Yifeng Pan

Reversion of multidrug resistance by co-encapsulation of doxorubicin and curcumin in chitosan/poly(butyl cyanoacrylate) nanoparticles

Co-encapsulated doxorubicin (DOX) and curcumin (CUR) in poly(butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared with emulsion polymerization and interfacial polymerization. The mean particle size and mean zeta potential of CUR-DOX-PBCA-NPs were 133 ± 5.34 nm in diameter and +32.23 ± 4.56 mV, respectively. The entrapment efficiencies of doxorubicin and curcumin were 49.98 ± 3.32% and 94.52 ± 3.14%, respectively. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs+CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX+CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of doxorubicin and curcumin achieved the highest reversal efficacy and down-regulation of P-glycoprotein in MCF-7/ADR cell lines, an MCF-7 breast carcer cell line resistant to adriamycin. Multidrug resistance can be enhanced by combination delivery of encapsulated cytotoxic drugs and reversal agents.

A novel PEGylation of chitosan nanoparticles for gene delivery

CS (chitosan) has emerged as a promising non‐viral vector for gene delivery because of its ability to form complexes with pDNA (plasmid DNA) and enhance its transport across cellular membranes through endocytosis. Complexes of CS and pDNA may improve transfection efficiency; however, they are not capable of sustained DNA release and prolonging gene transfer. In order to achieve prolonged delivery of CS–DNA complexes, we prepared CS NP (nanoparticle) and CS–DNA complexes. α‐Methoxy‐ω‐succinimidylpoly(ethylene glycol) was then conjugated to the surface of CS–DNA complexes using an active ester scheme; finally, the potential of PEGylation [poly(ethylene glycol)ylation] of CS NP as a non‐viral gene‐delivery vector to transfer exogenous genes in vitro and in vivo were examined. Electrophoretic analysis suggested that CS NPs could protect the DNA from nuclease degradation. The pDNA carried by CS NPs could enter and be expressed in HepG2 cells. However, the transfection efficiency was very low and the highest dose of DNA transferred was 1.6 μg. The transfection activities of CS–DNA–PEG were preserved and a higher dose (2.4 μg) of pDNA was transferred. This indicated that the transfection efficiency of the PEGylated complexes had been improved. In vivo experiments also showed that CS–DNA–PEG complexes mediated higher gene expression in tissues than did CS–DNA complexes, and that gene expression in tumours induced by CS–DNA–PEG complexes was the highest of all. These results suggested that PEGylation of CS–DNA complexes improves non‐viral gene delivery in vitro or in vivo and has the potential to deliver therapeutic genes directly into hepatoma tissues.

In vitro and in vivo antitumoral action of metformin on hepatocellular carcinoma

Aims:  Metformin is a biguanide that has been widely used to treat type 2 diabetes. Several studies have shown that metformin is also effective in treating cancer, including hepatocellular carcinoma (HCC). The objective of this study was to evaluate the antitumor effects of metformin in HCC, and to investigate the potential molecular target(s) of metformin‐mediated antitumor activity.

Meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were internalized by SW480 cells by a clathrin-mediated endocytosis pathway to induce high photocytotoxicity.

We studied the uptake of meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles by SW480 cells and carried out a systematic investigation of the cellular internalization mechanism of TCPP nanoparticles, also studied the photocytotoxicity of TCPP nanoparticles. At first, meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were prepared by the method of mixing solvent techniques. SW480 cellular uptakes of photosensitizers (TCPP nanoparticles, TCPP-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles and free TCPP) were analyzed by the method of fluorospectrophotometry. Endocytosis mechanism investigation was carried out by preincubating SW480 cells at 4 degrees C, and preincubating SW480 cells with sucrose, K+-free buffer solution and filipin. Clathrin HC expression after incubating SW480 cells with these three photosensitizers was analyzed by methods of Western blot and RT-PCR. At last, we analyzed the photo-cytotoxicity after incubating SW480 cells with photosensitizers and receiving irradiation. SW480 cells showed rapid uptake (0.0083fmoles TCPP/cell) of TCPP nanoparticles after 1h incubation. We also demonstrated that the uptake of TCPP nanoparticles by SW480 cells was a clathrin-mediated endocytosis pathway. As a result of rapid internalization of TCPP nanoparticles by SW480 cells, this special photosensitizer showed very high photocytotoxic effect on SW480 cells in vitro. The nano-sized photosensitizer with no matrix cover: TCPP nanoparticles, can produce higher photocytotoxicity than other photosensitizers (TCPP-loaded PLGA nanoparticles and free TCPP). The in vivo tumor growth inhibition experiment indicated that TCPP nanoparticles plus PDT treatment induced the most dramatic tumor-inhibiting efficacy in all TCPP treated groups. The results of this study suggest that TCPP nanoparticles represent a potential and powerful photodynamic therapy agent.

