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1.
Commun Biol ; 7(1): 556, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38730092

ABSTRACT

Lipid nanoparticles (LNPs) have emerged as promising platforms for efficient in vivo mRNA delivery owing to advancements in ionizable lipids. However, maintaining the thermostability of mRNA/LNP systems remains challenging. While the importance of only a small amount of lipid impurities on mRNA inactivation is clear, a fundamental solution has not yet been proposed. In this study, we investigate an approach to limit the generation of aldehyde impurities that react with mRNA nucleosides through the chemical engineering of lipids. We demonstrated that piperidine-based lipids improve the long-term storage stability of mRNA/LNPs at refrigeration temperature as a liquid formulation. High-performance liquid chromatography analysis and additional lipid synthesis revealed that amine moieties of ionizable lipids play a vital role in limiting reactive aldehyde generation, mRNA-lipid adduct formation, and loss of mRNA function during mRNA/LNP storage. These findings highlight the importance of lipid design and help enhance the shelf-life of mRNA/LNP systems.


Subject(s)
Lipids , Nanoparticles , Piperidines , RNA Stability , RNA, Messenger , Nanoparticles/chemistry , RNA, Messenger/metabolism , RNA, Messenger/genetics , Lipids/chemistry , Piperidines/chemistry , Humans , Temperature , Liposomes
2.
bioRxiv ; 2023 Nov 14.
Article in English | MEDLINE | ID: mdl-38014031

ABSTRACT

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.

3.
J Clin Invest ; 127(5): 1631-1645, 2017 May 01.
Article in English | MEDLINE | ID: mdl-28346230

ABSTRACT

Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel-Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate (αKG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL-/- RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL-/- cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor-induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra-S phase checkpoint, and suppressed the growth of VHL-/- RCC cells. These effects were rescued by administration of glutamate, αKG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL-/- cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.


Subject(s)
Glutaminase/antagonists & inhibitors , Kidney Neoplasms/drug therapy , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Animals , Carcinoma, Renal Cell , Glutamates/genetics , Glutamates/metabolism , Glutaminase/genetics , Glutaminase/metabolism , Glutamine/genetics , Glutamine/metabolism , Humans , Kidney Neoplasms/enzymology , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Mice , Mice, Nude , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Reactive Oxygen Species/metabolism , S Phase Cell Cycle Checkpoints/drug effects , S Phase Cell Cycle Checkpoints/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Xenograft Model Antitumor Assays
4.
PLoS One ; 10(5): e0126849, 2015.
Article in English | MEDLINE | ID: mdl-25978818

ABSTRACT

A lipopolysaccharide from Pantoea agglomerans (LPSpa) has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine) with LPSpa induced both HA-specific IgG (systemic) and IgA (mucosal) antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens.


Subject(s)
Adjuvants, Immunologic/pharmacology , Immunity, Humoral/drug effects , Immunity, Mucosal/drug effects , Influenza Vaccines/immunology , Lipopolysaccharides/pharmacology , Pantoea/immunology , Toll-Like Receptor 4/physiology , Adjuvants, Immunologic/administration & dosage , Administration, Sublingual , Animals , Enzyme-Linked Immunosorbent Assay , Female , Immunity, Humoral/immunology , Immunity, Mucosal/immunology , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/administration & dosage , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
5.
J Biomater Sci Polym Ed ; 21(2): 185-204, 2010.
Article in English | MEDLINE | ID: mdl-20092684

ABSTRACT

The objective of this study is to prepare a non-viral carrier of gene transfection from various polysaccharides and evaluate the feasibility in gene expression for mesenchymal stem cells (MSCs). Various amounts of spermine were chemically introduced into pullulan, dextran and mannan with a molecular weight of around 40 000 or pullulan with different molecular weights to prepare cationized polysaccharides with different extents of spermine introduced (spermine-polysaccharide). Each cationized polysaccharide was complexed with a plasmid DNA at various ratios and in vitro gene transfection was investigated for rat bone marrow-derived MSCs. The level of gene expression depended on the type of cationized polysaccharide. The highest level was observed for the complex of spermine-pullulan and plasmid DNA. Additionally, the level also depended on the molecular weight of pullulan and the extent of spermine introduced to pullulan. Suppression of gene expression with chlorpromazine and methyl-beta-cyclodextrin of endocytosis inhibitors demonstrated that the cellular uptake of spermine-pullulan-plasmid DNA complexes was mediated by clathrin- and raft/caveolae-dependent endocytic pathways. The cationized pullulan is a promising non-viral carrier of plasmid DNA for MSCs.


