葡聚糖分子量和接枝度对阿霉素/白蛋白-葡聚糖纳米粒子体外抗肿瘤效果的影响

doi:10.7503/cjcu20130708

摘要/Abstract

摘要：

以Maillard反应制备的牛血清白蛋白-葡聚糖共价接枝物作为载体, 通过调节混合溶液的pH值和温度制备负载阿霉素的白蛋白-葡聚糖纳米粒子. 利用分子量为5×10³, 10×10³和62×10³的葡聚糖制备了多种共价接枝物, 研究了共价接枝物分子量对载药纳米粒子的粒径和稳定性及载药量的影响. 用短链葡聚糖(分子量5×10³和10×10³)制备的纳米粒子粒径为60 nm左右, 用长链葡聚糖(分子量62×10³)制备的纳米粒子粒径约为200 nm; 阿霉素的包埋效率为81%~98%, 包埋量为7.4%~16.9%. 细胞实验结果表明, 共价接枝物具有很好的生物相容性; 与自由阿霉素相比, 纳米粒子可以促进阿霉素进入人口腔上皮癌细胞; 受缓释性质的影响, 纳米粒子在低浓度时的细胞毒性要小于自由阿霉素. 与长链葡聚糖纳米粒子相比, 接枝度高的短链葡聚糖纳米粒子由于具有较小的粒径、密集的葡聚糖分子刷表面、一定的自由阿霉素浓度和较快的阿霉素释放速率, 因而更容易进入细胞并具有更好的体外抗肿瘤活性.

关键词: 葡聚糖, 牛血清白蛋白, 药物释放, 纳米粒子, 抗肿瘤活性, 阿霉素

Abstract:

Bovine Sercurn albumin-dextran(BSA-dextran) conjugates were prepared with different molecular weights of dextran and different molar ratios of BSA to dextran via Maillard reaction. Doxorubicin loaded BSA-dextran nanoparticles were fabricated by changing the pH and then temperature of the mixture. The nanoparticles with 5×10³ and 10×10³ dextran have a size about 60 nm, and the nanoparticles with 62×10³ dextran have a size about 200 nm. The doxorubicin loading efficiency is in the range of 81%—98%, and the loading amount is 7.4%—16.9%. In vitro cell viability investigation confirms the excellent biocompatibility of BSA-dextran conjugates. Compared with free doxorubicin, the nanoparticles can enhance the cellular internalization of the loaded doxorubicin and they show lower anticancer activity at lower doxorubicin concentrations because of the slower release of the loaded doxorubicin. In comparison with the nanoparticles with 62×10³ and 10×10³ dextran, the nanoparticles with 5×10³ dextran and higher dextran conjugation degree show better cellular internalization and better anticancer activity in vitro due to their smaller size, denser dextran brush surface, certain free doxorubicin concentration, and faster release rate of the loaded doxorubicin.

Key words: Dextran, Bovine Sercurn albumin(BSA), Drug delivery, Nanoparticles, Anticancer activity, Doxorubicin

中图分类号:

O636
O629

TrendMD:

郝和群, 姚萍. 葡聚糖分子量和接枝度对阿霉素/白蛋白-葡聚糖纳米粒子体外抗肿瘤效果的影响. 高等学校化学学报, 2014, 35(3): 652.

HAO Hequn, YAO Ping. In Vitro Anticancer Effects of Doxorubicin Loaded BSA-dextran Nanoparticles with Different Molecular Weights of Dextran and Different Dextran Conjugation Degrees^†. Chem. J. Chinese Universities, 2014, 35(3): 652.

图/表 8

Table 1 BSA-DEX conjugates produced from different molecular weights of dextran and different molar ratios of BSA to dextran

Sample	M_w(DEX)	n(BSA):n(DEX) in feed	m(BSA):m(DEX) in feed	Average DEX number conjugated to each BSA
B2-D1-62k	62×10³	2:1	1:0.47	0.484^[25]
B1-D1-62k		1:1	1:0.94
B1-D2-62k		1:2	1:1.88
B1-D3-10k	10×10³	1:3	1:0.45	2.4
B1-D4-10k		1:4	1:0.61	2.7
B1-D5-10k		1:5	1:0.76	3.1
B1-D6-5k	5×10³	1:6	1:0.45
B1-D7-5k		1:7	1:0.53
B1-D8-5k		1:8	1:0.61	5.9
B1-D9-5k		1:9	1:0.68	7.5

Table 2 Size distribution, loading efficiency(LE), and loading amount(LA) results of DOX loaded nanoparticles before and after 30 d storagea

Sample	Fresh prepared				After 30 d
Sample	D_h/nm	PDI	LE(%)	LA(%)	D_h/nm	PDI
DOX/B2-D1-62k	198±14	0.13±0.04	92.4±1.9	15.7±0.5	194±15	0.11±0.02
DOX/B1-D1-62k^b	140±6	0.15±0.01	91.0±1.8	11.7±0.2	—	—
DOX/B1-D2-62k^b	108±8	0.14±0.02	84.8±0.8	7.4±0.1	—	—
DOX/B1-D3-10k	106±8	0.24±0.04	98.0±0.2	16.9±0.1	104±8	0.24±0.02
DOX/B1-D4-10k^c	68±8	0.24±0.02	95.9±0.4	14.9±0.1	72±10	0.22±0.04
DOX/B1-D5-10k	58±6	0.28±0.08	95.6±0.6	13.7±0.1	58±4	0.29±0.04
DOX/B1-D6-5k	198±2	0.31±0.01	84.3±0.6	14.5±0.3	208±8	0.34±0.02
DOX/B1-D7-5k	114±2	0.25±0.02	83.9±0.4	14.0±0.2	118±6	0.27±0.01
DOX/B1-D8-5k	76±2	0.24±0.01	82.8±0.3	13.3±0.2	74±6	0.25±0.02
DOX/B1-D9-5k	66±2	0.37±0.01	81.1±0.7	12.5±0.1	70±4	0.36±0.03

Fig.1 TEM images of DOX/B2-D1-62k(A), DOX/B1-D5-10k(B) and DOX/B1-D9-5k(C) nanoparticles Samples (B) and (C) were negative stained with phosphotungstic acid.

Fig.2 Accumulative release of DOX from DOX/B2-D1-62k(a), DOX/B1-D5-10k(b) and DOX/B1-D9-5k(c) nanoparticles in PBS(A) and 0.1 mol/L pH=5.0 acetate buffer(B) Free DOX solution was assayed as a control(curve d).

Fig.3 Flow cytometry analysis of the cellular uptake of control(a), free DOX(b), DOX/B2-D1-62k(c), DOX/B1-D5-10k(d) and DOX/B1-D9-5k(e) nanoparticles with a fixed DOX concentration of 2 mg/mL after 2 h incubation at 37 ℃

Fig.4 Confocal fluorescence microscopy images of cellular uptake of free DOX(A, B) and DOX/B1-D9-5k nanoparticles(C, D) with a fixed DOX concentration of 2 μg/mL after 2 h incubation at 37 ℃(A, C) and 4 ℃(B, D)

Fig.5 Cell viability against B2-D1-62k, B1-D5-10k and B1-D9-5k conjugates after 48 h incubation with different BSA concentrations

Fig.6 Cell viability against free DOX solution as well as DOX/B2-D1-62k, DOX/B1-D5-10k and DOX/B1-D9-5k nanoparticles after 48 h(A) and 72 h(B) incubation with different DOX concentrations* P>0.05 compared with DOX/B2-D1-62k group; ** P<0.05 compared with DOX/B2-D1-62k group; *** P<0.01 compared with DOX/B2-D1-62k group.

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