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The Anti-Viral Activity And Toxicity Of Poly I:C, Preparation And Evaluation Of Poly I:C Microcapsules

Posted on:2010-05-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y J LvFull Text:PDF
GTID:1103360305986988Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
As a copolymer of polyinosinic acid and polycytidylic acid, Poly I:C (polyinosinic:polycytidylic acid) is an efficient inducer of interferon and plays a role on the broad-spectrum antiviral, provoking swallow, adjusting immunity, and activity of antineoplastic, antibacterium and antitozoon. In human clinical medicine, Poly I:C exhibit obvious effect especially in treating hepatitis, herpesvirus infection and some neoplastic disease. Only a few Poly I:C reports have been reported in treating swine plague and duck plague in veterinary clinical medicine. In this study the anti-viral activities and toxicity were investigated in vitro and in vivo, and in order to slove the expensive cost, easily destroyed in serum and stress reaction of injection of Poly I:C in veterinary clinic, Poly I:C microcapsules which have sustained-release ability were prepared and evaluted in vitro and in vivo.Anti-virus activity induced by Polyinosinic:polycytidylic acid copolymer in in vitro chick embryo fibroblast cells The effect of a polyinosinic:polycytidylic acid copolymer (Poly I:C) on chick embryo fibroblast (CEF) cell viability, anti-Newcastle disease virus (NDV) activity and on IFN-αand IFN-βlevels were determined in vitro. Poly I:C inhibited NDV growth in CEF cells in the range of 30-500μg/mL and over 0.75-12 h of treatment. The anti-NDV activity was maximal for 60μg/mL of Poly I:C treatment at 12 h, after which it declined. The result of VSV protection assay which was used to determined IFN-activity showed that Poly I:C inhibited VSV growing on CEF cells, indicating that Poly I:C had immune-stimulating activities. The results of the amount of IFN-βin cell supernatant and the transcription levels of IFN-αmRNA and IFN-βmRNA increased after Poly I:C treatment, indicating that the anti-viral activity was relatived to the production of typeⅠIFN. However Poly I:C also showed a toxic effect by inhibitaing CEF cell proliferation in a dose-dependent and time-dependent manner. The results of this study indicate that the dosage level of Poly I:C must be carefully considered when used in a clinical setting.Antiviral activities of Poly I:C on the chick embryos and the broilers infected by NDV To investigate the anti-NDV activity of Poly I:C in vivo, SPF embryos and broilers infected by NDV were used to study the effect of Poly I:C in this paper. The result of antiviral effects of Poly I:C on SPF embryos showed that after the dosages of 5 mg·mL-1 Poly I:C (0.1 mL per egg) NDV proliferation was significantly inhibited, the weight of chick embryos increased and the NDV hemagglutination and the mortality rate of chick embryos reduced compared to positive control. The result of antiviral effects of Poly I:C on broilers exhibited that the mortality of 0.02 mg/kg-bw,0.01 mg/kg-bw and 0.005 mg/kg-bw dose groups is 40%,20% and 26.7%, respectively, which are all lower significantly (P<0.01) than that of positive control group (63.3%).Apoptosis induced by a polyinosinic:polycytidylic acid copolymer on chicken embryo fibroblast cells in vitro In this study apoptosis induced by the toxic effects of the Polyinosinic:polycytidylic acid copolymer (Poly I:C) was investigated on CEF cells. Along with cell viability and morphological changes, other indicators including phosphatidylserine translocation, the formation of DNA fragments, activity of caspase-3, caspase-8 and caspase-9, and the expression levels of RIPK1 mRNA and TNRSF8 mRNA, were measured in vitro, after incubation of the cells with Poly I:C. An inhibition of proliferation was found with increased levels of Poly I:C, showing that the Poly I:C was toxic to the cells. The finding of phosphatidylserine translocation and formation of DNA fragmentation suggested that Poly I:C induced apoptosis. With increased incubation time, the activities of caspase-3 and caspase-8 increased, while there was no significant change in caspase-9 activity. Accordingly it is concluded that the apoptosis induced by Poly I:C involves a cell death receptor-mediated pathway. The transcription level of RIPK1 mRNA decreased, while that of TNFRSF8 mRNA increased, indicating that Poly I:C-induced apoptosis was related to an upregulation of TNFRSF8. These observations provide insight into the potential mechanism of Poly I:C-induced toxicity.Preparation of polyinosinic:polycytidylic acid copolymer microcapsules and evaluation in in vitro The purpose of this research is to prepare polyinosinic: polycytidylic acid (poly I:C) microcapsules by intra-liquid desiccation with ethylcellulose (EC) as the polymer coating material. An orthogonal test was used to develop an optimal combination. The optimal combination of encapsulating factors for poly I:C microcapsules was found to be as follows:a rotation speed of 700 rpm,0.4 g magnesium stearate,2% Span-80 in acetone,2.5% EC in acetone, and an acetone/paraffin ratio of 30:30 (v/v). The pharmaceutical characteristics of size, morphological characterization, drug loading efficiency, encapsulation yield, and in vitro release rate were evaluated. The in vitro release rate was directly proportional to the amount of PEG-2000, i.e., with an increase in the amount of PEG-2000, poly I:C release from the microcapsules was more rapidInductive effects of IFN-a and immune activities by poly I:C microcapsule In Newcastle Disease Virus infected broilers To evaluate anti-virus effect of Poly I:C microcapsule, IFN-a level and the specific and non-specific immune indicators were detected in Newcastle disease virus (NDV) infected broilers after oral administration of Poly I:C microcapsule. The results showed that the therapeutic efficacy of Poly I:C Microcapsule is similar to that of Poly I:C injection. It reveal that nucleinase in digestive tract do not show obvious effect on the degradation of Poly I:C microcapsule during absorption of Poly I:C. IFN-a concentration increased with dosage of Poly I:C microcapsule in a limited range, while IFN-a level decreased overen the dosage of 0.2 mg/kg-bw. Poly I:C microcapsule administration display obvious effect on improvement of cell immunity but no exhibition on adjusting humoral immunity.
Keywords/Search Tags:Poly I:C, anti-virus activity, apoptosis, toxicity, microcapsule
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