Subject(s)
Bone Marrow Cells/cytology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Transfection/methods , Animals , Cell Survival/drug effects , DNA/genetics , DNA/metabolism , Endocytosis/drug effects , Glucans/chemistry , Glucans/metabolism , Glucans/toxicity , Male , Mesenchymal Stem Cells/drug effects , Molecular Weight , Plasmids/genetics , Polysaccharides/toxicity , Rats , Rats, Wistar , Spermine/chemistry
6.
J Pharm Sci ; 97(5): 1843-52, 2008 May.
Article in English | MEDLINE | ID: mdl-17828749

ABSTRACT

The purpose of the present study was to construct the theoretical dissolution model of poly-disperse drug particles in biorelevant media containing bile salt/ lecithin aggregates (micelles or vesicles). The effective diffusion coefficient in the biorelevant medium and the particle size distribution of drug particles were simultaneously factored into the Nernst-Brunner equation. The effective diffusion coefficient of a drug in the biorelevant medium was calculated to be smaller than that in the blank buffer, since the diffusion coefficient of a drug bound to the aggregates became similar to that of the aggregates. The particle size distribution of a drug powder was simulated as the sum of mono-disperse fractions covering the particle size range. To verify the modified equation, the dissolution profile of griseofulvin and danazol in a taurocholic acid/egg lecithin (4:1 mixture, taurocholic acid = 0-30 mM) system was investigated. It was clearly demonstrated that both modifications on the Nernst-Brunner equation improved the prediction accuracy. When the effect of the particle size distribution was neglected, the theoretical curve underestimated the observed value at the early phase of dissolution process. When the diffusion coefficient of a free drug was used instead of the effective diffusion coefficient, the theoretical curve overestimated the observed value. The results of the present study suggested that the effect of the particle size distribution and the effective diffusion coefficient should be taken into consideration.


Subject(s)
Pharmaceutical Preparations/chemistry , Solubility , Diffusion , Models, Theoretical , Particle Size
7.
Drug Metab Pharmacokinet ; 22(4): 225-54, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17827779

ABSTRACT

The purposes of the review are to: a) Provide a comprehensible introduction of the-state-of-the-art sciences of solubility and dissolution, b) introduce typical technologies to assess solubility and dissolution, and c) propose the best practice strategy. The theories of solubility and dissolution required in drug discovery were reviewed especially from the view point of oral absorption. The physiological conditions in the gastrointestinal fluid in humans and animals were then briefly summarized. Technologies to assess solubility and dissolution in drug discovery were then introduced. Recently, these technologies have been improved by the laboratory automation and computational technologies. Finally, the strategies to apply these technologies for a drug discovery project were discussed.


Subject(s)
Pharmaceutical Preparations/chemistry , Solubility , Algorithms , Bile Acids and Salts/chemistry , Chemistry, Pharmaceutical , Computer Simulation , Diffusion , Drug Design , Drug Industry , Humans , Hydrogen-Ion Concentration , Kinetics , Particle Size , Thermodynamics
8.
J Biomater Sci Polym Ed ; 18(7): 883-99, 2007.
Article in English | MEDLINE | ID: mdl-17688746

ABSTRACT

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 x 10(3). Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.


Subject(s)
Gene Expression , Genetic Vectors/chemistry , Glucans/chemistry , Plasmids/chemistry , Transfection/methods , Asialoglycoproteins/pharmacology , Cations/chemistry , Cell Line, Tumor , DNA/chemistry , DNA/genetics , Fetuins , Genetic Vectors/drug effects , Genetic Vectors/genetics , Humans , Liver/metabolism , Luciferases/analysis , Luciferases/genetics , Molecular Weight , Plasmids/drug effects , Plasmids/genetics , Spermine/chemistry , alpha-Fetoproteins/pharmacology
9.
Tissue Eng ; 13(2): 245-51, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17518561

ABSTRACT

A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.


