技术领域technical field
本发明涉及医药技术领域，具体地说，是一种新的制备载盐霉素的聚乙二醇-神经酰胺胶束的方法，它可以靶向肝癌干细胞，以达到根治肝癌的目的。The invention relates to the technical field of medicine, in particular to a new method for preparing polyethylene glycol-ceramide micelles loaded with salinomycin, which can target liver cancer stem cells to achieve the goal of radically curing liver cancer.
背景技术Background technique
肝癌是全球第五常见的癌症，亦是我国癌症第二大常见的杀手，全球50％以上的肝癌发生在我国。它是临床上最常见的恶性肿瘤之一，目前发病率有逐年上升的趋势。肝癌是高侵袭性的恶行肿瘤，其恶性程度高、病情进展快，且早期无明显症状，导致大多数就诊较晚，使得其治疗难度大、疗效差，病死率高。因此，肝癌的预防和治疗是我国医药研究的一项重要而紧迫的任务。Liver cancer is the fifth most common cancer in the world and the second most common cancer killer in my country. More than 50% of liver cancer in the world occurs in my country. It is one of the most common malignant tumors clinically, and its incidence rate is increasing year by year. Liver cancer is a highly invasive malignant tumor with high malignancy, rapid disease progression, and no obvious symptoms in the early stage, which leads to late treatment for most patients, making its treatment difficult, poor curative effect, and high mortality rate. Therefore, the prevention and treatment of liver cancer is an important and urgent task of medical research in our country.
现今肝癌的治疗手段主要包括外科治疗(即手术切除)、介入治疗、放射治疗、中医中药及免疫治疗，而手术切除至今仍是肝癌治疗的首选方案。对于大部分不能手术切除的中晚期肝癌患者，采用多种方法综合、序贯治疗是目前最有效的措施。但由于个体的特殊性，使得多种手段如何选择，如何搭配从而达到最佳疗效成为目前要解决的问题。目前用于肝癌治疗的一线药物针仅针对已分化的肿瘤细胞，它可以在较短时间内杀灭这类细胞，使肿瘤体积减小甚至消失，然而难以根除肝癌的复发，不能从根源上对肝癌进行治疗。因此治疗后肿瘤复发和转移仍是肝癌治疗中的一大难题。Current treatment methods for liver cancer mainly include surgical treatment (ie, surgical resection), interventional therapy, radiotherapy, traditional Chinese medicine and immunotherapy, and surgical resection is still the first choice for liver cancer treatment. For most patients with unresectable middle-advanced liver cancer, comprehensive and sequential treatment with multiple methods is currently the most effective measure. However, due to the particularity of the individual, how to choose and match various means to achieve the best curative effect has become a problem to be solved at present. The current first-line drug needles for the treatment of liver cancer only target differentiated tumor cells. It can kill such cells in a relatively short period of time and reduce or even eliminate the tumor volume. However, it is difficult to eradicate the recurrence of liver cancer and cannot treat the root causes. liver cancer treatment. Therefore, tumor recurrence and metastasis after treatment are still a major problem in the treatment of liver cancer.
肿瘤干细胞(CSCs)是肿瘤组织中存在的一小部分具有高度增殖能力与自我更新能力的细胞群体，是能产生肿瘤细胞的一个亚群，在肿瘤的发生、转移、复发中起着关键作用。因此，在肿瘤的治疗过程中对肿瘤干细胞的作用是非常关键的。尽管目前研究者们对此提出了很多策略，肿瘤干细胞的消除仍然是很困难的。传统的化疗药物如多柔比星和紫杉醇可以杀死多数肿瘤细胞，但是对肿瘤干细胞作用极弱甚至耐药，从而导致肿瘤中肿瘤干细胞比例的升高。研究人员通过高通量筛选等方法鉴定出几种旧的药物(如盐霉素、二甲双胍、姜黄素、戒酒硫)具有对抗肿瘤干细胞的作用。本方法中选取盐霉素作为抗肿瘤干细胞药物，将其包载于聚乙二醇-神经酰胺胶束中，有效杀伤肿瘤干细胞。Cancer stem cells (CSCs) are a small group of cells with high proliferative ability and self-renewal ability existing in tumor tissues. Therefore, the role of tumor stem cells in the treatment of tumors is very critical. Although researchers have proposed many strategies, the elimination of cancer stem cells is still very difficult. Traditional chemotherapeutic drugs such as doxorubicin and paclitaxel can kill most tumor cells, but have very weak or even drug-resistant effects on tumor stem cells, resulting in an increase in the proportion of tumor stem cells in tumors. The researchers identified several older drugs (such as salinomycin, metformin, curcumin, disulfiram) that have anti-tumor stem cell effects through methods such as high-throughput screening. In this method, salinomycin is selected as an anti-tumor stem cell drug, and it is encapsulated in polyethylene glycol-ceramide micelles to effectively kill tumor stem cells.
