The therapeutic potential of antisense oligonucleotides (ASODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptakein vivo. The present study focuses on designing mannosylated low molecular weight (LMW) chitosan nanoconstructs for safe ODNs delivery by macrophage targeting. Mannose groups were coupled with LMW chitosan and characterized spectroscopically. Mannosylated chitosan ODN nanoparticles (MCHODN NPs) were formulated by self-assembled method using variousN/Pratio (moles of amine groups of MCH to phosphate moieties of ODNs) and characterized for gel retardation assay, physicochemical characteristics, cytotoxicity and transfection efficiency, and antisense assay. Complete complexation of MCH/ODN was achieved at charge ratio of 1:1 and above. On increasing theN/Pratio of MCH/ODN, particle size of the NPs decreased whereas zeta potential (ZV) increased. MCHODN NPs displayed much higher transfection efficiency into Raw 264.7 cells (bears mannose receptors) than Hela cells and no significant toxicity was observed at all MCH concentrations. Antisense assay revealed that reduction in lipopolysaccharide (LPS) induced serum TNF-αis due to antisense activity of TJU-2755 ODN (sequence complementary to 3′-UTR of TNF-α). These results suggest that MCHODN NPs are acceptable choice to improve transfection efficiencyin vitroandin vivo.

2005-02-01·Biochemical Pharmacology2区 · 医学

Inhibition of tumor necrosis factor alpha secretion and prevention of liver injury in ethanol-fed rats by antisense oligonucleotides

2区 · 医学

Article

作者: Yedy Israel ; Yiyang Hu ; Maria Aini ; Qing-na Cao ; Feng Zhou ; Rachel Russ ; Raphael Rubin ; Biddanda C. Ponnappa

Elevated serum tumor necrosis factor alpha (TNF-alpha) levels predict mortality in patients with alcoholic liver disease. Administration of anti-TNF-alpha antibodies, obliteration of Kupffer cells or gut sterilization protect against ethanol-induced hepatocellular injury in animal models. In this study, we evaluated the in vivo efficacy of an antisense phosphorothioate oligodeoxynucleotide (S-ODN) targeted against TNF-alpha mRNA (TJU-2755). Naive rats that were administered TJU-2755 (10 mg/(kg body weight (BW)/day) for 2 days) in the free form were challenged with LPS to induce TNF-alpha secretion. Antisense TJU-2755 treatment reduced serum TNF-alpha levels by 62%. A comparison of the efficacies of mismatched and random S-ODNs with that of TJU-2755 showed that some non-specific inhibition might accompany the sequence-specific effects of TJU-2755. To optimize the targeting of the S-ODN, TJU-2755 was encapsulated in pH-sensitive liposomes for in vivo delivery to macrophages. The efficacy of liposome-encapsulated TJU-2755 was assessed in ethanol-fed animals that were administered LPS to induce liver injury. Liposomal delivery of TJU-2755 allowed a much lower dose (1.9 mg/kg BW/day, for 2 days) of the S-ODN to reduce LPS-induced serum TNF-alpha (by 54%) and liver injury (by 60%) in ethanol-fed rats. These data indicate that liposome-encapsulated S-ODNs targeted against TNF-alpha have therapeutic potential in the treatment of alcoholic liver disease.

2004-04-01·Antioxidants & Redox Signaling2区 · 生物学

Effects of Acute γ-Hexachlorocyclohexane Intoxication in Relation to the Redox Regulation of Nuclear Factor-κB, Cytokine Gene Expression, and Liver Injury in the Rat

2区 · 生物学

Article

作者: Varela, Patricia ; Fernández, Virginia ; Tapia, Gladys ; Videla, Luis A. ; Guerrero, Julia ; Cornejo, Pamela ; Israel, Yedy

gamma-Hexachlorocyclohexane-induced hepatotoxicity is associated with oxidative stress. We tested the hypothesis that gamma-hexachlorocyclohexane triggers the redox activation of nuclear factor-kappaB (NF-kappaB), leading to proinflammatory cytokine expression. Liver NF-kappaB activation (electrophoretic mobility shift assay), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha) mRNA expression (reverse transcription-polymerase chain reaction), and their serum levels (enzyme-linked immunosorbent assay) were measured at different times after gamma-hexachlorocyclohexane treatment (50 mg/kg). The relationship between these and hepatic O(2) uptake, glutathione and protein carbonyl levels, and sinusoidal lactate dehydrogenase (LDH) efflux in liver perfusion studies was determined. gamma-Hexachlorocyclohexane increased liver NF-kappaB DNA binding at 14-22 h after treatment, concomitantly with significant glutathione depletion and an increase in the rate of O(2) consumption, the content of protein carbonyls, and the sinusoidal LDH efflux. In these conditions, the expression of TNF-alpha and IL-1alpha is enhanced, with maximal increases in their respective mRNA content and serum levels of the cytokines being elicited at 18 h after gamma-hexachlorocyclohexane treatment. All these changes are suppressed by the administration of alpha-tocopherol (100 mg/kg) or the Kupffer cell inactivator gadolinium chloride (10 mg/kg) prior to gamma-hexachlorocyclohexane. gamma-Hexachlorocyclohexane-induced TNF-alpha levels in serum are suppressed by pretreatment with an antisense oligonucleotide (ASO TJU-2755; daily doses of 10 mg/kg for 2 days) targeting the primary transcript for the cytokine, whereas those of IL-1alpha are not modified. It is concluded that gamma-hexachlorocyclohexane-induced liver oxidative stress triggers the DNA binding activity of NF-kappaB, with the consequent increase in the expression of NF-kappaB-dependent genes for TNF-alpha and for IL-1alpha, factors that may mediate the hepatotoxicity of the insecticide.

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