负载表柔比星壳聚糖微球的制备及单纯与联合微波消融治疗小鼠皮下肝移植瘤疗效观察
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摘要
背景：
癌症已严重危害人类的健康,成为人类杀手之一,居常见死亡原因的首位。世界卫生组织(WHO)统计,每年世界上有1000万人患上癌症,而死于癌症的人数约600万,占全球死亡人数的12%。我国每年新增癌症患者180万,死亡140万,平均每3分钟就有1.3人死于癌症,而且癌症的发病率呈急剧上升趋势。原发性肝癌是常见的恶性肿瘤之一,其发病率及死亡均较高,据统计每年全球肝癌发病率位居恶性瘤发病率的第5位,死亡率位居第3位,而我国是肝癌高发区,据统计55%发生在我国,发病率及死亡率均较高,其进展迅速、生存质量差,是迄今全部肿瘤中治疗效果较差的恶性肿瘤之一,而且大部分确诊已属晚期,手术切除率低,对全身化疗及放疗均不敏感,且全身化疗存在靶向性差、疗效不佳、毒副作用大等缺点,肝癌的局部、联合治疗成为重要治疗途径及研究热点。微波是一种高频电磁波,组织中的极性分子(主要是水分子、带电离子和胶体蛋白)在微波作用下发生振荡摩擦产生局部高温,使肿瘤组织凝固性坏死,肝癌组织具有含水丰富的特点,在超声引导下将微波天线左右,插入肝癌组织内部,可短时间内产生高达65℃-100℃左右的高温,从而使肝脏肿瘤组织坏死。但临床上往往存在消融不彻底、较易出现局部病灶残留及复发等弊端,因此微波联合治疗成为肝脏肿瘤治疗的重要局部治疗方法。
控释缓释剂(controlled-and sustained-release drugs)属于第三代药物剂型,将传统的药物包埋于基质或载体,利用基质的吸附、增粘、支架粘连或膜屏障等作用,提高药物稳定性,降低血浓峰谷现象,延缓药物释放速度,提高药物靶向定位,使药物吸收变慢、作用延长、毒性降低,提高药效和安全度,解决了传统药物尤其是抗肿瘤药物半衰期短、难于长期间维持血药浓度、全身毒副作用大、病人较难耐受等缺点,是目前抗肿瘤药物研究的重要发展趋势,并可为寻找新的治疗方法提供方向。药物的靶向作用根据其机制不同可分为以类：①主动靶向,将抗体或配体等特异性的靶向分子偶联至粒子表面而使药物定向分布到靶组织,是根据生物免疫靶向理论设计靶向给药系统；②物理化学靶向,是根据药物微粒或者药物载体微粒表面电荷、表面疏水和表面吸附大分子的不同,可以达到不同的器官以实现靶向；③被动靶向,依据载体的理化性质(大小、形状、亲水性、表面电荷和囊壁孔径等)进行药物的分布和释放,用对机体各种组织和病变亲和力不同的微粒也可达到靶向的目的,是根据机体的组织生理学特性对不同大小微粒的滞留性不同,选择性地聚集于肝、脾、肺、淋巴部位释放药物而发挥疗效,以将药物制成不同大小的微粒实现对于不同器官组织的生物物理靶向,或者将药物包裹于可生物降解的生物相容性高分子粒子中以实现缓释与生物物理靶向。靶向给药可以减少抗肿瘤药物与非靶向器官、组织及细胞的非特异性相互作用,从而增加靶部位／非靶部位的药物比率,提高了抗肿瘤药物疗效的同时,降低了药物的毒副反应。
本实验以壳聚糖为载体,包裹表柔比星从而制备成壳聚糖载药微球。壳聚糖微球是目前实际应用较多的药物缓释形式,壳聚糖微球系指药物溶解或分散在载体中形成的微小球型实体,粒径范围在1～250μm。壳聚糖(Chitosan, CS)是一种广泛存在于自然界的聚阳离子多糖衍生物,又称可溶性甲壳质、甲壳胺、几丁聚糖等,由D-氨基葡萄糖和N-乙酰-D-氨基葡萄糖组成,是以β-(1,4)糖苷键连接起来的直链多糖,化学名为(1,4)-2-氨基-2-脱氧-β-D-葡萄糖,其结构类似于纤维素。壳聚糖是天然多糖中唯一的碱性多糖,具有良好的生物相容性和可降解性,具有独特的物理化学特性和生物学功能,其分解产物无毒。用CS制备的靶向制剂具有缓释、控释、靶向释放药物、增加药物吸收、提高药物的生物利用度,降低药物毒副作用等特点,并可选择性地与肿瘤细胞聚集,发挥其抗肿瘤作用。多柔比星为蒽环类抗肿瘤抗生素,因其抗瘤谱广其主要毒副作用为心脏毒性.可导致严重的心肌损伤和心力衰竭,且损伤程度和剂量有密切关系,减少其心脏毒性的主要方法就是应用药物载体,改变的生物分布,减少在全身特别是心脏组织中的分布,提高其在局部肿瘤中的含量。
本研究选用CS作为原材料,采用W／O型乳化-固化法制备表柔比星-壳聚糖微球,检测微球的理化性质,通过单纯瘤内注射来治疗小鼠肝移植瘤,观察其对肿瘤的抑制作用,进一步与微波消融治疗相结合,探讨其联合治疗肝移植瘤的疗效,以期寻找新的抗肝肿瘤联合治疗方法,并为今后临床应用研究提供理论依据。
目的：
1.制备负载模型药物表柔比星(EPI)的壳聚糖微球(CS)并检测载药微球的粒径大小和体外释药性能。
2.对移植瘤小鼠进行体内抑瘤试验,证明负载表柔比星的壳聚糖微球较普通抗癌药物具有更好的体内抑瘤作用。
3.壳聚糖微球联合微波消融治疗小鼠移植瘤,证实其与单独微波治疗相比能获得较高的抑瘤率；探讨壳聚糖载药微球联合微波消融治疗肝癌的可行性。
方法：
1.采用W／O型乳化-固化法制备表柔比星-壳聚糖微球,扫描电镜观察壳聚糖微球的表面形态,并计算粒径大小。利用紫外分光光度计通过动态透析法分析载药微球的包封率、载药量及药物累积释放率。
2.采用传代腹水H22细胞接种昆明小鼠皮下,制备H22皮下肝癌荷瘤小鼠模型,设载药壳聚糖微球、游离药物、壳聚糖和生理盐水四组,给药后开始监测肿瘤体积大小,2周后处死小鼠,剥离瘤体、称重,计算抑瘤率。
