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J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (3): 364-374    DOI: 10.3785/j.issn.1008-9292.2020.06.03
Synthesis of folate modified chitosan-based nanomicelles and its in vitro anti-tumor activity
LIU Lu1,2(),HUANG Guojun1,BAI Hongzhen1,TANG Guping1,*()
1. Department of Chemistry, Zhejiang University, Hangzhou 310028, China
2. Zhejiang Shengda Bio-pharm Co., Ltd., Taizhou 317200, Zhejiang Province, China
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Objective: To design and synthesize folate-modified pH-responsive chitosan-based nanomicelles and investigate the in vitro anti-tumor activity of the drug-loaded micelles. Methods: CHI-DMA was obtained by reductive amination reaction of aldehyde-based chitosan and hydrophilic amine compounds, and CHI-DMA-LA was obtained by condensation reaction with lauric acid; FA-CHI-DMA-LA was obtained after modification with folic acid (FA). The drug-loaded nanomicelles FA-CHI-DMA-LA/DOX were assembled by solvent change method. The physicochemical properties of polymers were characterized by hydrogen nuclear magnetic resonance and transmission electron microscope. The particle size and surface potential were determined by dynamic light scattering method. Folic acid access rate, doxorubicin (DOX) loading rate and entrapped efficiency were measured by UV-vis spectrophotometer. The drug release properties of DOX-loaded micelles in vitro were monitored by fluorescence spectrophotometer at different pHs (7.4, 6.5, 5.0). The cytotoxicity against human oral cancer KB cells was detected by MTT assay. Fluorescence microscope and flow cytometry were applied to investigate the phagocytosis of DOX-loaded micelles on KB cells. Results: FA-CHI-DMA-LA was synthesized. The particle sizes of FA-CHI-DMA-LA-1 and FA-CHI-DMA-LA-2 micelles which used for the subsequent experiments were (73±14) nm and (106±15) nm, zeta potential were (15.59±1.98) mV and (21.20±2.35) mV, respectively. The drug loading rates of drug-loaded micelles FA-CHI-DMA-LA-1/DOX and FA-CHI-DMA-LA-2/DOX are (4.08±1.12)%and (4.12±0.44)%, respectively. In vitro drug release is pH-responsive, with cumulative release of DOX up to 37%and 36%at pH 5.0, which is about 1.5 times higher than that of pH 7.4. For FA-CHI-DMA-LA micelles with 1.25 to 125 μg/mL concentration, the survival rate of KB cells is more than 70%after incubation for 24 hours. The cell uptake of FA-CHI-DMA-LA/DOX micelles was enhanced compared to CHI-DMA-LA/DOX, and the cell uptake was higher in incubation without FA medium than that with FA. Compared with free DOX or CHI-DMA-LA/DOX, FA-CHI-DMA-LA/DOX nanomicelles showed higher cyctoxicity to KB cells, especially the FA-CHI-DMA-LA-2/DOX nanomicelles, the cell survival rate was about 17% after incubation for 24 hours. Conclusion: FA-modified chitosan-based nanomicelle with good biocompatibility was successfully prepared, which exhibits tumor microenvironmental pH responsive drug release and tumor targeting.

Key wordsDrug delivery systems      Nanoparticles      Antineoplastic agents      Folic acid      pH responsive     
Received: 16 September 2019      Published: 24 July 2020
CLC:  R943  
Corresponding Authors: TANG Guping     E-mail:;
Cite this article:

LIU Lu,HUANG Guojun,BAI Hongzhen,TANG Guping. Synthesis of folate modified chitosan-based nanomicelles and its in vitro anti-tumor activity. J Zhejiang Univ (Med Sci), 2020, 49(3): 364-374.

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目的: 设计并合成叶酸修饰酸碱度响应壳聚糖纳米胶束,考察材料及其载药胶束对肿瘤细胞的体外生物活性。方法: 醛基化的壳聚糖与亲水性的胺类化合物经还原胺化反应得CHI-DMA,进一步与月桂酸(LA)经缩合反应得CHI-DMA-LA;叶酸(FA)修饰后得FA-CHI-DMA-LA;包载阿霉素(DOX)后得载药纳米胶束FA-CHI-DMA-LA/DOX。采用氢核磁共振测定材料结构;透射电子显微镜考察材料形态;动态光散射法测定粒径和表面电位;紫外分光光度法测定材料中叶酸的接入率、载药胶束的载药率和包封率;荧光分光光度法测试不同酸碱度下载药胶束体外释放药物的性能;MTT法检测胶束对KB细胞的毒性;荧光显微镜和流式细胞仪考察载药胶束在KB细胞中的吞噬情况。结果: 合成了叶酸修饰的壳聚糖胶束材料FA-CHI-DMA-LA,后续用于实验的FA-CHI-DMA-LA-1和FA-CHI-DMA-LA-2胶束的粒径分别为(73±14)nm和(106±15)nm,表面电位分别为(15.59±1.98)mV和(21.20±2.35)mV。其载药胶束FA-CHI-DMA-LA-1/DOX和FA-CHI-DMA-LA-2/DOX的载药率分别为(4.08±1.12)%和(4.12±0.44)%,体外药物在酸碱度5.0下累积释放分别达37%和36%,是酸碱度7.4下的1.5倍左右。FA-CHI-DMA-LA在1.25~125 μg/mL浓度下作用24 h后KB细胞存活率均在70%以上。FA-CHI-DMA-LA/DOX载药胶束的细胞摄取较CHI-DMA-LA/DOX增强,且在无叶酸培养基孵育下细胞摄取比含叶酸培养基孵育下增强。与游离阿霉素和CHI-DMA-LA/DOX比较,FA-CHI-DMA-LA/DOX对KB细胞杀伤力更高,其中FA-CHI-DMA-LA-2/DOX给药孵育24 h后细胞存活率约17%。结论: 成功制备了作为药物输送载体的叶酸修饰壳聚糖纳米胶束材料,其生物相容性良好,具有适应肿瘤微环境酸碱度的药物释放和肿瘤靶向效果。

关键词: 药物释放系统,  纳米粒子,  抗肿瘤药,  叶酸,  酸碱度响应 
Fig 1 Synthesis of FA-CHI-DMA-LA
Tab 1 Folic acid access rate, particle sizes and zeta potential of FA-CHI-DMA-LA micelles  ($\bar x \pm s$, n=3)
Fig 3 TEM spectra of FA-CHI-DMA-LA
Fig 4 DOX release from drug-loaded FA-CHI-DMA-LA/DOX micelles
Fig 5 Cytotoxicity test of FA-CHI-DMA-LA micelles
Fig 6 Microscope image of KB cells after treated with FA-CHI-DMA-LA/DOX drug-loaded micelles
Fig 7 Detection of cell uptake of FA-CHI-DMA-LA/DOX drug-loaded micelles by flow cytometry
Fig 8 Cell viability results of FA-CHI-DMA-LA/DOX micelles
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