[Sorafenib regulates vascular endothelial growth factor by runt-related transcription factor-3 to inhibit angiogenesis in hepatocellular carcinoma].

Objective: To investigate the molecular mechanism of sorafenib against hepatocellular carcinoma. Methods: Sorafenib efficacy was screened and verified by the hepatocellular carcinoma patient-derived tumor xenograft (PDX) model. Veterinary B-mode ultrasonography and in vivo confocal laser scanning microscopy were used to observe PDX angiogenesis. Immunohistochemistry was used to observe the expression of proliferation and angiogenesis-related proteins in PDX tissue. Real-time quantitative PCR technology was used to observe the RUNX3 gene in PDX tissues. SPSS 17.0 statistical software was used for statistical analysis. Results: Four cases of PDX were used to screen the efficacy of sorafenib. PDX1 had a significant response to sorafenib, with an inhibition rate of 68.07%. Compared with the control group, sorafenib had significantly inhibited PDX1 relative tumor volume (5.76±2.14 vs. 11.71±2.87, P<0.05). Cell division index (39.50±7.72 vs. 67.10±9.14, P<0.05) and Ki67 expression (288.6±43.40 vs. 531.70±55.60, P<0.05) were significantly decreased. Veterinary B-mode ultrasonography showed evident blood flow signals in PDX1 tumors. In vivo confocal laser scanning microscopy results showed that sorafenib had significantly reduced the total vessel length (1573.00±236.21 vs. 2675.03±162.00, P<0.05) and area (11 145.33±1931.97 vs. 20 105.37±885.93, P<0.05)) of PDX1 tumors. Immunohistochemical results showed that sorafenib had significantly down-regulated the protein expressions of CD34 (27.55±3.76 vs. 45.47±5.57, P<0.05), VEGF (16.33±2.86 vs. 22.77±3.20, P<0.05) and MVD (38.75±6.01 vs. 55.50±8.61, P<0.05). Real-time PCR results showed that sorafenib had significantly up-regulated RUNX3 gene expression (2.14±0.71 vs. 1.00±0.36, P<0.05). However, there was a negative correlation between the expression of RUNX3 gene and the ratio of VEGF-positive cells in sorafenib group (R2=0.509 7). Conclusion: Sorafenib may inhibit the PDX angiogenesis and the growth of hepatocellular carcinoma by regulating the RUNX3-VEGF pathway.

[Construction of apoptosis-stimulating of p53 protein 2 gene knockout mice and its effect on diethylnitrosamine-induced liver cancer].

Objective: To construct apoptosis-stimulating of p53 protein 2 (ASPP2) gene knockout mice using diethylnitrosamine (DEN)-induced liver cancer model to study the biological functions of ASPP2. Methods: The sgRNA oligonucleotides were constructed, and ASPP2 knockout mice were prepared with the CRISPR/Cas9 system. PCR and sequencing methods were used to identify the genotypes of F0 and F1 generations and their progeny. DEN was used to induce ASPP2+/- mice to establish liver cancer model. Results: PCR and sequencing results showed that ASPP2 gene was successfully knocked out in F0 generation mice. The genotype of F1 generation mice was accorded with ASPP2+/- and had obtained stable heredity. The success rate of DEN-induced liver cancer model (7/8 and 3 / 8) of ASPP2 + /-mice obtained by self-hybridization of F1 generation was significantly higher than that of wild-type mice. Conclusion: ASPP2 knockout mice were successfully constructed based on the CRISPR/Cas9 system. The success rate of DEN-induced liver cancer model of ASPP2 knockout mice was significantly higher than that of the wild-type mice.

Supramolecular host-guest pseudocomb conjugates composed of multiple star polycations tied tunably with a linear polycation backbone for gene transfection.

A series of novel supramolecular pseudocomb polycations (l-PGEA-Ad/CD-PGEAs) were synthesized by tying multiple low-molecular-weight ß-cyclodextrin (CD)-cored, ethanolamine-functionalized poly(glycidyl methacrylate) (PGEA) star polymers (CD-PGEAs) with an adamantine-modified linear PGEA (l-PGEA-Ad) backbone via the host-guest interaction. The pseudocomb carriers were studied in terms of their DNA binding capabilities, cytotoxicities, and gene transfection efficiencies in the HepG2 and HEK293 cell lines. The pseudocomb l-PGEA-Ad/CD-PGEAs exhibited better plasmid DNA-condensing abilities than their counterparts, CD-PGEA and l-PGEA. Meanwhile, the pseudocomb carriers displayed low cytotoxicity, similar to CD-PGEA and l-PGEA. Moreover, the gene transfection efficiencies of the pseudocomb carriers were much higher than those of CD-PGEA and l-PGEA at various PGEA nitrogen/DNA phosphate molar ratios. Such supramolecular preparation of pseudocomb gene carriers could provide a flexible approach for adjusting the structure and functionality of supramolecular polymers via the proper use of non-covalent interactions.

Aminated poly(glycidyl methacrylate)s for constructing efficient gene carriers.

