论文总字数：20089字

摘 要

：本文用脱氧胆酸对羧甲基壳聚糖进行疏水改性形成两亲性胶束载药系统，包载人参皂苷CK，并对其体外抗肿瘤活性进行了研究。首先，用催化剂EDC和NHS将脱氧胆酸连接到羧甲基壳聚糖上，对羧甲基壳聚糖用红外光谱，核磁共振氢谱，X射线衍射进行表征，用元素分析仪对聚合物的取代度进行定量分析。用芘作为荧光探针，用荧光分光光度计测定脱氧胆酸-羧甲基壳聚糖胶束（DA-OCMC）的临界胶束浓度（CMC）， DA-OCMC在较低浓度下即可以在水中形成胶束。用超声法制备DA-OCMC胶束的纳米粒子后，用马尔文粒径仪，透射电子显微镜和扫描射电子显微镜观察纳米粒子的形态和大小。结果表明，形成的纳米粒子分散性良好，呈球形，大小在150 nm左右。用超声法将人参皂苷CK包裹在DA-OCMC胶束中，马尔文粒径仪测得载药胶束的粒径比空白胶束稍大，但不影响其分布的均一性和胶束的稳定性。用高效液相法测载药量和包封率。MTT法对载药胶束体外抗肿瘤活性的研究发现，载药胶束的浓度越大，对细胞活性的抑制就越强。本实验制备的两亲性壳聚糖衍生物成功地提高了人参皂苷CK的水溶性，证明了DA-OCMC是肿瘤细胞靶向抗肿瘤药物的一种很有前途的载体。

关键词：人参皂苷CK，脱氧胆酸羧甲基壳聚糖，聚合物胶束，抗癌

Abstract: In this paper， carboxymethyl chitosan was hydrophobically modified with deoxycholic acid to form an amphiphilic micelle carrier drug system， which contained Ginsenoside CK， and its antitumor activity was studied in vitro. First， deoxycholic acid was conjugated to carboxymethyl chitosan using catalysts EDC and NHS. The carboxymethyl chitosan was characterized by IR， 1H NMR， and X-ray diffraction， using an elemental analyzer for the polymer. The degree of substitution was quantitatively analyzed. The critical micelle concentration (CMC) of deoxycholic acid-carboxymethyl chitosan micelles (DA-OCMC) was measured with a fluorescence spectrophotometer using hydrazine as a fluorescent probe. The results showed that DA-OCM can be used at a certain concentration. Micelles form in water. After the nanoparticles of DA-OCMC micelles were prepared by ultrasonic method， the morphology and size of the nanoparticles were characterized by Malvern particle size analyzer， scanning electron microscope and transmission electron microscope. The results show that the formed nanoparticles are spherical and uniformly distributed， with a size of about 150 nm. The Ginsenoside CK was entrapped in the DA-OCMC micelles by the ultrasonic method. The particle size of the drug-loaded micelles measured by the Malvern particle size analyzer was slightly larger than that of the blank micelles， but it did not affect the uniformity of the distribution and the stability of the micelles. The drug loading and entrapment efficiency were measured by HPLC. The in vitro anti-tumor activity of drug-loaded micelles by MTT assay showed that the greater the concentration of drug-loaded micelles， the stronger the inhibition of cellular activity. The amphiphilic chitosan derivatives prepared in this experiment successfully improved the water solubility of ginsenoside CK， and proved that DA-OCMC is a promising carrier for tumor cell targeted anti-tumor drugs.

Key words: Ginsenoside CK， deoxycholic acid carboxymethyl chitosan， polymer micelle， antitumor

目录

1 前言 6

1.1 人参皂苷 6

1.2 纳米载药系统 6

1.3 壳聚糖纳米载体的研究 7

1.4 研究的目的和意义 8

2 实验材料和设备 8

2.1 实验材料和试剂 8

2.2 实验仪器和设备 8

3.脱氧胆酸-O-羧甲基壳聚糖的制备与表征 9

3.1 脱氧胆酸-O-羧甲基壳聚糖的制备 9

3.2载体的结构表征 9

3.3 DA-OCMC临界胶束浓度的测定 9

3.4脱氧胆酸-羧甲基壳聚糖（DA-OCMC）自聚集纳米粒的制备和结构表征 10

3.5 结果与讨论 10

4.包载人参皂苷CK脱氧胆酸-O-羧甲基壳聚糖纳米粒的载药性能和体外抗肿瘤活性 16

4.1纳米粒子制备的方法 16

4.2 包载人参皂苷CK的脱氧胆酸-O-羧甲基壳聚糖纳米粒的表征 16

4.3 载药胶束的载药量和包封率的测定 16

4.4 体外缓释性能的测定 17

4.5.载药胶束的体外抗肿瘤活性测试 17

4.6结果与讨论 19

5.结论 25

参考文献 26

致谢 29

1 前言

1.1 人参皂苷

1.1.1人参皂苷的生物活性

人参是一种传统中草药，属于五加科人参属，主要产于中国东北，日本，朝鲜，俄罗斯，在亚洲已有5000多年历史^[1]。人参有美容之效，并能够调整血压，恢复心脏功能，强身健体。人参的活性成分为人参皂苷，目前已从人参的根、叶、茎，花，果实中分离出150多种天然人参皂苷^[2]，其具有大量生物活性，包括增强心血管健康^[3]，免疫抵抗力^[4]，抗压能力^[5]，学习记忆能力^[6]。人参皂苷是一种固醇类化合物，包括人参二醇型，人参三醇型，齐墩果型。人参皂苷Compound K(CK)是稀有人参皂苷，属于原人参二醇型皂苷，在自然界中并不存在，是由人参皂苷Rb1和Rb2经过肠道微生物的作用转化而来^[7]，对人体无毒无害并且具有多种生物学活性。

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