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CN103611168A - Composite magnetic nanoparticles for targeted therapy of liver cancer and preparation method thereof

Publication number

CN103611168A

CN103611168A CN201310626550.XA CN201310626550A CN103611168A CN 103611168 A CN103611168 A CN 103611168A CN 201310626550 A CN201310626550 A CN 201310626550A CN 103611168 A CN103611168 A CN 103611168A

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CN

China

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afp

plasmid

hre

magnetic nanoparticles

liver cancer

Prior art date

2013-12-02

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The invention relates to composite magnetic nanoparticles for targeted therapy of liver cancer and a preparation method thereof. The method comprises the following steps: respectively preparing recombinant eukaryon expression plasmid p[HRE]AFP-p53 and polyethyleneimine-modified ferroferric oxide magnetic nanoparticles, evenly mixing the recombinant eukaryon expression plasmid p[HRE]AFP-p53 and polyethyleneimine-modified ferroferric oxide magnetic nanoparticles in a mass ratio of 2:1-16:1, and incubating for 30 minutes to obtain the p[HRE]AFP-p53/polyethyleneimine ferroferric oxide magnetic nanoparticle compound. The compound prepared by the method is used for combination of targeted gene therapy of liver cancer and magnetic fluid thermotherapy, and has specific therapeutic effect for liver cancer in in-vivo and in-vitro therapeutic tests.

技术领域 technical field

本发明属于基因工程领域，具体涉及一种靶向治疗肝癌的复合磁性纳米粒及其制备方法。   The invention belongs to the field of genetic engineering, and in particular relates to a composite magnetic nanoparticle for targeted treatment of liver cancer and a preparation method thereof. the

背景技术 Background technique

肝细胞癌（Hepatocellular  carcinoma, HCC）是常见的恶性肿瘤，每年全世界发病率为5.5-14.9/10万人，有60-100万人死于肝细胞癌，我国是肝细胞癌发病率最高的国家，每年发病率超过30.3/10万人。肝细胞癌侵袭性强、发病隐匿，大部分患者（80%）不能手术切除，仅能依赖化疗、放疗等治疗方法，而肝细胞癌对很多传统的化疗药物都有耐药性，且肝脏放射耐受量低，治疗效果有限。因此人们逐渐把更多的目光投向肿瘤的第四种治疗模式：生物治疗。生物治疗包括免疫治疗和基因治疗，其中基因疗法被认为有希望能从根源上治愈肿瘤，在肿瘤治疗方面显示出良好的前景。  Hepatocellular carcinoma (HCC) is a common malignant tumor, with an annual incidence of 5.5-14.9 per 100,000 people worldwide, and 600,000-1 million deaths from hepatocellular carcinoma. China has the highest incidence of hepatocellular carcinoma In my country, the annual incidence rate exceeds 30.3/100,000 people. Hepatocellular carcinoma is highly invasive and insidious. Most patients (80%) cannot be surgically removed and can only rely on chemotherapy, radiotherapy and other treatment methods. However, hepatocellular carcinoma is resistant to many traditional chemotherapeutic drugs, and liver radiation The tolerated dose is low and the therapeutic effect is limited. Therefore, people are gradually turning more attention to the fourth treatment mode of tumors: biological therapy. Biological therapy includes immunotherapy and gene therapy, among which gene therapy is considered to have the hope of curing tumors from the root, and shows good prospects in tumor treatment. the

常用的治疗基因包括抑癌基因、自杀基因和反义基因。抑癌基因p53是与肿瘤相关性最强的基因，超过50%的人类恶性肿瘤和60%的肝细胞癌中都存在p53的突变，选用野生型p53作为治疗基因，能调节细胞周期,修复DNA,诱导细胞凋亡,抑制肿瘤血管生成，控制肿瘤转移，还有研究显示p53基因治疗可以提高肿瘤组织对热疗、化疗和放疗的敏感性。但是目前基因治疗还不能在临床广泛开展，限制其应用的瓶颈在于其安全性问题，尤其是基因表达不可控制性所引起的正常细胞损伤，通过对基因的表达进行调控，提高基因治疗的靶向性，能最小程度的减少其治疗的毒性和副作用。  Commonly used therapeutic genes include tumor suppressor genes, suicide genes and antisense genes. The tumor suppressor gene p53 is the gene most related to tumors. More than 50% of human malignant tumors and 60% of hepatocellular carcinomas have p53 mutations. Wild-type p53 is used as a therapeutic gene, which can regulate cell cycle and repair DNA , induce apoptosis, inhibit tumor angiogenesis, control tumor metastasis, and studies have shown that p53 gene therapy can improve the sensitivity of tumor tissue to hyperthermia, chemotherapy and radiotherapy. However, at present, gene therapy cannot be widely used clinically. The bottleneck limiting its application lies in its safety, especially the normal cell damage caused by the uncontrollable gene expression. By regulating gene expression, the targeting of gene therapy can be improved. Sex, can minimize the toxicity and side effects of its treatment.