3.建立皮下移植瘤模型后,对局部皮下肝移植瘤进行微波消融治疗,其后瘤内注射上述四组药物,瘤内给药后开始监测肿瘤体积大小,2周后处死小鼠,剥离瘤体、称重,计算抑瘤率,免疫组化监测凋亡因子Bcl-2、bax表达。
4.采用SPSS 13.0统计软件包,试验结果表示为(x±s),组间差异性统计采用one-way ANOVA,经Levene方差齐性检验后,若方差齐,采用LSD法进行多重比较,若方差不齐,采用Dunnett's法进行多重比较,以P<0.05表示差异有统计学意义。
结果：
1.配制2%的壳聚糖溶液,CS/EPI质量比为8：1,制备的载药微球呈圆形或椭圆形,分散性良好,平均粒径105μm,大小较一致,包封率约为80%,载药率约为11%,2周的累积缓释率为84%。
2.瘤内注射生理盐水、壳聚糖、表柔比星、载药微球后,通过观察肿瘤体积变化大小、抑瘤率、检测凋亡因子,表明壳聚糖微球是一种有效的表柔比星局部缓释剂型,具有较好的抗肿瘤作用。
3.微波联合瘤内注射生理盐水、表柔比星、载药微球的抑瘤率分别为8%,20%,47%。表明壳聚糖微球联合微波消融治疗有较强的抑瘤作用。
结论：
1.W／O型乳化-固化法制备表柔比星-壳聚糖微球形状规则、粒度分布均匀、具有较高包封率和较好缓释性能。
2.与单纯化疗药物相比,载药壳聚糖微球对移植瘤小鼠的体内抑瘤作用强,壳聚糖微球适合作为化疗药物的新型载体。
3.微波联合局部瘤内注载药壳聚糖微球治疗鼠移植瘤能明显缩小肿瘤体积,可获得较高的抑瘤率；微波联合瘤内注射药物缓释微球这种治疗模式值得进一步探讨,有可能成为治疗中晚期肝癌的有效方法。
Background
The World Health Organization (WHO) statistics 1,000 people each year in the world sufferred from cancer, while the number of deaths is about 60,000 accounting for 12% of global deaths. There is about 1.8 million new cancer patients each year,1.4 million dead.an average of 1.3 every 3 minute, people died of cancer. Primary liver cancer is a common malignant tumor, its incidence and mortality are high. Liver cancer incidence locates the fifth. Mortality rate ranked the third[1]. According to statistics 55% liver cancer occurred in China, while the incidence and death rates are higher. Its progress is rapid and life quality is poor,which is far less effective disease in all tumors.And most patients'diagnosis is late.Hepatic carcinoma is not sensitive to chemotherapy and radiotherapy. Also chemotherapy is poor targeting, poor efficacy, toxic side effects and other defects.The local or Combination therapy to liver cancer is a major focus. a high-frequency electromagnetic wave, the organization polar molecule (mainly water molecules, ions and charged colloidal protein) under the action of the microwave generates friction heat, leading to coagulation necrosis of tumor tissue, and liver tissue is rich of water. The features in the ultrasound-guided inserted within the tumor tissue, can quickly produce up to about 65℃-100℃temperature. However, there is often kinds of problems,such as incompleted ablation,residual tumor tissue and recurrence and so on. The microwave treatment combined with other treatment is an important local method to liver tumor treatment.
