Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. METHODS: The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N, N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and 1HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. RESULTS: MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%+/-4.03%, which were comparable to Biotrans (8.94%+/-3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. CONCLUSION: MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.

Preparation and preliminary characterization of rabbit monoclonal antibodies against human midkine.

We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0 × 10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.

[Expression characteristics of nuclear factor kappa B in hepatocellular carcinoma tissues].

OBJECTIVE: To investigate the expression characteristics of nuclear factor kappa B (NF-kB) in hepatocellular carcinoma (HCC) tissues and its correlation with tumor necrosis factor alpha (TNF alpha) and clinical pathological features. METHODS: Thirty liver specimens from HCC patients were collected by self-control method. The localization and expression of NF-kappaB in HCC and their surrounding tissues were detected by immunohistochemistry and enzyme linked immunosorbent assay (ELISA), respectively. And the levels of TNF alpha in these tissues were analyzed by ELISA. RESULTS: The expressed NF-kappaB was localized in nucleus and cytoplasm in HCC, whereas only in cytoplasm in the surrounding tissues. The expression level and density of NF-kappaB in HCC tissues were obviously higher than those in the surrounding tissues (P < 0.01), which was positively correlated with increased TNF alpha in HCC tissues (r = 0.964, P < 0.01). No positive correlation was found between NF-kappaB expression and histological differentiation grade, number of tumor, size of tumor, and HBsAg positive (P > 0.05). CONCLUSION: The expression and localization of NF-kappaB in HCC tissues are obviously different from those in the surrounding normal liver tissues, and the level of nucleoprotein NF-kappaB in HCC tissues is correlated with expressed TNF alpha, suggesting that TNF alpha can activate NF-kB, the activated NF-kB then translocates to the nucleus and plays important role in the carcinogenesis of HCC.

Antisense oligonucleotide targeting p53 increased apoptosis of MCF-7 cells induced by ionizing radiation.

AIM: To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells. METHODS: Western blotting and RTPCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells. RESULTS: Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, AS3 transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines. CONCLUSION: AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.