pDNA/ protamine6.4 must be explained by interaction of cationic protein with a negative charge cellular membrane. 24,25) On the other hand, the morphology of pDNA condensates may be important for gene expression.…”

Section: Discussionmentioning

confidence: 99%

Ternary Complex of Plasmid DNA with Protamine and γ-Polyglutamic Acid for Biocompatible Gene Delivery System

Kanda

Kodama

Kurosaki

et al. 2013

Biological & Pharmaceutical Bulletin

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The purpose of the present study was to investigate the usefulness of the ternary complex with protamine and γ-polyglutamic acid (γ-PGA), which are biodegradable materials for foods and medical products, as a safe gene delivery vector. We formed cationic binary complexes (plasmid DNA (pDNA)/protamine complexes) with high transfection efficiency. The binary complex showed slight toxicity probably related to its total cationic charge. Then, we formed ternary complexes (pDNA/protamine/γ-PGA complexes) by addition of anionic polymer, γ-PGA, and they showed no cytotoxicity. The transfection efficiency of the pDNA/protamine/γ-PGA complexes was as high as that of the pDNA/protamine complexes, although their zeta potentials were different. Inhibition study of the gene expressions in B16-F10 cells suggested that pDNA/protamine complexes were taken up by caveolae-mediated endocytosis and macropinocytosis. On the other hand, pDNA/protamine/ γ-PGA complexes were taken up by clathrin-mediated endocytosis and macropinocytosis. Thus, we succeeded in developing the ternary complex as a safe gene delivery vector with biocompatible materials.

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“…According to previous studies, amiloride, chlorpromazine, and geneistein can inhibit macropinocytosis and clathrin-mediated and caveolae-mediated endocytosis, respectively. 24 To compare the endocytotic pathway of PPG1 supermolecule/pDNA polyplex and PEI 25K/pDNA polyplex in HEK293A cells, the cells were pretreated with 1.0 mM amiloride, 0.014 mM chlorpromazine, 0.2 mM genistein, or coinhibitors (0.014 mM chlorpromazine and 0.2 mM genistein) for 1 h before transfection. The PPG1 supermolecule/pDNA polyplex at the weight ratio of 5 and PEI 25K/pDNA polyplex at the N/P ratio of 10 containing 1.0 μg of pDNA were added to cells in the presence of the inhibitors according to the transfection experiment mentioned above.…”

Section: Characterization Of Ppg1 Supermoleculs and Ppg1mentioning

confidence: 99%

Dilution-Stable PAMAM G1-Grafted Polyrotaxane Supermolecules Deliver Gene into Cells through a Caveolae-Dependent Pathway

et al. 2014

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Numerous preclinical studies have demonstrated that polycation mediated gene delivery systems successfully achieved efficient gene transfer into cells and animal models. However, results of their clinical trials to date have been disappointing. That self-assembled gene and polycation systems should be stable undergoing dilution in the body is one of the prerequisites to ensuring efficiency of gene transfer in clinical trials, but it was neglected in most preclinical studies. In this account, we developed the dilution-stable PAMAM G1-grafted polyrotaxane (PPG1) supermolecules in which PAMAM G1-grafted α-cyclodextrins are threaded onto a PEG chain capped with hydrophobic adamantanamine. The PPG1/pDNA polyplex (approximate 100 nm in diameter) was very stable and kept its initial particle size and a uniform size distribution at ultrahigh dilution, whereas DNA/PEI 25K polyplex was above three times bigger at a 16-fold dilution than the initial size and their particle size distribution indicated multiple peaks mainly due to forming loose and noncompacted aggregates. PPG1 supermolecules showed significantly superior transfection efficiencies compared to either PEI 25K or Lipofectamine 2000 in most cell lines tested including normal cells (HEK293A) and cancer cells (Bel7402, HepG2, and HeLa). Furthermore, we found that the PPG1 supermolecules delivered DNA into HEK293A through a caveolae-dependent pathway but not a clathrin-dependent pathway as PEI 25K did. These findings raised the intriguing possibility that the caveolae-dependent pathway of PPG1 supermolecule/pDNA polyplex avoiding lysosomal degradation was attributed to their high transfection efficiency. The dilution-stable PPG1 supermolecule polyplex facilitating caveolae-dependent internalization has potential applications to surmount the challenges of high dilutions in the body and lysosomal degradation faced by most gene therapy clinical trials.

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Nanoparticles Electrostatically Coated with Folic Acid for Effective Gene Therapy

Cited by 43 publications

References 31 publications

Splenic Gene Delivery System Using Self-assembling Nano-complex with Phosphatidylserine Analog

Splenic Gene Delivery System Using Self-assembling Nano-complex with Phosphatidylserine Analog

Ternary Complex of Plasmid DNA with Protamine and γ-Polyglutamic Acid for Biocompatible Gene Delivery System

Dilution-Stable PAMAM G1-Grafted Polyrotaxane Supermolecules Deliver Gene into Cells through a Caveolae-Dependent Pathway

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