Controlled-and sustained-release drugs are the third generation.The traditional drugs are embedded in the matrix or vector, using matrix adsorption, viscosity, adhesion or membrane barriers stand, improving drug stability, reducing blood concentration peak and delaying the drug release rate and improving drug targeting positioning. It solves the problems sucn as the traditional drugs, especially anticancer drugs with short half-life, difficult to maintain the plasma concentration during long, systemic toxic side effects. Most patients are suffering from tolerating such shortcomings, which is an important trends and in finding new treatments In the treatment, the study found that the use of nano-scale polymer particles as a new drug delivery and controlled release carrier, because of its small size super-micro through targeting the role of positioning and release into the body's cells, of nucleic acids, proteins, etc. material for the molecular level of life treatment, not only improved the efficacy of existing drugs, but also significantly reduced the adverse reactions of their own, the role of nano-drug targeting mechanism in accordance with its three different types:①active targeting, the antibodies or ligand-specific targeting molecules, such as coupling to the surface of particle orientation distribution of drugs to target tissue;②passive targeting., by control and modified the physical or chemical properties of the vector (size, shape, hydro-philicity, surface charge and the wall aperture, etc.) can control its distribution and drug release characteristics in the body, or to choose the nanoparticles which targeted to the body of various organizations and different disease affinity can also achieve the purpose;③physical and chemistry targeting, such as:use of local magnetic field in vitro, with super paramagnetic iron nanoparticles as a carrier at the external magnetic field under the directional distribution in a predetermined target tissue. By changing the physical and chemical properties of the nanoparticles, such as size, mass, charge and water affinity, can reduced non-specific interactions with non-targeting organs, tissues and cells, thus increasing the target position/non-target ratio of the drug.