利用肿瘤细胞的特异性启动子，在转录水平实现基因表达的调控，可使治疗基因仅在靶细胞内表达，从而保护基因治疗中正常的组织和细胞不受损伤。使用AFP（alpha-fetoprotein）基因启动子能靶向AFP阳性的肝细胞癌，但AFP启动子较弱，介导的治疗体系很难达到理想的治疗效果，且在AFP阴性以及低AFP表达的HCC细胞中仅能介导痕量的基因表达。经研究发现，AFP启动子的表达活性在大程度上依赖于其上游增强子的作用，若将来源于血管内皮细胞生长因子（vascular endothelial growth factor, VEGF）基因的乏氧反应序列（hypoxia responsive element，HRE）作为增强子与AFP启动子5’端融合，于乏氧诱导的条件下，在高AFP和低AFP产生的HCC细胞内都能高效的表达。由于肿瘤组织生长速度远远快于新生血管的速度，瘤体内缺乏血供，人类实体肿瘤包括肝癌中都不同程度的存在乏氧环境，而对于正常细胞，如代表肝脏恢复和储备能力的肝干细胞以及肝脏的基质细胞，即使有微弱的AFP表达，由于不存在乏氧环境，HER及AFP启动子介导的治疗体系对它们没有细胞毒性，可以在很大程度上保留肝脏的修复和储备能力。  Using tumor cell-specific promoters to regulate gene expression at the transcriptional level can enable therapeutic genes to be expressed only in target cells, thereby protecting normal tissues and cells from damage during gene therapy. The AFP (alpha-fetoprotein) gene promoter can be used to target AFP-positive hepatocellular carcinoma, but the AFP promoter is weak, and the treatment system mediated by it is difficult to achieve the desired therapeutic effect, and in HCC with AFP-negative and low AFP expression Only trace amounts of gene expression can be mediated in cells. Studies have found that the expression activity of the AFP promoter largely depends on the function of its upstream enhancer. If the hypoxia response sequence ( hypoxia responsive element , HRE ) as an enhancer fused with the 5' end of the AFP promoter, under the condition of hypoxia induction, it can be expressed efficiently in HCC cells with high AFP and low AFP production. Because the growth rate of tumor tissue is much faster than the speed of new blood vessels, the tumor body lacks blood supply, and human solid tumors, including liver cancer, have hypoxic environments to varying degrees. For normal cells, such as liver stem cells that represent the recovery and reserve capacity of the liver As well as the stromal cells of the liver, even if there is a weak expression of AFP, since there is no hypoxic environment, the treatment system mediated by HER and AFP promoters has no cytotoxicity to them, and the repair and reserve capacity of the liver can be preserved to a large extent.

目前将治疗基因导入体内的运载体包括两类：病毒载体和非病毒载体。病毒载体转运效率高，但是存在不够安全（如产生免疫原性、可能致宿主基因突变等），携带基因量有限，载体功能单一等不足之处。   Currently, there are two types of carriers for introducing therapeutic genes into the body: viral vectors and non-viral vectors. Viral vectors have high transfer efficiency, but they are not safe enough (such as immunogenicity, possible mutation of host genes, etc.), limited number of genes carried, and single function of the vector. the

发明内容 Contents of the invention

技术问题：本发明提供了一种得到用于肝癌的体内外治疗，具有肝癌特异性的治疗效果的靶向治疗肝癌的复合磁性纳米粒的制备方法。  Technical problem: The present invention provides a method for preparing composite magnetic nanoparticles used for in vivo and in vitro treatment of liver cancer and having liver cancer-specific therapeutic effects for targeted therapy of liver cancer.

技术方案：本发明的靶向治疗肝癌的复合磁性纳米粒的制备方法，包括下述步骤：  Technical solution: The preparation method of the composite magnetic nanoparticles for targeted therapy of liver cancer of the present invention comprises the following steps:

（1）质粒p[HRE]AFP-p53的制备：将甲胎蛋白启动子序列插入质粒pCDNA3.1 的MluI-HindIII质粒限制性内切酶位点，得到质粒pCDNA3.1-AFP；将5个连续的乏氧反应序列元件插入质粒pCDNA3.1-AFP的质粒限制性内切酶M1uI位点，得到质粒pCDNA3.1-HRE-AFP；将野生型p53片段亚克隆至质粒pCDNA3.1-HRE-AFP的质粒限制性内切酶EcoRⅠ-XhoⅠ位点，得到质粒p[HRE]AFP-p53； (1) Preparation of plasmid p[HRE]AFP-p53: Insert the alpha-fetoprotein promoter sequence into the MluI-HindIII plasmid restriction endonuclease site of plasmid pCDNA3.1 to obtain plasmid pCDNA3.1-AFP; A continuous hypoxia response sequence element was inserted into the plasmid restriction enzyme M1uI site of plasmid pCDNA3.1-AFP to obtain plasmid pCDNA3.1-HRE-AFP; the wild-type p53 fragment was subcloned into plasmid pCDNA3.1-HRE- The plasmid restriction endonuclease EcoRI-XhoI site of AFP to obtain the plasmid p[HRE]AFP-p53;

