Preparation Of Panda Interferon Gamma Loaded Polybutylcyanoacrylate Nanoparticles

Giant panda as a hightly endangered species is a unique and rare wildlife in our country. With the increasing number of pandas in captivity, they have more chance to get close to people, which made the prevalent background of panda’s diseases became increasingly complicated and the viral diseases rose gradually. Interferon-gamma (IFN-γ) mainly secreted by T cells and NK cells, is a key member of the cytokine family. IFN-［ known as immune interferon is the only member of the type II interferon. IFN-γ not only capable of regulatig the relationship of macrophages, T cells and B cells, enhancing the immune response, but also has the bio-activities of antiviral and antitumor and has been widely used in the treatment of several diseases. However, since its half-life is short, low bioavailability, prone to be enzymolyzed, and multiple-dose is needed. That brings a great burden to clinical treatment. Nanoparticles drug delivery system is a novel drug delivery system that developed in recent decades, it can not only increase the penetrating power, targeting and stability, but also protect the albuminoidal drug from being damaged. As a highly promising nanometer material, polybutylcyanoacrylate has good biocompatibility, biodegradability and with no immunogenicity.In order to optimize the preparation process of IFNy-PBCA-NS, we selected polybutylcyanoacrylate as a carrier material and panda’s recombinant IFN-y as a drug model. The aim of this research is to provide reference for the further study on the preparation of the panda’s albuminoidal drug nanoparticles, and offer new ideas for the diagnosis and treatment of diseases of giant panda. The main contents are as follows:1.In this study, total RNA was extracted from lymphocytes of giant panda that stimulated with ConA, interferon-gamma gene were amplified by RT-PCR with Primer5.0 and the products of PCR were cloned into PMD19-T vector. The sequencing results showed that the IFN-y gene was 465bp longth, compared to other panda IFN-y gene in GenBank, the matching rate was above 96% and no gene mutation. After identification by restriction enzyme and sequencing, IFN-y was cloned into prokaryotic expression plasmids PET-32(a)+ vector and transformed into Ecoli Rosetta(DE3) to be cultured and induced. The results showed that the IPTG-induced recombinant protein was approximately 33.5 kD, and most of them were inclusion body. After refolding, purification by Ni column, the purity of the concentration of recombinant protein was 0.46mg/mL. The result of VSV-WISH showed that the antiviral activity of recombinant IFN-y was 3.2× 105U/mg.2.IFNy-PBCA-NS were prepared using an emulsion polymerization method with polybutylcyanoacrylate as a carrier material and freeze drying powder of recombinant IFN-y as a drug model. The results indicated that the nanopartieles of IFN-y were globular in appearance and with uniform particle size, diameters of IFNy-PBCA-NS were range from 50 to 200nm, the span was 0.55, the encapsulation efficiency of the particles was 56.7% and the drug loading was 0.86%. The best condition of preparation for IFNy-PBCA-NS was established, namely pH6-6.5, BCA 5-10g/L, the usage of poloxamerl88 is 5-10g/L and IFN-y smaller than 0.8g/L. With these conditions IFNy-PBCA-NS were good disperse and no adhesion. The results of pharmocodynamic test showed that the group of sc-IFNy-PBCA-NS worked best, and the group of IFNy-PBCA-NS was better than group of IFN-y. Results of variance analysis indicated that the group of ig-IFN-y is significantly different with the group of ig-IFNy-PBCA (P<0.01), the difference between the group of sc-IFN-y and group of sc-IFNy-PBCA was significant (P<0.05) in RLML. In summary, IFNy-PBCA-NS which were prepared using an emulsion polymerization method showed good sustained release function and antiviral activity in vivo.