In this study, chitosan is as a carrier, packaging epirubicin to prepare the drug into chitosan microspheres. Chitosan microspheres is a more practical forms of drug delivery. Chitosan microspheres means of drug dissolved or dispersed in the carrier in the form of small spherical solid particle whose size ranges from 1 to 250μm.Chitosan (CS) is a polycationic polysaccharide derivatives which is widely exist in nature, also known as soluble chitin, chitosan, etc. from D-glucosamine and N-acetyl-D-glucosamine composition, based onβ-(1,4)glycosidic bond linking the straight-chain polysaccharide, the chemical known as (1,4)-2-amino-2-deoxy-β-D-glucose, its structure similar to cellulose, chitosan is non-toxic, has good biocompatibility and degradability. Chitosan microspheres can selectively gathered to the tumor cells and play its anti-tumor effects.Doxorubicin is the anthracycline antitumor antibiotics, because of its broad spectrum anti-tumor drug whose main side effects is its cardiac toxicity. It Can lead to severe myocardial injury and heart failure, and the extent of damage is closely related to dose.to The main method of reducing their cardiac toxicity is the application of drug delivery,biodistribution changes to reduce the distribution in heart tissue, increasing local dose in tumor.
This study use CS as a raw material used in this study.Using W/O type emulsion-Curing Preparation epirubicin-chitosan microspheres to detect physical and chemical properties of microspheres,by a simple injection to treat tumors in mice liver tumor. It was observed by treating tumor proliferation. After that, we do next experiment. It is combined with microwave ablation to explore the combined effect in treating liver tumor, and to find a new combination therapy against liver cancer.
Objective:
1.Explore the drug-loaded chitosan microspheres as anti-cancer drugs combined with microwave treatment of hepatic tumor is feasible.
2.Preparation of the load model drug epirubicin (EPI) of the chitosan microspheres (CS) and to detect drug-loaded microspheres and in vitro release properties of the particle size.
3.Pairs of mice in vivo tumor inhibition test to prove that the load epirubicin-chitosan microspheres as compared with ordinary anti-cancer drugs have a better anti-tumor effect in vivo, is more suitable as a carrier of chemotherapeutic drugs.
4.Chitosan microspheres combined with microwave treatment of transplanted tumors in mice to prove that microwave therapy alone compared with other method the tumor inhibition rate compared to higher.
Methods:
1.Epirubicin-loaded chitosan microspheres were prepared by using emulsion-chemical cross linking technique.
2.The surface morphology and particles size of chitosan microspheres were observed by scanning electron microscope. Ultraviolet spectrophotometer was used to analyze the entrapment efficiency, entrapment efficiency and cumulative release rates of epirubicin-loaded chitosan microspheres.
3.Totally 24 mice with transplanted subcutaneous H22 HCC were divided into 4 groups, which were respectively treated by microwave coagulation therapy, intratumoral injected with physiological saline after microwave microwave coagulation therapy, intratumoral injected with epirubicin after microwave coagulation therapy, intratumoral injected with CS-EPI after microwave coagulation therapy.The tumor inhibitory rate was calculated.
Results:
1.The microspheres'size distribution was Uniform. The average diameter was 105um, The ratio of drug loading was 11% and the entrapment was 80%. The drug cumulative release rate in vitro after 2weeks was 84%.
2.Chitosan microspheres is an effective epirubicin local sustained release formulation, has good anti-tumor effect.
3.Compared with the pure microwave group, the tumour-inhibition rate of other groups were 11%,20%,47%.
Conclusion:
4.1. W/O type emulsion-Preparation of curing Epirubicin-chitosan microspheres shape the rules of uniform particle size distribution with high encapsulation efficiency and good release properties.
2. With chemotherapy alone compared with drug-loaded chitosan microspheres on tumor in vivo anti-tumor effect in mice strong, chitosan microspheres is more suitable as a carrier of new chemotherapy drugs.
3. Microwave combined with local intratumoral injection drug containing chitosan microspheres of treatmenting mice reduces tumor volume, gets a higher inhibition rate; microwave and intratumoral injection of drug delivery microspheres of this treatment model is worthy of further study.There may be an effective treatment method for advanced liver cancer.