（2）制备复合磁性纳米粒：将质粒p[HRE]AFP-p53和聚乙烯亚胺修饰的四氧化三铁磁性纳米粒分别用无血清培养基稀释，然后将两者按照磁性纳米粒与p[HRE]AFP-p53质量比为2：1至16：1混和均匀，孵育后即获得靶向治疗肝癌的复合磁性纳米粒。 (2) Preparation of composite magnetic nanoparticles: Dilute the plasmid p[HRE]AFP-p53 and polyethyleneimine-modified ferric iron tetroxide magnetic nanoparticles with serum-free medium, respectively, and then mix the two with the magnetic nanoparticles and p [HRE] The mass ratio of AFP-p53 is 2:1 to 16:1, mixed evenly, and the composite magnetic nanoparticles for targeted therapy of liver cancer can be obtained after incubation.

本发明方法的一个优选方案的步骤（2）中，磁性纳米粒与p[HRE]AFP-p53质量比为8：1。  In step (2) of a preferred embodiment of the method of the present invention, the mass ratio of magnetic nanoparticles to p[HRE]AFP-p53 is 8:1. the

本发明的靶向治疗肝癌的复合磁性纳米粒，是按照上述方法制备得到。  The composite magnetic nanoparticles for targeted treatment of liver cancer of the present invention are prepared according to the above method. the

有益效果：本发明与现有技术相比，具有以下优点：  Beneficial effect: compared with the prior art, the present invention has the following advantages:

试验证实，在本发明中，受到甲胎蛋白启动子介导和乏氧反应序列增强的p53蛋白仅仅局限在肝癌细胞内表达，所以对人体正常细胞和组织不会产生毒副作用，与其它的基因治疗方案相比，本发明的安全性和可控性极大提高。与其它靶向治疗方案相比，本发明不仅可以靶向原发肝细胞癌的病灶，对于原发性肝癌的转移灶也有靶向治疗作用。在本发明中，使用的基因载体是磁性四氧化三铁纳米粒，相比较于常用的病毒载体，纳米载体具有很有优势：其一，安全性高，可反复注射而不产生抗原性，不会导致细胞的转化。第二，携带基因数量多，对非增殖期的细胞也能有效转染,能与其他功能性材料相复合。第三。研究表明磁性纳米颗粒制备简单，生物相容性好，转染效率高，易于表面修饰，能在交变磁场中可控升温，可用于肿瘤的磁流体热疗。并且，随着纳米生物技术的发展，纳米粒子作为探针和造影剂被用于肿瘤的分子成像，可以在很大程度上提高肿瘤显像的灵敏度，对肿瘤的早期诊断具有重大的意义。因此，同时具备成像和基因转运功能的纳米粒子将能实现肿瘤的诊断和治疗相结合。此外，本发明可以在一个系统内实现靶向基因治疗与肿瘤磁流体热疗的结合。体内外试验数据显示，相对于单一治疗，肿瘤的磁流体热疗联合靶向基因治疗具有最大的肿瘤抑制率，取得的治疗效果要远远优于单一疗法。  Experiments have confirmed that in the present invention, the p53 protein mediated by the alpha-fetoprotein promoter and enhanced by the hypoxic response sequence is only expressed in liver cancer cells, so it will not produce toxic side effects on normal cells and tissues of the human body. Compared with the treatment scheme, the safety and controllability of the present invention are greatly improved. Compared with other targeted treatment schemes, the present invention not only can target the focus of primary liver cell carcinoma, but also has a targeted treatment effect on the metastatic focus of primary liver cancer. In the present invention, the gene carrier used is magnetic iron ferric oxide nanoparticles. Compared with commonly used viral vectors, nanocarriers have great advantages: first, they are highly safe and can be injected repeatedly without generating antigenicity and lead to cell transformation. Second, it carries a large number of genes, can effectively transfect non-proliferating cells, and can be combined with other functional materials. third. Studies have shown that magnetic nanoparticles are easy to prepare, have good biocompatibility, high transfection efficiency, are easy to modify the surface, can controllably heat up in an alternating magnetic field, and can be used for magnetic fluid hyperthermia of tumors. Moreover, with the development of nanobiotechnology, nanoparticles are used as probes and contrast agents for molecular imaging of tumors, which can greatly improve the sensitivity of tumor imaging, which is of great significance to the early diagnosis of tumors. Therefore, nanoparticles with both imaging and gene transfer functions will be able to realize the combination of tumor diagnosis and treatment. In addition, the present invention can realize the combination of targeted gene therapy and tumor magnetic fluid hyperthermia in one system. In vivo and in vitro test data show that compared with monotherapy, magnetic fluid hyperthermia therapy combined with targeted gene therapy has the largest tumor inhibition rate, and the therapeutic effect is far better than monotherapy. the