Subject(s)
Adult Stem Cells/physiology , DNA/administration & dosage , DNA/pharmacokinetics , Drug Carriers/chemistry , Genetic Engineering/methods , Glucans/chemistry , Spermine/chemistry , Transfection/methods , Adult Stem Cells/cytology , Animals , Biotechnology/methods , Cells, Cultured , Coated Materials, Biocompatible/chemistry , DNA/chemistry , Male , Rats , Rats, Wistar , Tissue Engineering/methods
10.
J Control Release ; 118(3): 389-98, 2007 Apr 23.
Article in English | MEDLINE | ID: mdl-17320235

ABSTRACT

The objective of this study is to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Various amounts of spermine were chemically introduced into pullulan with molecular weights of 22,800, 47,300, and 112,000 to prepare cationized pullulan derivatives with different percentages of spermine introduced. Each cationized pullulan derivative was complexed with a plasmid DNA at various ratios and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the percent spermine introduced of cationized pullulan derivatives and the molecular weight of pullulan. However, when compared at the complexation molar ratio of pullulan derivative to the plasmid DNA, the expression level became maximum around the ratio of 10(2), irrespective of the pullulan molecular weight. Pre-treatment of cells with asialofetuin of asialoglycoprotein receptor ligand decreased the level of gene expression by the complexes. The cationized pullulan derivative with an appropriate physicochemical character is a promising non-viral carrier which promotes the receptor-mediated internalization of plasmid DNA and consequently enhances the expression level.


Subject(s)
DNA/genetics , Gene Expression Profiling/methods , Glucans/genetics , Plasmids/genetics , Spermine , Animals , Cell Survival/drug effects , Cell Survival/physiology , DNA/administration & dosage , DNA/biosynthesis , Glucans/administration & dosage , Glucans/biosynthesis , Luciferases, Firefly/biosynthesis , Luciferases, Firefly/genetics , Plasmids/administration & dosage , Plasmids/biosynthesis , Spermine/administration & dosage , Spermine/biosynthesis
11.
J Control Release ; 116(1): 75-82, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17055606

ABSTRACT

The feasibility and mechanism of gene delivery by pullulan-spermine, a recently developed cationic polysaccharide, were investigated. Pullulan-spermine-mediated transfection of plasmid DNA resulted in greatly reduced cytotoxicity and a 10-fold increase in the level of gene expression when compared to Lipofectamine 2000, a commercially available cationic lipid. Additionally, after transfection of p53-expressing plasmid DNA by pullulan-spermine but not Lipofectamine 2000, the in vitro proliferation of T24 cells was significantly reduced. Pullulan-spermine-mediated gene expression was inhibited by both chlorpromazine of clathrin-mediated endocytosis inhibitor and methyl-beta-cyclodextrin and filipin of raft/caveolae inhibitors. We conclude that pullulan-spermine is a promising carrier for gene transfection, and that cellular uptake of pullulan-spermine-plasmid DNA complexes is mediated by clathrin- and raft/caveolae-dependent endocytotic pathways.


Subject(s)
Caveolae/physiology , Clathrin/physiology , Gene Transfer Techniques , Glucans/chemistry , Spermine/chemistry , Asialoglycoprotein Receptor/genetics , Caveolae/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Clathrin/drug effects , DNA/administration & dosage , DNA/genetics , Drug Carriers , Flow Cytometry , Genes, p53/genetics , Humans , Luciferases/genetics , Microscopy, Confocal , Plasmids/genetics , RNA/biosynthesis , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection
12.
Nucleic Acids Symp Ser (Oxf) ; (48): 79-80, 2004.
Article in English | MEDLINE | ID: mdl-17150487

ABSTRACT

We synthesized a photoreactive hairpin-type oligodeoxynucleotide (P-ODN) possessing an o-nitrobenzyl chromophore and a triplet quencher of 1-aminonaphthalene. Photoirradiation of the hybrid of H-ODN with its complementary DNA led to release of drug in an efficient amount, while the photo-induced drug release was remarkably suppressed in the absence of complementary DNA.


Subject(s)
Drug Delivery Systems/methods , Electrons , Light , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/radiation effects , Base Sequence , Benzoic Acid , DNA, Complementary/radiation effects , Molecular Sequence Data , Nucleic Acid Conformation/radiation effects , Oligodeoxyribonucleotides/genetics
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