癌症已严重危害人类的健康,成为人类杀手之一,居常见死亡原因的首位。世界卫生组织(WHO)统计,每年世界上有1000万人患上癌症,而死于癌症的人数约600万,占全球死亡人数的12%。我国每年新增癌症患者180万,死亡140万,平均每3分钟就有1.3人死于癌症,而且癌症的发病率呈急剧上升趋势。原发性肝癌是常见的恶性肿瘤之一,其发病率及死亡均较高,据统计每年全球肝癌发病率位居恶性瘤发病率的第5位,死亡率位居第3位,而我国是肝癌高发区,据统计55%发生在我国,发病率及死亡率均较高,其进展迅速、生存质量差,是迄今全部肿瘤中治疗效果较差的恶性肿瘤之一,而且大部分确诊已属晚期,手术切除率低,对全身化疗及放疗均不敏感,且全身化疗存在靶向性差、疗效不佳、毒副作用大等缺点,肝癌的局部、联合治疗成为重要治疗途径及研究热点。微波是一种高频电磁波,组织中的极性分子(主要是水分子、带电离子和胶体蛋白)在微波作用下发生振荡摩擦产生局部高温,使肿瘤组织凝固性坏死,肝癌组织具有含水丰富的特点,在超声引导下将微波天线左右,插入肝癌组织内部,可短时间内产生高达65℃-100℃左右的高温,从而使肝脏肿瘤组织坏死。但临床上往往存在消融不彻底、较易出现局部病灶残留及复发等弊端,因此微波联合治疗成为肝脏肿瘤治疗的重要局部治疗方法。
控释缓释剂(controlled-and sustained-release drugs)属于第三代药物剂型,将传统的药物包埋于基质或载体,利用基质的吸附、增粘、支架粘连或膜屏障等作用,提高药物稳定性,降低血浓峰谷现象,延缓药物释放速度,提高药物靶向定位,使药物吸收变慢、作用延长、毒性降低,提高药效和安全度,解决了传统药物尤其是抗肿瘤药物半衰期短、难于长期间维持血药浓度、全身毒副作用大、病人较难耐受等缺点,是目前抗肿瘤药物研究的重要发展趋势,并可为寻找新的治疗方法提供方向。药物的靶向作用根据其机制不同可分为以类：①主动靶向,将抗体或配体等特异性的靶向分子偶联至粒子表面而使药物定向分布到靶组织,是根据生物免疫靶向理论设计靶向给药系统；②物理化学靶向,是根据药物微粒或者药物载体微粒表面电荷、表面疏水和表面吸附大分子的不同,可以达到不同的器官以实现靶向；③被动靶向,依据载体的理化性质(大小、形状、亲水性、表面电荷和囊壁孔径等)进行药物的分布和释放,用对机体各种组织和病变亲和力不同的微粒也可达到靶向的目的,是根据机体的组织生理学特性对不同大小微粒的滞留性不同,选择性地聚集于肝、脾、肺、淋巴部位释放药物而发挥疗效,以将药物制成不同大小的微粒实现对于不同器官组织的生物物理靶向,或者将药物包裹于可生物降解的生物相容性高分子粒子中以实现缓释与生物物理靶向。靶向给药可以减少抗肿瘤药物与非靶向器官、组织及细胞的非特异性相互作用,从而增加靶部位／非靶部位的药物比率,提高了抗肿瘤药物疗效的同时,降低了药物的毒副反应。
本实验以壳聚糖为载体,包裹表柔比星从而制备成壳聚糖载药微球。壳聚糖微球是目前实际应用较多的药物缓释形式,壳聚糖微球系指药物溶解或分散在载体中形成的微小球型实体,粒径范围在1～250μm。壳聚糖(Chitosan, CS)是一种广泛存在于自然界的聚阳离子多糖衍生物,又称可溶性甲壳质、甲壳胺、几丁聚糖等,由D-氨基葡萄糖和N-乙酰-D-氨基葡萄糖组成,是以β-(1,4)糖苷键连接起来的直链多糖,化学名为(1,4)-2-氨基-2-脱氧-β-D-葡萄糖,其结构类似于纤维素。壳聚糖是天然多糖中唯一的碱性多糖,具有良好的生物相容性和可降解性,具有独特的物理化学特性和生物学功能,其分解产物无毒。用CS制备的靶向制剂具有缓释、控释、靶向释放药物、增加药物吸收、提高药物的生物利用度,降低药物毒副作用等特点,并可选择性地与肿瘤细胞聚集,发挥其抗肿瘤作用。多柔比星为蒽环类抗肿瘤抗生素,因其抗瘤谱广其主要毒副作用为心脏毒性.可导致严重的心肌损伤和心力衰竭,且损伤程度和剂量有密切关系,减少其心脏毒性的主要方法就是应用药物载体,改变的生物分布,减少在全身特别是心脏组织中的分布,提高其在局部肿瘤中的含量。
本研究选用CS作为原材料,采用W／O型乳化-固化法制备表柔比星-壳聚糖微球,检测微球的理化性质,通过单纯瘤内注射来治疗小鼠肝移植瘤,观察其对肿瘤的抑制作用,进一步与微波消融治疗相结合,探讨其联合治疗肝移植瘤的疗效,以期寻找新的抗肝肿瘤联合治疗方法,并为今后临床应用研究提供理论依据。
目的：
1.制备负载模型药物表柔比星(EPI)的壳聚糖微球(CS)并检测载药微球的粒径大小和体外释药性能。
2.对移植瘤小鼠进行体内抑瘤试验,证明负载表柔比星的壳聚糖微球较普通抗癌药物具有更好的体内抑瘤作用。
3.壳聚糖微球联合微波消融治疗小鼠移植瘤,证实其与单独微波治疗相比能获得较高的抑瘤率；探讨壳聚糖载药微球联合微波消融治疗肝癌的可行性。
方法：
1.采用W／O型乳化-固化法制备表柔比星-壳聚糖微球,扫描电镜观察壳聚糖微球的表面形态,并计算粒径大小。利用紫外分光光度计通过动态透析法分析载药微球的包封率、载药量及药物累积释放率。
2.采用传代腹水H22细胞接种昆明小鼠皮下,制备H22皮下肝癌荷瘤小鼠模型,设载药壳聚糖微球、游离药物、壳聚糖和生理盐水四组,给药后开始监测肿瘤体积大小,2周后处死小鼠,剥离瘤体、称重,计算抑瘤率。
3.建立皮下移植瘤模型后,对局部皮下肝移植瘤进行微波消融治疗,其后瘤内注射上述四组药物,瘤内给药后开始监测肿瘤体积大小,2周后处死小鼠,剥离瘤体、称重,计算抑瘤率,免疫组化监测凋亡因子Bcl-2、bax表达。
4.采用SPSS 13.0统计软件包,试验结果表示为(x±s),组间差异性统计采用one-way ANOVA,经Levene方差齐性检验后,若方差齐,采用LSD法进行多重比较,若方差不齐,采用Dunnett's法进行多重比较,以P<0.05表示差异有统计学意义。
结果：
1.配制2%的壳聚糖溶液,CS/EPI质量比为8：1,制备的载药微球呈圆形或椭圆形,分散性良好,平均粒径105μm,大小较一致,包封率约为80%,载药率约为11%,2周的累积缓释率为84%。
2.瘤内注射生理盐水、壳聚糖、表柔比星、载药微球后,通过观察肿瘤体积变化大小、抑瘤率、检测凋亡因子,表明壳聚糖微球是一种有效的表柔比星局部缓释剂型,具有较好的抗肿瘤作用。
3.微波联合瘤内注射生理盐水、表柔比星、载药微球的抑瘤率分别为8%,20%,47%。表明壳聚糖微球联合微波消融治疗有较强的抑瘤作用。