附图说明 Description of drawings

图1是肝癌靶向复合磁性纳米颗粒介导肝细胞癌选择性的细胞毒效应示意图；  Figure 1 is a schematic diagram of the hepatocellular carcinoma selective cytotoxic effect mediated by liver cancer-targeted composite magnetic nanoparticles;

图中，HepG2 (产甲胎蛋白) 和SMMC7721 (不产甲胎蛋白)是肝癌细胞系。Lovo (肠癌细胞) 和 L929 (成纤维细胞)是非肝癌细胞系。所有的细胞系的细胞经复合磁性纳米粒转染后72小时后，计算细胞相对增值率（RPR %）。 In the figure, HepG2 (alpha-fetoprotein-producing) and SMMC7721 (non-alpha-fetoprotein-producing) are liver cancer cell lines. Lovo (colon carcinoma cells) and L929 (fibroblasts) are non-hepatoma cell lines. 72 hours after the cells of all cell lines were transfected with the composite magnetic nanoparticles, the relative cell proliferation rate (RPR %) was calculated.

图2是体内抑瘤试验的分组效果图；  Figure 2 is a grouping effect diagram of the tumor suppression test in vivo;

图中，每个治疗组的瘤块质量以 mean ± SD (n=5)的形式表示，*治疗组相对阴性对照组 p<0.001。 In the figure, the mass of tumor mass in each treatment group is expressed in the form of mean ± SD (n=5), *p<0.001 in the treatment group versus the negative control group.

图3是体内移植瘤经热疗联合基因治疗后细胞的超微结构TEM图；  Figure 3 is a TEM image of the ultrastructural cells of transplanted tumors in vivo after hyperthermia combined with gene therapy;

图中可见细胞中染色质聚集，边聚，典型的凋亡小体形成。 In the figure, it can be seen that the chromatin in the cell is aggregated and edge-assembled, and typical apoptotic bodies are formed.

具体实施方式 Detailed ways

下面通过实施例对本发明做进一步具体说明。  The present invention will be further specifically described below by way of examples. the

本发明用于肝细胞癌的靶向基因治疗与热疗的联合治疗，在具体实施例中，不论是体外细胞试验还是体内肿瘤抑制试验的数据，都证明了该靶向肝癌的磁性纳米粒具有对肝癌组织和细胞的特异性抑制作用，同时结合磁流体热疗，具有协同治疗效果，肿瘤抑制率优于单一治疗方法。  The present invention is used for combined treatment of targeted gene therapy and hyperthermia for hepatocellular carcinoma. In specific embodiments, whether it is the data of in vitro cell test or in vivo tumor suppression test, it has been proved that the magnetic nanoparticles targeting liver cancer have The specific inhibitory effect on liver cancer tissues and cells, combined with magnetic fluid hyperthermia, has a synergistic therapeutic effect, and the tumor inhibition rate is better than that of a single treatment. the

实施例1：  Example 1:

1. 复合磁性纳米粒的制备 1. Preparation of Composite Magnetic Nanoparticles

（1）质粒p[HRE]AFP-p53的制备：p[HRE]AFP-p53为一种重组真核表达质粒的命名，其中HRE是指代乏氧反应序列元件，在质粒表达中起到增强子的作用，AFP是指代甲胎蛋白启动子，作用是介导下游的抑癌基因p53（一种基因的名称）仅能在肝癌细胞内表达。甲胎蛋白启动子序列通过碱基合成法制备，合成的序列两端分别含有质粒限制性内切酶MluI（MluI是内切酶的名称，以下相同）和HindIII位点，分别将真核表达质粒pCDNA3.1（pCDNA3.1是一个常用的基因克隆质粒的名称，最早由西方生物公司合成并应用，目前尚没有对应的中文名称）和甲胎蛋白启动子片段用内切酶MluI-HindIII双酶切后，纯化的片段用质粒连接酶连接，既将甲胎蛋白启动子序列插入质粒pCDNA3.1的MluI-HindIII质粒限制性内切酶位点，替换原有的启动子，得到质粒pCDNA3.1-AFP；含有5个连续的乏氧反应序列元件也采用碱基合成法合成，两端均有质粒限制性内切酶M1uI的酶切位点，将其和质粒pCDNA3.1-AFP均用M1uI酶切后，纯化片段用质粒连接酶连接，即将5个连续的乏氧反应序列元件插入质粒pCDNA3.1-AFP的质粒限制性内切酶M1uI位点，得到质粒pCDNA3.1-HRE-AFP；将野生型p53片段亚克隆至质粒pCDNA3.1-HRE-AFP的质粒限制性内切酶EcoRⅠ-XhoⅠ位点，得到质粒p[HRE]AFP-p53，经酶切鉴定及测序分析，合成质粒序列正确。 (1) Preparation of plasmid p[HRE]AFP-p53: p[HRE]AFP-p53 is the name of a recombinant eukaryotic expression plasmid, where HRE refers to the hypoxia response sequence element, which enhances the expression of the plasmid AFP refers to the alpha-fetoprotein promoter, which mediates the downstream tumor suppressor gene p53 (the name of a gene) that can only be expressed in liver cancer cells. The alpha-fetoprotein promoter sequence was prepared by base synthesis, and the two ends of the synthesized sequence contained plasmid restriction endonuclease MluI (MluI is the name of the endonuclease, the same below) and HindIII sites, respectively, and the eukaryotic expression plasmid pCDNA3.1 (pCDNA3.1 is the name of a commonly used gene cloning plasmid, which was first synthesized and applied by Western Biological Company, and there is no corresponding Chinese name at present) and alpha-fetoprotein promoter fragment with endonuclease MluI-HindIII double enzyme After cleavage, the purified fragments were ligated with plasmid ligase, and the alpha-fetoprotein promoter sequence was inserted into the MluI-HindIII plasmid restriction endonuclease site of plasmid pCDNA3.1 to replace the original promoter to obtain plasmid pCDNA3.1 -AFP; Containing 5 consecutive hypoxia response sequence elements also synthesized by base synthesis method, both ends of the plasmid restriction endonuclease M1uI restriction endonuclease site, it and the plasmid pCDNA3.1-AFP are used M1uI After digestion, the purified fragment was ligated with plasmid ligase, that is, five consecutive hypoxia response sequence elements were inserted into the plasmid restriction endonuclease M1uI site of plasmid pCDNA3.1-AFP to obtain plasmid pCDNA3.1-HRE-AFP; The wild-type p53 fragment was subcloned into the plasmid restriction endonuclease EcoRI-XhoI site of the plasmid pCDNA3.1-HRE-AFP to obtain the plasmid p[HRE]AFP-p53, which was identified by enzyme digestion and sequencing analysis, and the plasmid sequence was synthesized correct.

（2）聚乙烯亚胺修饰的四氧化三铁纳米磁粒的制备：用化学共沉淀法制备四氧化三铁磁性纳米颗粒：在氮气保护下，于250毫升容积的三口烧瓶中将氯化亚铁(1.0摩尔每升)和氯化铁(1.0摩尔每升)以摩尔比5：3的比例溶于150毫升纯水，300转/分边搅拌边加入氨水调整溶液pH值至9.5±0.1，50℃反应30分钟后得到磁性四氧化三铁纳米粒，磁分离，水洗至pH为7.0，真空冷冻干燥。0.5克磁性纳米颗粒溶于50ml纯水中，超声分散60分钟后，逐滴滴入2ml 体积比为20%的聚乙烯亚胺水溶液，得到聚乙烯亚胺修饰的四氧化三铁纳米磁粒。  (2) Preparation of ferroferric oxide magnetic nanoparticles modified by polyethyleneimine: preparation of ferroferric oxide magnetic nanoparticles by chemical co-precipitation method: under the protection of nitrogen, FeCl Iron (1.0 moles per liter) and ferric chloride (1.0 moles per liter) were dissolved in 150 milliliters of pure water at a molar ratio of 5:3, and ammonia water was added while stirring at 300 rpm to adjust the pH value of the solution to 9.5±0.1, After reacting at 50° C. for 30 minutes, magnetic ferric oxide nanoparticles were obtained, which were separated by magnetic field, washed with water until the pH was 7.0, and vacuum freeze-dried. Dissolve 0.5 g of magnetic nanoparticles in 50 ml of pure water, and after ultrasonic dispersion for 60 minutes, add 2 ml of polyethyleneimine aqueous solution with a volume ratio of 20% dropwise to obtain polyethyleneimine-modified iron ferric oxide nanoparticles. the

（3）制备复合磁性纳米粒：将质粒p[HRE]AFP-p53和聚乙烯亚胺修饰的四氧化三铁磁性纳米粒分别用无血清培养基稀释，然后将两者按照磁性纳米粒与p[HRE]AFP-p53质量比为8：1混和均匀，孵育后即获得靶向治疗肝癌的复合磁性纳米粒。  (3) Preparation of composite magnetic nanoparticles: Dilute the plasmid p[HRE]AFP-p53 and polyethyleneimine-modified ferric iron tetroxide magnetic nanoparticles with serum-free medium respectively, and then mix the two with the magnetic nanoparticles and p [HRE] The mass ratio of AFP-p53 is 8:1, mixed evenly, and the composite magnetic nanoparticles for targeted therapy of liver cancer can be obtained after incubation. the

