Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment
Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment
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DOI：
10.1006/mthe.2000.0105
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发表时间：
2000-08-01
影响因子：
12.4
通讯作者：
Kim, SW
中科院分区：
文献类型：
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作者：
Maheshwari, A;Mahato, RI;Kim, SW
直接注射编码细胞因子基因（例如 mIL-12 和 mIFN-gamma）的质粒 DNA 后，转基因表达和肿瘤消退仍然非常低。本研究的目的是开发基于无毒可生物降解聚合物的细胞因子基因传递系统，该系统应增强 mIL-12 的表达，从而增加完全消除肿瘤的可能性。我们通过熔融缩合合成了聚[α-(4-氨基丁基)-L-乙醇酸](PACA)，一种可生物降解的无毒聚合物。本研究中使用的质粒编码荧光素酶 (pLuc) 和鼠白细胞介素 12 (pmIL-12) 基因。 PAGA/质粒复合物以不同的 (+/-) 电荷比制备，并在粒径、zeta 电位、渗透压、表面形态和细胞毒性方面进行表征。将 PAGA 与 pmIL-12 以及 pLuc 复合制备的复合物用于转染培养的 CT-26 结肠腺癌细胞以及携带 CT-26 肿瘤的 BALB/c 小鼠。通过荧光素酶测定（对于 pLuc）、酶联免疫吸附测定（对于 mIL-12、p70 和 p40）和逆转录酶聚合酶链反应（RT-PCR）（对于 Luc 和 mIL-12 p35）测定体外和体内转染效率。 PACA 浓缩并保护质粒免遭核酸酶降解。在 5% (w/v) 葡萄糖中以 3:1 (+/-) 电荷比制备的复合物的平均粒径和 zeta 电位分别约为 100 nm 和 20 mV。通过原子力显微镜测定的聚合物复合物的表面特征显示DNA在Z方向上完全缩合并具有椭圆体结构。 PAGA/pmIL-12 复合物的 mIL-12 p40、mIL-12 p70 和 mIFN-gamma 水平显着高于裸露 pmIL-12。这与 RT-PCR 数据非常一致，RT-PCR 数据显示了显着水平的 mIL-12 p35 表达。 3-{4,5-二甲基噻唑-2-基}-2,5-二苯基四唑溴化物测定表明，PAGA/pmIL-12 复合物不会在 CT-26 细胞中诱导任何细胞毒性，并且与裸露的 pmIL-12 相比，显示出增强的体内抗肿瘤活性。 PACA/pmIL-12 复合物无毒，并且在体外和体内显着增强 mIL-12 mRNA 和蛋白质水平的表达。
Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-L-glycolic acid] (PACA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PACA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PACA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.