结论：
1.W／O型乳化-固化法制备表柔比星-壳聚糖微球形状规则、粒度分布均匀、具有较高包封率和较好缓释性能。
2.与单纯化疗药物相比,载药壳聚糖微球对移植瘤小鼠的体内抑瘤作用强,壳聚糖微球适合作为化疗药物的新型载体。
3.微波联合局部瘤内注载药壳聚糖微球治疗鼠移植瘤能明显缩小肿瘤体积,可获得较高的抑瘤率；微波联合瘤内注射药物缓释微球这种治疗模式值得进一步探讨,有可能成为治疗中晚期肝癌的有效方法。
Background
The World Health Organization (WHO) statistics 1,000 people each year in the world sufferred from cancer, while the number of deaths is about 60,000 accounting for 12% of global deaths. There is about 1.8 million new cancer patients each year,1.4 million dead.an average of 1.3 every 3 minute, people died of cancer. Primary liver cancer is a common malignant tumor, its incidence and mortality are high. Liver cancer incidence locates the fifth. Mortality rate ranked the third[1]. According to statistics 55% liver cancer occurred in China, while the incidence and death rates are higher. Its progress is rapid and life quality is poor,which is far less effective disease in all tumors.And most patients'diagnosis is late.Hepatic carcinoma is not sensitive to chemotherapy and radiotherapy. Also chemotherapy is poor targeting, poor efficacy, toxic side effects and other defects.The local or Combination therapy to liver cancer is a major focus. a high-frequency electromagnetic wave, the organization polar molecule (mainly water molecules, ions and charged colloidal protein) under the action of the microwave generates friction heat, leading to coagulation necrosis of tumor tissue, and liver tissue is rich of water. The features in the ultrasound-guided inserted within the tumor tissue, can quickly produce up to about 65℃-100℃temperature. However, there is often kinds of problems,such as incompleted ablation,residual tumor tissue and recurrence and so on. The microwave treatment combined with other treatment is an important local method to liver tumor treatment.
Controlled-and sustained-release drugs are the third generation.The traditional drugs are embedded in the matrix or vector, using matrix adsorption, viscosity, adhesion or membrane barriers stand, improving drug stability, reducing blood concentration peak and delaying the drug release rate and improving drug targeting positioning. It solves the problems sucn as the traditional drugs, especially anticancer drugs with short half-life, difficult to maintain the plasma concentration during long, systemic toxic side effects. Most patients are suffering from tolerating such shortcomings, which is an important trends and in finding new treatments In the treatment, the study found that the use of nano-scale polymer particles as a new drug delivery and controlled release carrier, because of its small size super-micro through targeting the role of positioning and release