Aminated poly(glycidyl methacrylate) (PGMA) vectors could efficiently mediate gene delivery. Recently, we reported that ethanolamine (EA)-functionalized PGMA could provide high transfection efficiency, while exhibiting very low toxicity. Herein, different amine species, including 1-amino-2-propanol (AP1), 3-amino-2-propanol (AP2), EA, and N,N,-dimethylethylenediamine (DED), and its quaternized DED, were proposed to aminate PGMA. The DNA condensation abilities, pH buffering capacities, cytotoxicities, and gene transfection efficiencies of the resultant aminated PGMA vectors were systematically compared. Compared with EA, AP1 (or AP2) contains an additional methyl (or methylene) group. EA-, AP1-, and AP2-functionalized PGMA vectors exhibited similar condensation abilities. The methyl (from AP1) and methylene (from AP2) species could benefit the gene delivery. The transfection performance mediated by AP1-functionalized PGMA is best. DED possesses a tertiary amine group, which could be quaternized to further enhance the DNA condensation ability of aminated PGMA. No obvious increase in cytotoxicity of quaternized DED-aminated PGMA was observed. But both DED- and its quaternized counterpart-functionalized PGMA vectors exhibited very low pH buffering capacities, making them exhibit poor gene transfection performances. The current study would provide useful information for constructing better PGMA-based delivery systems with good biophysical properties.

Facilitation of gene transfection with well-defined degradable comb-shaped poly(glycidyl methacrylate) derivative vectors.

Recently, we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) vectors (PGEAs) can produce good transfection efficiency, while exhibiting very low toxicity. Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers. Herein, the degradable high-molecular-weight comb-shaped PGEA vectors (c-PGEAs) composed of the low-molecular-weight PGEA backbone and side chains were prepared by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions. The PGEA side chains were linked with the PGEA backbones via hydrolyzable ester bonds. Such comb-shaped c-PGEA vectors possessed the degradability, good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the comb-shaped c-PGEA vectors could enhance the gene expression levels. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with some poly(poly(ethylene glycol)ethyl ether methacrylate) species to further improve the gene delivery system.

Functionalized cotton via surface-initiated atom transfer radical polymerization for enhanced sorption of Cu(II) and Pb(II).

The surface-initiated atom transfer radical polymerization (ATRP) was used to successfully prepare the aminated cotton and polyacrylic acid sodium (P(AA-Na))-grafted cotton for the efficient removal of Cu(II) and Pb(II) from aqueous solution in this study. The modified cotton surfaces were characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). The grafted long polymers with high density of amine and carboxyl groups on the cotton surfaces were responsible for the enhanced adsorption of heavy metals. The sorption behaviors including sorption kinetics, isotherms and pH effect were investigated. The sorption equilibrium of Cu(II) and Pb(II) was achieved within 1h on the P(AA-Na)-grafted cotton, much faster than 8h on the aminated cotton. According to the Langmuir fitting, the maximum sorption capacities of Cu(II) and Pb(II) on the P(AA-Na)-grafted cotton were 2.45 and 2.44 mmol/g, respectively, higher than many adsorbents reported in the literature. The P(AA-Na)-grafted cotton had better adsorption behaviors for Cu(II) and Pb(II) than the aminated cotton.

Comparison of ethanolamine/ethylenediamine-functionalized poly(glycidyl methacrylate) for efficient gene delivery.

Cationic polymers with low cytotoxicity and high transfection efficiency have attracted considerable attention as non-viral carriers for gene delivery. Recently we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) (termed PGEA) vectors can have excellent transfection efficiency, while exhibiting very low toxicity. Herein different EA- and ethylenediamine (ED)-functionalized PGMA (termed PGEAED) vectors, as well as ED-functionalized PGMA (termed PGED) vectors, are proposed and compared for efficient gene delivery. In addition to the cationic pendant secondary amine and hydroxyl groups of PGEA, PGEAED, and PGED also contain flanking primary amine groups. PGEAED and PGED exhibited a substantially enhanced ability to condense pDNA into complex nanoparticles at the 100 nm level with positive zeta potentials of about 30 mV at nitrogen/phosphate (N/P) ratios of 10 or higher. More interestingly, no obvious change in the cytotoxicity of PGEAED was observed with a substantial increase in ED content. Moreover, the flanking primary amine groups induced by ED could be readily functionalized by glycyrrhetinic acid or cholic acid to improve the biophysical properties of the gene vectors.

Well-defined poly(2-hydroxyl-3-(2-hydroxyethylamino)propyl methacrylate) vectors with low toxicity and high gene transfection efficiency.

Successful gene delivery vectors for clinical translation should have high transfection efficiency and minimal toxicity. In this work, well-defined poly(2-hydroxyl-3-(2-hydroxyethylamino)propyl methacrylate) (PGEA) vectors with flanking cationic secondary amine and nonionic hydroxyl units were prepared via the ring-opening reaction of the pendant epoxide groups of poly(glycidyl methacrylate) with the amine moieties of ethanolamine. It was found that PGEA carriers possess very low toxicity (<10% of the toxicity of branched polyethylenimine (PEI, 25 kDa), while exhibiting surprisingly excellent transfection efficiency (higher than or comparable to that of PEI (25 kDa)) in different cell lines. A series of transfection and cytotoxicity assays revealed that PGEAs are highly promising as a new class of safe and efficient gene delivery vectors for future clinical gene therapies.