Temperature- and pH-sensitive core-shell nanoparticles self-assembled from poly(n-isopropylacrylamide-co-acrylic acid-co-cholesteryl acrylate) for intracellular delivery of anticancer drugs.

Temperature- and pH-sensitive amphiphilic polymer poly(N-isopropylacrylamide-co-acrylic acid-co-cholesteryl acrylate) (P(NIPAAm-co-AA-co-CHA)) has been synthesized and employed to encapsulate paclitaxel, a highly hydrophobic anticancer drug, in core-shell nanoparticles fabricated by a membrane dialysis method. The nanoparticles are spherical in shape, and their size can be made below 200 nm by varying fabrication parameters. The lower critical solution temperature (LCST) of the nanoparticles is pH-dependent. Under the normal physiological condition (pH 7.4), the LCST is well above the normal body temperature (37 degrees C) but it is below 37 degrees C in an acidic environment (e.g. inside the endosome or lysosome). The critical association concentration of the polymer is determined to be 7 mg/L. Paclitaxel can be easily encapsulated into the nanoparticles. Its encapsulation efficiency is affected by fabrication temperature, initial drug loading and polymer concentration. In vitro release of paclitaxel from the nanoparticles is responsive to external pH changes, which is faster in a lower pH environment. Cytotoxicity of paclitaxel-loaded nanoparticles against MDA-MB-435S human breast carcinoma cells is slightly higher than that of free paclitaxel. In addition, doxorubicin is used as a probe to study cellular uptake using a confocal laser scanning microscope (CLSM). Doxorubicin molecules are able to enter the cytoplasm after escaping from the endosome and/or the lysosome. The temperature- and pH-sensitive nanoparticles would make a promising carrier for intracellular delivery of anticancer drugs.

[Fusion expression, purification and bioactivity detection of human defensinalpha (HDalpha)].

AIM: To obtain recombinant human defensin alpha(HDalpha) and detect its biological activity, so as to facilitate further research. METHODS: The HDalpha gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4-HDalpha. The constructed vector which was confirmed to be correct by sequencing was transformed into E.coli DH5alpha and the IL-4-HDalpha fusion protein was expressed under temperature induction. After fusion protein was cleaved to remove IL-4 by hydroxylamine, purification and renaturation was performed. HDalpha's characteristics were identified by SDS-PAGE and bioactivity detection. RESULTS: After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body. After cleaving by hydroxylamine, the purity of obtained HDalpha was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDalpha could obviously inhibit the growth of bacteria. CONCLUSION: The recombinant expression plasmid for HDalpha gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDalpha.

[Cloning and expression of a novel gene restin and preparation of antisera against the recombinant restin].

AIM: To clone a new human gene, restin, from retinoic acid-treated promyelocytic cell line HL-60, express the protein in E.coli and prepare the antisera against the protein. METHODS: The restin gene was amplified from retinoic acid-treated promyelocytic cell line HL-60 by RT-PCR and cloned into a prokaryotic expression vector. The recombinant restin was induced to express in E. coli by temperature. After preliminary purification by SDS-PAGE, the restin protein was used to immunize rabbits to obtain antisera. The titers and specificity of the rabbit anti-restin antisera were tested by Western blot and immunofluorescence analysis. RESULTS: Recombinant restin with M(r) being about 26,000 was highly expressed in E. coli. The titers of the anti-sera to restin ranged from 1:100 to 1:800. Immunofluorescence analysis showed that restin distributed mainly in the nuclei of COS-7 cells. CONCLUSION: We successfully prepared the antisera against restin, which are useful for further investigation of biological functions of restin.