2. 纳米材料的表征：  2. Characterization of nanomaterials:

磁性纳米材料用透射电镜（SEM,HITACHI-600）和扫描电镜(SEM,JEOLJSM-6360LV)观察形貌、粒径、分散性；扫描电镜能谱仪（SEM-EDS，GENESIS2000XMS60）对材料成分进行分析；用傅立叶红外光谱分析仪（FTIR,Nicolet 560）分析表面官能团的变化；用X射线衍射仪（ARL-X’TRA）分析样品的晶体结构；结果显示磁性纳米粒构建成功。 Magnetic nanomaterials were observed with transmission electron microscope (SEM, HITACHI-600) and scanning electron microscope (SEM, JEOLJSM-6360LV) for morphology, particle size, and dispersion; scanning electron microscope energy spectrometer (SEM-EDS, GENESIS2000XMS60) was used to analyze the composition of the material ; The change of surface functional groups was analyzed by Fourier transform infrared spectrometer (FTIR, Nicolet 560); the crystal structure of the sample was analyzed by X-ray diffractometer (ARL-X'TRA); the results showed that the magnetic nanoparticles were constructed successfully.

3. 纳米粒子DNA结合能力的检测：将纳米粒子与质粒DNA按0：1，3：1，5：1，10：1的质量比混合，终体积50μl，质粒浓度恒定在0.01μg/μl,室温静置结合30分钟后，用琼脂糖凝胶电泳检测结合情况，以未加材料的等浓度质粒作为对照，若材料与质粒充分结合将能阻止质粒电泳，即电泳抑制试验阳性。结果显示，在纳米粒子与质粒DNA质量比高于2：1之后，质粒电泳均被抑制，表明以高于纳米粒子和质粒质量比2：1的比例混合时，质粒均能被纳米粒子完全结合。  3. Detection of DNA binding ability of nanoparticles: mix nanoparticles and plasmid DNA at a mass ratio of 0:1, 3:1, 5:1, 10:1, the final volume is 50 μl, and the concentration of plasmid is constant at 0.01 μg/μl. After binding at room temperature for 30 minutes, use agarose gel electrophoresis to detect the binding situation. The equal concentration of plasmid without material is used as a control. If the material is fully combined with the plasmid, it will prevent the plasmid from electrophoresis, that is, the electrophoresis inhibition test is positive. The results showed that plasmid electrophoresis was inhibited after the mass ratio of nanoparticles to plasmid DNA was higher than 2:1, indicating that plasmids could be completely bound by nanoparticles when mixed at a ratio higher than the mass ratio of nanoparticles to plasmid DNA of 2:1 . the

4. 体外热动力学试验：将测试材料用生理盐水配置成浓度为1.0mg/mL的磁流体溶液，各取5ml加入直径25mm的平底试管中，置于230kHZ，30A的SPG-06A高频磁感应加热设备平板线圈上加热1h，起始室温25℃，试管底距线圈中心0.5cm，每5分钟用TM902C数字测温器测温一次，绘制磁流体的升温曲线图。实验结果显示，前30分钟，温度上升速度很快，30分钟时达到42摄氏度，然后在接下来的30分钟内，温度仅有缓慢上升，稳定在44摄氏度左右，显示出磁性纳米粒子在交变磁场中升温的可控性，用于体内肿瘤热疗时，可以达到有效温度，且不会过度加温伤害正常组织。  4. In vitro thermodynamic test: Prepare the test material with physiological saline to form a magnetic fluid solution with a concentration of 1.0mg/mL, take 5ml each into a flat-bottomed test tube with a diameter of 25mm, and place it in 230kHZ, 30A SPG-06A high-frequency magnetic induction The heating equipment is heated on a flat coil for 1h, the initial room temperature is 25°C, the bottom of the test tube is 0.5cm away from the center of the coil, and the temperature is measured with a TM902C digital thermometer every 5 minutes, and the temperature rise curve of the magnetic fluid is drawn. The experimental results show that the temperature rises rapidly in the first 30 minutes, reaching 42 degrees Celsius in 30 minutes, and then in the next 30 minutes, the temperature only rises slowly and stabilizes at around 44 degrees Celsius, showing that the magnetic nanoparticles are alternating. The controllability of the temperature rise in the magnetic field can reach an effective temperature when used in the hyperthermia of tumors in the body, and the normal tissue will not be damaged by excessive heating. the

5. 体外靶向肝癌的治疗试验：  5. In vitro targeted liver cancer treatment test:

选用肝癌细胞系HepG2 (产甲胎蛋白) 和SMMC7721 (不产甲胎蛋白)。非肝癌细胞系Lovo (肠癌细胞) 和 L929 (成纤维细胞)进行试验。所有的细胞系的细胞经复合磁性纳米粒转染后72小时后，计算细胞相对增值率（RPR %）。试验结果显示，该复合磁性纳米粒仅对肝癌细胞HepG2  和SMMC7721的增殖有抑制作用，且该抑制作用可以被乏氧培养诱导增强，而对非肝癌细胞没有显示出明显的抑制作用。该试验显示出复合磁性纳米粒对肝癌的治疗效果是具有靶向性的。 The liver cancer cell lines HepG2 (producing alpha-fetoprotein) and SMMC7721 (not producing alpha-fetoprotein) were selected. Non-hepatoma cell lines Lovo (intestinal cancer cells) and L929 (fibroblasts) were tested. 72 hours after the cells of all cell lines were transfected with the composite magnetic nanoparticles, the relative cell proliferation rate (RPR %) was calculated. The test results showed that the composite magnetic nanoparticles only had an inhibitory effect on the proliferation of liver cancer cells HepG2 and SMMC7721, and the inhibitory effect could be induced and enhanced by hypoxic culture, but did not show significant inhibitory effect on non-liver cancer cells. This test shows that the therapeutic effect of composite magnetic nanoparticles on liver cancer is targeted.

6.体外联合治疗试验：  6. In vitro combination therapy test:

将HepG2细胞分为阴性对照组，单独热疗组，单独基因治疗组，热疗联合基因治疗组，阴性对照组的细胞不采用任何操作，各处理组细胞MTT吸光度和对照组的比值为增值率，并用流式细胞检测凋亡细胞率，与空白对照组相比较。体外靶向试验结果显示，热疗联合基因治疗组细胞增殖抑制率为76.11%，单独热疗组为35.22%，单独基因治疗组为50.18%。联合治疗组在体外试验中显示出良好的协同效应。 The HepG2 cells were divided into negative control group, hyperthermia alone group, gene therapy alone group, hyperthermia combined with gene therapy group, and the cells in the negative control group were not subjected to any operation, and the ratio of the MTT absorbance of cells in each treatment group to the control group was the value-added rate , and the rate of apoptotic cells was detected by flow cytometry, compared with the blank control group. The results of in vitro targeting experiments showed that the cell proliferation inhibition rate was 76.11% in the hyperthermia combined with gene therapy group, 35.22% in the hyperthermia alone group, and 50.18% in the gene therapy alone group. The combination therapy group showed a good synergistic effect in the in vitro test.

7. 荷瘤模型动物的建立：  7. Establishment of tumor-bearing model animals:

取培养的HepG2细胞，经计数后调整浓度为6.0×10⁶细胞/ml，裸鼠称重后，皮下注射细胞培养液0.2ml，所有裸鼠在SPF条件下的层流架中饲养。试验后观察动物存活量和生理状态。 The cultured HepG2 cells were taken, and the concentration was adjusted to 6.0×10 ⁶ cells/ml after counting. After the nude mice were weighed, 0.2 ml of cell culture medium was subcutaneously injected, and all nude mice were raised in a laminar flow rack under SPF conditions. After the test, the survival rate and physiological state of the animals were observed.

8. 体内基因治疗联合热疗：  8. In vivo gene therapy combined with hyperthermia:

荷瘤动物手术两周后按体外试验分组，分为阴性对照组，单独热疗组，单独基因治疗组，热疗联合基因治疗组，每组5只裸鼠，治疗后6周，各组动物活杀，剥离瘤体称重，取平均值。结果显示，联合治疗组对肿瘤生长抑制率为85.18%，单独基因治疗组抑制率为42.85%，单独热疗组抑制率为46.93%。体内试验的结果与体外试验结果相符合。  Two weeks after the operation, the tumor-bearing animals were grouped according to the in vitro test, and divided into negative control group, hyperthermia alone group, gene therapy alone group, hyperthermia combined with gene therapy group, 5 nude mice in each group, and 6 weeks after treatment, the animals in each group were They were killed alive, the tumors were stripped off and weighed, and the average value was taken. The results showed that the tumor growth inhibition rate was 85.18% in the combination therapy group, 42.85% in the gene therapy group alone, and 46.93% in the hyperthermia group alone. The results of the in vivo test were consistent with those of the in vitro test. the

实施例2： Example 2:

基本步骤和流程同实施例1，不同之处在于，制备复合磁性纳米粒时：将质粒p[HRE]AFP-p53和聚乙烯亚胺修饰的四氧化三铁磁性纳米粒分别用无血清培养基稀释，然后将两者按照磁性纳米粒与p[HRE]AFP-p53质量比为2：1混和均匀，孵育后即获得靶向治疗肝癌的复合磁性纳米粒。 The basic steps and process are the same as in Example 1, except that when preparing composite magnetic nanoparticles: the ferroferric oxide magnetic nanoparticles modified with plasmid p[HRE]AFP-p53 and polyethyleneimine are respectively used in serum-free medium Dilute, and then mix the two evenly according to the mass ratio of magnetic nanoparticles to p[HRE]AFP-p53 of 2:1, and obtain composite magnetic nanoparticles for targeted treatment of liver cancer after incubation.

