Anionic Complex with Efficient Expression and Good Safety Profile for mRNA Delivery

We previously found that a complex comprising plasmid DNA (pDNA), polyethylenimine (PEI), and γ-polyglutamic acid (γ-PGA) had high transgene efficiency without cytotoxicity in vitro and in vivo. However, messenger RNA (mRNA) remains an attractive alternative to pDNA. In this study, we developed a safe and effective delivery system for mRNA to prevent its degradation and efficiently deliver it into target cells. Various cationic and anionic complexes were produced containing PEI, γ-PGA, and an mRNA encoding firefly luciferase. Their physicochemical properties and cytotoxicities were analyzed and the in vitro and in vivo protein expression were determined. The cationic mRNA/PEI complex showed high in vitro protein expression with strong cytotoxicity. The anionic complex was constructed as mRNA/PEI8/γ-PGA12 complex with a theoretical charge ratio of 1:8:12 based on the phosphate groups of the mRNA, nitrogen groups of PEI, and carboxylate groups of γ-PGA. It was stable and showed high in vitro protein expression without cytotoxicity. After intravenous administration of mRNA/PEI8/γ-PGA12 complex to mice, high protein expression was observed in the spleen and liver and slight expression was observed in the lung over 24 h. Thus, the newly constructed mRNA/PEI8/γ-PGA12 complex provides a safe and effective strategy for the delivery of mRNA

Delivery of pDNA to the Lung by Lipopolyplexes Using N-Lauroylsarcosine and Effect on the Pulmonary Fibrosis

In a previous study, we constructed a lung-targeting lipopolyplex containing polyethyleneimine (PEI), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and N-lauroylsarcosine (LS). The lipopolyplex exhibited an extremely high gene expression in the lung after intravenous administration. Here, we optimized the lipopolyplex and used it to deliver a TGF-β1 shRNA to treat refractory pulmonary fibrosis. We constructed several lipopolyplexes with pDNA, various cationic polymers, cationic lipids, and LS to select the most effective formulation. Then, the pDNA encoding shRNA against mouse TGF-β1 was encapsulated in the lipopolyplex and injected into mice with bleomycin-induced pulmonary fibrosis. After optimizing the lipopolyplex, dendrigraft poly-L-lysine (DGL) and DOTMA were selected as the appropriate cationic polymer and lipid, respectively. The lipopolyplex was constructed with a pDNA, DGL, DOTMA, and LS charge ratio of 1:2:2:4 showed the highest gene expression. After intravenous administration of the lipopolyplex, the highest gene expression was observed in the lung. In the in vitro experiment, the lipopolyplex delivered pDNA into the cells via endocytosis. As a result, the lipopolyplex containing pDNA encoding TGF-β1 shRNA significantly decreased hydroxyproline in the pulmonary fibrosis model mice. We have successfully inhibited pulmonary fibrosis using a novel lung-targeting lipopolyplex

Effects of changing the timing of warfarin administration in combination with fluconazole on prolongation of the PT-INR: a case report

Abstract Background Fluconazole (FLCZ) inhibits cytochrome P450 (CYP) 2C9, 2C19, and 3A4 and has a drug-drug interaction that potentiates the effects of warfarin and prolong the prothrombin time-international normalized ratio (PT-INR). Although a drug-drug interaction have been reported between FLCZ and warfarin, the effects of the timing of their administration on this interaction have not yet been investigated. Case presentation A female patient in her 30s with Marfan syndrome had undergone the Bentall procedure with a mechanical valve and total arch replacement for acute aortic dissection Stanford A type and rupture of the ascending aorta. Warfarin was administered to prevent thromboembolism. She was hospitalized 1 year ago for graft infection caused by Candida albicans, and treatment with FLCZ was initiated. She received FLCZ 200 mg once a day in the morning and warfarin 1.75 mg once a day in the evening, and the PT-INR remained stable at approximately 2.0 and within the therapeutic range. However, 42 days after changing the timing of administration of warfarin from evening to morning, the PT-INR was prolonged by approximately 3-fold to 6.25. The PT-INR then decreased to the previous level by changing the timing of administration of warfarin from morning to evening. Conclusions The timing of administration of FLCZ and warfarin may affect the magnitude of drug-drug interaction

Methotrexate-Coated Complexes of Plasmid DNA and Polyethylenimine for Gene Delivery

Folate receptors are overexpressed on the surface cancer cells. We successfully constructed a new gene delivery vector of methotrexate (MTX)-coated plasmid DNA-polyethylenimine (pDNA-PEI) complexes (PEI complexes) by electrostatic binding. The stable anionic nanoparticle was optimized at MTX charge ratios of 120 or more. pDNA-PEI-MTX complexes (MTX complexes) demonstrated gene expression efficiency as high as cationic pDNA-PEI complexes in the mouse melanoma cell line, B16-F10. The MTX complexes were taken up by the cell-specific uptake mechanisms via the folate receptor. MTX-coated complexes are useful as endocytosis ligands. The MTX120 complexes exhibited no blood aggregation. The transgene efficiency of MTX120 complexes in the liver and spleen after their intravenous administration was higher than that of PEI complexes. Therefore, MTX complexes are expected as a new gene vector in the future

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

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