FTC-CD_(133)纳米微粒抑制肝癌干细胞耐药性的研究

Research of FTC-CD_(133) Nanoparticles Inhibiting the Drug Resistance of Liver Cancer Stem Cell

目的制备包裹乳腺癌耐药蛋白(BCRP/ABCG2)多药转运体的特异抑制剂FTC且连接CD_(133)抗体的纳米微粒,并评价其特性和治疗作用。方法运用复乳蒸发法制备以聚乳酸-聚乙醇酸(PLGA)为载体的包封FTC的载药纳米微粒,通过碳化二亚胺法在其表面连接CD_(133)抗体,得到纳米微粒FTC-PLGA-NP-C;分析制备得到的纳米微粒的粒径分布、FTC包封率、载药量和纳米微粒的抗体连接效率;CD_(133)^+免疫磁珠法分选出CD_(133)^+SP细胞,MTT法检测CD_(133)^+SP细胞的增殖能力,Western blotting检测SP细胞中ABCG2的表达;荧光显微镜下观察FTC-PLGA-NP-C与CD_(133)^+SP细胞的结合效率;将CD_(133)^+SP细胞加入不同浓度的FTC-PLGA-NP-C纳米微粒,Western blotting检测其ABCG2的表达;以同体积PBS为对照,在CD_(133)^+SP细胞中加入FTC-PLGA-NP-C纳米微粒,同时加入阿霉素,HPLC检测孵育不同时间后细胞内阿霉素的浓度。裸鼠腋下荷瘤,成瘤后尾静脉注射FTC-PLGA-NP-C或者0.9%氯化钠溶液,48 h后再由尾静脉注射阿霉素,测量瘤体直径,并采用高效液相色谱法检测裸鼠肿瘤组织中阿霉素的浓度。结果 FTC-PLGA-NPC呈规则球形,粒径分布为60~120 nm,载药量为11.27%,包封率为37.18%,与CD_(133)^+SP细胞有较好的结合能力;Western blotting检测显示ABCG2在CD_(133)^+SP细胞中表达,且随着FTC-PLGA-NP-C纳米微粒剂量的增加,ABCG2的表达量逐渐降低,到4 mg/ml FTC-PLGA-NP-C时已几乎检测不到ABCG2的表达。从24 h开始,PBS中CD_(133)^+SP细胞内阿霉素浓度很快下降至1μg/ml及以下;而FTC-PLGA-NP-C中CD_(133)^+SP细胞内阿霉素浓度一直维持在2~3μg/ml。生理盐水组小鼠瘤体直径为(1.845±0.076)cm,高于FTC-PLGA-NP-C组的(1.238±0.072)cm(t=5.802,P<0.001);生理盐水组瘤内阿霉素浓度为(2.005±0.154)μg/ml,低于FTC-PLGA-NP-C组的(3.853±0.261)μg/ml(t=6.088,P<0.001)。结论制备的载药纳米微粒FTC-PLGA-NP-C可较好地靶向肝癌干细胞,并抑制其耐药蛋白ABCG2的表达,降低肿瘤细胞对常规治疗药物的耐药性,提高化疗药物的治疗效果。

Objective To prepare the nanoparticles packing FTC, a specific inhibitor of multidrug transporter BCRP/ ABCG2, and connecting CDI33 antibody, and to evaluate its features and therapeutic effect. Methods Nanoparticles packing FTC and with PLGA as carrier were prepared using multiple emulsion - solvent evaporation method, and carbodiimide reaction was undertaken to connect CD133 antibody on the surface of the nanopartieles, forming the nanoparticles of FTC-PLGA-NP-C ; the particle size distribution, FTC encapsulation efficiency, drug loading capacity and the antibody connection efficiency of nanoparticles were analyzed ; CD133 SP cells were sorted by CDI133 magnetic activated cell sorting （ MACS ）, MTT method was used to measure the multiplication capacity of CD133 SP ceils, and western blotting method was employed to detect the expression of ABCG2 in SP cells ; the combination efficiency of FTC-PLGA-NP-C and CDI^3 SP was observed under fluorescence microscope ;CD133 SP cells were added into nanoparticles of FTC-PLGA-NP-C of different concentrations, and western blotting was used to detect the expression of ABCG2; with PBS of the same volume taken as control, FTC-PLGA-NP-C nanoparticles were added into CD133 SP ceils with adriamycin added at the same time, and the concentration of adriamycin in the ceils was determined by HPLC at different time points of incubation. Tumor was transplanted to the armpit of nude mice, and tail intravenous injection of FTC- PLGA-NP-C or 0. 9% sodium chloride solution was undertaken after the formation of tumor; 48 hours later, tail intravenous injection of adriamycin was conducted, tumor diameter was measured, and high efficiency liquid chromatography was employed to measure the concentration of adriamycin in the tumor tissue of mude mice. Results FFC-PLGA-NP-C was in sphere shape, with a size distribution of 60 -120 nm, a drug loading capacity of 11.27% and an encapsulation efficiency of 37.18% , and had a better combining capacity with CD133 SP cells; western blotting showed that ABCG2 expression existed in CD＋133 SP cells and decreased with the increase of the dosage of FTC-PLGA-NP-C nanoparticles with no ABCG2 expression detected when the dosage of FTC-PLGA-NP-C was 4 mg/ml. At 24 hours of incubation, the concentration of adriamycin began to decrease quickly to below 1 μg/ml in the CD＋133 SP cells in PBS, while the concentration of adriamycin in the CD＋133 SP cells in FTC-PLGA-NP-C remained at 2 - 3 μg/ml. The diameter of the mice tumor of normal saline group was （ 1. 845 ± 0. 076） cm, higher than （ 1. 238 ±0. 072） cm of FTC-PLGA-NP-C group （t = 5. 802, P 〈 0. 001 ） ; the concentration of adriamycin in the tumor of normal saline group was （2. 005 ± 0. 154） μg/ml, lower than （ 1. 238± 0. 072） cm of FTC-PLGA-NP-C group （ t = 6. 088, P 〈 0. 001 ）. Conclusion Prepared drug - loading nanoparticle FTC-PLGA-NP-C could effectively target liver cancer stem cells, inhibit the expression of drug resistance protein ABCG2, reduce the drug resistance of tumor cells to conventional drugs and improve the therapeutical effect of chemotherapeutic drug.