into the body's cells, of nucleic acids, proteins, etc. material for the molecular level of life treatment, not only improved the efficacy of existing drugs, but also significantly reduced the adverse reactions of their own, the role of nano-drug targeting mechanism in accordance with its three different types:①active targeting, the antibodies or ligand-specific targeting molecules, such as coupling to the surface of particle orientation distribution of drugs to target tissue;②passive targeting., by control and modified the physical or chemical properties of the vector (size, shape, hydro-philicity, surface charge and the wall aperture, etc.) can control its distribution and drug release characteristics in the body, or to choose the nanoparticles which targeted to the body of various organizations and different disease affinity can also achieve the purpose;③physical and chemistry targeting, such as:use of local magnetic field in vitro, with super paramagnetic iron nanoparticles as a carrier at the external magnetic field under the directional distribution in a predetermined target tissue. By changing the physical and chemical properties of the nanoparticles, such as size, mass, charge and water affinity, can reduced non-specific interactions with non-targeting organs, tissues and cells, thus increasing the target position/non-target ratio of the drug.
In this study, chitosan is as a carrier, packaging epirubicin to prepare the drug into chitosan microspheres. Chitosan microspheres is a more practical forms of drug delivery. Chitosan microspheres means of drug dissolved or dispersed in the carrier in the form of small spherical solid particle whose size ranges from 1 to 250μm.Chitosan (CS) is a polycationic polysaccharide derivatives which is widely exist in nature, also known as soluble chitin, chitosan, etc. from D-glucosamine and N-acetyl-D-glucosamine composition, based onβ-(1,4)glycosidic bond linking the straight-chain polysaccharide, the chemical known as (1,4)-2-amino-2-deoxy-β-D-glucose, its structure similar to cellulose, chitosan is non-toxic, has good biocompatibility and degradability. Chitosan microspheres can selectively gathered to the tumor cells and play its anti-tumor effects.Doxorubicin is the anthracycline antitumor antibiotics, because of its broad spectrum anti-tumor drug whose main side effects is its cardiac toxicity. It Can lead to severe myocardial injury and heart failure, and the extent of damage is closely related to dose.to The main method of reducing their cardiac toxicity is the application of drug delivery,biodistribution changes to reduce the distribution in heart tissue, increasing local dose in tumor.