Panning and Identification of a Colon Tumor Binding Peptide from a Phage Display Peptide Library

Tumor-targeting therapy can be an efficacious way to cure a malignant tumor in clinical trials. Phage display is a molecular diversity technology that allows the presentation of a large number of peptides or proteins on the surface of filamentous phage for various applications. In this study, we report on using phage display to generate peptide libraries that bind to colon cancer tissues. To accomplish this, we developed a screening protocol that contained 3 rounds of in vitro positive panning on colon cancer cells (SW480) and 2 rounds of subtractive screening in vitro on normal human intestinal epithelial cells with a phage display-7 peptide library. After several rounds of panning, both phage titer and recovery efficiency were significantly improved. Through a cell-based enzyme-linked immunosorbent assay, immunofluorescence, in vivo binding assay, immunocytochemical staining, and immunohistochemical staining, peptide CP15 (VHLGYAT) was demonstrated to be the most effective peptide in targeting tumor cells (SW480 and HT29 cells) and tumor tissues but not the normal human intestinal epithelial cells and control colon tissue. These studies suggest that peptide CP15 may be a promising lead candidate in the development of a useful colon tumor diagnostic and targeted drug delivery agent. (Journal of Biomolecular Screening 2007:429-435)

Cationic Polybutyl Cyanoacrylate Nanoparticles for DNA Delivery

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA) and chitosan to prepare PBCA nanoparticles (NPs) by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2) cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.

Prognostic Value of Matrix Metalloproteinase-1/ Proteinase-Activated Receptor-1 Signaling Axis in Hepatocellular Carcinoma

Matrix metalloproteinase-1 (MMP-1) is proposed to be involved in both tumor cell invasion and metastasis. MMP-1 proteolytically activates protease activated receptor-1 (PAR-1), which also plays an important role in tumor development and progression. However, it is currently unknown whether MMP-1 activation of PAR-1 has relevance to the progression of hepatocellular carcinoma (HCC). To address this problem, we investigate the clinicopathological and prognostic value of MMP-1/PAR-1 signaling axis in HCC. Immunohistochemistry assay was used to determine the expression of MMP-1 and PAR-1 proteins in normal and HCC tissues. The correlations of MMP-1 and PAR-1 expression with clinicopathological parameters were assessed by Chi-squared test. Patient survival and their differences were determined by Kaplan-Meier method and log-rank test. Cox regression was adopted for multivariate analysis of prognostic factors. MMP-1 and PAR-1 immunoreactivities were negative or low in normal liver tissues, but high in HCC tissues. PAR-1 expression was significantly correlated with that of MMP-1 (r = 0.896, p < 0.0001). The overexpression of MMP-1 and PAR-1 was significantly associated with recurrence, TNM staging and portal vein invasion of HCC. Patients with high MMP-1 and PAR-1 expression had significantly poorer overall survival (OS) (both P < 0.001) and disease-free survival (DFS) (both P < 0.001) when compared with patients with the low expression of MMP-1 and PAR-1. On multivariate analysis, MMP-1 and PAR-1 expression patterns were found to be independent prognostic factors for OS (both P < 0.001) and DFS (both P < 0.001). Our results suggest for the first time that the MMP-1/PAR-1 signaling axis might be applied as a novel marker for the prediction of recurrence and metastasis potency and a significant indicator of poor prognosis for patients with HCC.

Galangin (GG) combined with cisplatin (DDP) to suppress human lung cancer by inhibition of STAT3-regulated NF-κB and Bcl-2/Bax signaling pathways

Lung cancer represents a significant problem for public health worldwide. Galangin (GG), a natural active compound 3, 5, 7-trihydroxyflavone, is a type of bioflavonoid, which is isolated from the Alpinia galangal root and suggested to induce apoptosis in various cancers. We investigated the ability of Galangin (GG) to attenuate the drug resistance of human lung cancer cells, resistant to treatment of cisplatin (DDP). DDP is a pyrimidine analog, widely used in cancer treatment. Galangin and DDP co-treatment resulted in a dose-dependent suppression of the cell proliferation. Decreasing of p-STAT3 was included in p65 suppression by GG with DDP in combination. Additionally, the presence of GG potentiated the effects of DDP on apoptosis induction through suppressing Bcl-2 in DDP-resistant lung cancer cells. The pro-apoptotic proteins of Bax and Bid were up-regulated, accompanied with Caspases cleavage, leading to apoptosis. Moreover, in mice xenograft models, the combined therapy inhibited tumor growth compared to the GG or DDP treatment alone. Our data indicated a novel therapeutic strategy to potentiate DDP-induced anti-tumor effect in lung cancer cells with DDP resistance by GG through inactivating p-STAT3/p65 and Bcl-2 pathways.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU.

The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2(1)2(1)2(1) and tetragonal P4(1)2(1)2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 A resolution and the VP8*(65-224) structure was determined by molecular replacement.