其余实施部分与实施例1相同。  All the other implementation parts are the same as in Example 1. the

实施例3：  Example 3:

基本步骤和流程同实施例1，不同之处在于，制备复合磁性纳米粒时：将质粒p[HRE]AFP-p53和聚乙烯亚胺修饰的四氧化三铁磁性纳米粒分别用无血清培养基稀释，然后将两者按照磁性纳米粒与p[HRE]AFP-p53质量比为16：1混和均匀，孵育后即获得靶向治疗肝癌的复合磁性纳米粒。 The basic steps and process are the same as in Example 1, except that when preparing composite magnetic nanoparticles: the ferroferric oxide magnetic nanoparticles modified with plasmid p[HRE]AFP-p53 and polyethyleneimine are respectively used in serum-free medium Dilute, and then mix the two evenly according to the mass ratio of magnetic nanoparticles to p[HRE]AFP-p53 of 16:1, and obtain the composite magnetic nanoparticles for targeted treatment of liver cancer after incubation.

其余实施部分与实施例1相同。  All the other implementation parts are the same as in Example 1. the

1.一种靶向治疗肝癌的复合磁性纳米粒的制备方法，其特征在于，该方法包括下述步骤： 1. a method for preparing a composite magnetic nanoparticle for targeted therapy of liver cancer, characterized in that the method may further comprise the steps: （1）质粒p[HRE]AFP-p53的制备：将甲胎蛋白启动子序列插入质粒pCDNA3.1 的MluI-HindIII质粒限制性内切酶位点，得到质粒pCDNA3.1-AFP；将5个连续的乏氧反应序列元件插入质粒pCDNA3.1-AFP的质粒限制性内切酶M1uI位点，得到质粒pCDNA3.1-HRE-AFP；将野生型p53片段亚克隆至质粒pCDNA3.1-HRE-AFP的质粒限制性内切酶EcoRⅠ-XhoⅠ位点，得到质粒p[HRE]AFP-p53； (1) Preparation of plasmid p[HRE]AFP-p53: Insert the alpha-fetoprotein promoter sequence into the MluI-HindIII plasmid restriction endonuclease site of plasmid pCDNA3.1 to obtain plasmid pCDNA3.1-AFP; A continuous hypoxia response sequence element was inserted into the plasmid restriction enzyme M1uI site of plasmid pCDNA3.1-AFP to obtain plasmid pCDNA3.1-HRE-AFP; the wild-type p53 fragment was subcloned into plasmid pCDNA3.1-HRE- The plasmid restriction endonuclease EcoRI-XhoI site of AFP to obtain the plasmid p[HRE]AFP-p53; （2）制备复合磁性纳米粒：将质粒p[HRE]AFP-p53和聚乙烯亚胺修饰的四氧化三铁磁性纳米粒分别用无血清培养基稀释，然后将两者按照磁性纳米粒与p[HRE]AFP-p53质量比为2：1至16：1混和均匀，孵育后即获得靶向治疗肝癌的复合磁性纳米粒。 (2) Preparation of composite magnetic nanoparticles: Dilute the plasmid p[HRE]AFP-p53 and polyethyleneimine-modified ferric iron tetroxide magnetic nanoparticles with serum-free medium, respectively, and then mix the two with the magnetic nanoparticles and p [HRE] The mass ratio of AFP-p53 is 2:1 to 16:1, mixed evenly, and the composite magnetic nanoparticles for targeted therapy of liver cancer can be obtained after incubation. 2.根据权利要求1所述的靶向治疗肝癌的复合磁性纳米粒的制备方法，其特征在于，所述的步骤（2）中，磁性纳米粒与p[HRE]AFP-p53质量比为8：1。 2. The preparation method of composite magnetic nanoparticles for targeted therapy of liver cancer according to claim 1, characterized in that, in the step (2), the mass ratio of magnetic nanoparticles to p[HRE]AFP-p53 is 8 :1. 3.一种靶向治疗肝癌的复合磁性纳米粒，其特征在于，该方法复合磁性纳米粒是按照权利要求1或2所述方法制备得到。 3. A composite magnetic nanoparticle for targeted treatment of liver cancer, characterized in that the composite magnetic nanoparticle is prepared according to the method described in claim 1 or 2.

• 2013
  • 2013-12-02 CN CN201310626550.XA patent/CN103611168A/en active Pending

Application publication date: 20140305

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