This study use CS as a raw material used in this study.Using W/O type emulsion-Curing Preparation epirubicin-chitosan microspheres to detect physical and chemical properties of microspheres,by a simple injection to treat tumors in mice liver tumor. It was observed by treating tumor proliferation. After that, we do next experiment. It is combined with microwave ablation to explore the combined effect in treating liver tumor, and to find a new combination therapy against liver cancer.
Objective:
1.Explore the drug-loaded chitosan microspheres as anti-cancer drugs combined with microwave treatment of hepatic tumor is feasible.
2.Preparation of the load model drug epirubicin (EPI) of the chitosan microspheres (CS) and to detect drug-loaded microspheres and in vitro release properties of the particle size.
3.Pairs of mice in vivo tumor inhibition test to prove that the load epirubicin-chitosan microspheres as compared with ordinary anti-cancer drugs have a better anti-tumor effect in vivo, is more suitable as a carrier of chemotherapeutic drugs.
4.Chitosan microspheres combined with microwave treatment of transplanted tumors in mice to prove that microwave therapy alone compared with other method the tumor inhibition rate compared to higher.
Methods:
1.Epirubicin-loaded chitosan microspheres were prepared by using emulsion-chemical cross linking technique.
2.The surface morphology and particles size of chitosan microspheres were observed by scanning electron microscope. Ultraviolet spectrophotometer was used to analyze the entrapment efficiency, entrapment efficiency and cumulative release rates of epirubicin-loaded chitosan microspheres.
3.Totally 24 mice with transplanted subcutaneous H22 HCC were divided into 4 groups, which were respectively treated by microwave coagulation therapy, intratumoral injected with physiological saline after microwave microwave coagulation therapy, intratumoral injected with epirubicin after microwave coagulation therapy, intratumoral injected with CS-EPI after microwave coagulation therapy.The tumor inhibitory rate was calculated.
Results:
1.The microspheres'size distribution was Uniform. The average diameter was 105um, The ratio of drug loading was 11% and the entrapment was 80%. The drug cumulative release rate in vitro after 2weeks was 84%.
2.Chitosan microspheres is an effective epirubicin local sustained release formulation, has good anti-tumor effect.
3.Compared with the pure microwave group, the tumour-inhibition rate of other groups were 11%,20%,47%.
Conclusion:
4.1. W/O type emulsion-Preparation of curing Epirubicin-chitosan microspheres shape the rules of uniform particle size distribution with high encapsulation efficiency and good release properties.
2. With chemotherapy alone compared with drug-loaded chitosan microspheres on tumor in vivo anti-tumor effect in mice strong, chitosan microspheres is more suitable as a carrier of new chemotherapy drugs.
3. Microwave combined with local intratumoral injection drug containing chitosan microspheres of treatmenting mice reduces tumor volume, gets a higher inhibition rate; microwave and intratumoral injection of drug delivery microspheres of this treatment model is worthy of further study.There may be an effective treatment method for advanced liver cancer.
引文
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[3]McCarron PA, Woolfson AD, Keating SM. Sustained release of 5-fluorouracil from polymeric nanoparticles. J Pharm Pharmacol.2000;52(12):1451-1459.
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[6]Liu F, Liu L, Li X,et al. Preparation of chitosan-hyaluronate double-walled microspheres by emulsification-coacervation method. J Mater Sci Mater Med. 2007;18(11):2215-2224.
[7]Gupta KC, Jabrail FH. Glutaraldehyde and glyoxal cross-linked chitosan microspheres for controlled delivery of centchroman. Carbohydr Res. 2006;341(6):744-756.
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