盐霉素是一类广谱抗菌药，临床上主要用于预防和治疗家畜、家禽的球虫病，还常作为饲料添加剂在动物生产中长期应用。盐霉素(Salinomycin,SAL)又叫沙利霉素、优素精、萨里诺马辛，它是由白色链球菌(Streptomycesaldus)经发酵培养产生的一种一元羧酸聚醚类离子载体型抗生素类，具有杀菌、抑菌、抗球虫的作用,对大多数革兰氏阳性菌有抑制作用。最近研究显示，盐霉素不仅可以用于牲畜的抗球虫病，对乳腺肿瘤干细胞也具有很强的杀伤作用。2009年，《Cell》上发表文章，发现从16,000个化合物中筛选得到的盐霉素能高选择性杀死小鼠身上的人乳腺癌CSC，并且其效力比传统化疗药物紫杉醇高100倍。后续研究发现，盐霉素不仅对乳腺癌CSC具有杀灭作用，对其他许多肿瘤干细胞(肝癌，前列腺癌)均具有杀灭作用。盐霉素成为一种广谱抗肿瘤干细胞药物，受到研究人员的广泛关注，表现出很强的临床应用前景。Salinomycin is a kind of broad-spectrum antibacterial drug, which is mainly used clinically to prevent and treat coccidiosis in livestock and poultry, and is often used as a feed additive in animal production for a long time. Salinomycin (Salinomycin, SAL), also known as Salinomycin, Yousujing, and Salinomacin, is a monocarboxylic acid polyether ionophore produced by fermentation and culture of Streptococcus albicans (Streptomycesaldus). Antibiotics have bactericidal, bacteriostatic and anticoccidial effects, and have inhibitory effects on most Gram-positive bacteria. Recent studies have shown that salinomycin can not only be used for anti-coccidiosis in livestock, but also has a strong killing effect on breast cancer stem cells. In 2009, an article was published in "Cell", and it was found that salinomycin, which was screened from 16,000 compounds, could kill human breast cancer CSCs in mice with high selectivity, and its potency was 100 times higher than that of the traditional chemotherapy drug paclitaxel. Subsequent studies have found that salinomycin not only has a killing effect on breast cancer CSC, but also has a killing effect on many other tumor stem cells (liver cancer, prostate cancer). Salinomycin has become a broad-spectrum anti-tumor stem cell drug, which has attracted extensive attention from researchers and has shown strong clinical application prospects.
尽管盐霉素是一种有效的抗肿瘤干细胞药物，然而其对普通肿瘤细胞仍然有待加强，而且其毒副作用较大，这两个缺点导致了盐霉素的临床应用受到严重阻碍。联合给药是一种将多种化疗药物进行联合的给药方式，临床治疗常常需要联合给药才能达到提高化疗药物疗效和降低化疗药物毒副作用。而神经酰胺(Ceramide)是一种由鞘氨醇和脂肪酸构成的脂质分子，具有显著的化疗增敏效果。多项研究显示其能显著增强化疗药物的治疗效果，我们前期研究结果显示聚乙二醇化神经酰胺(PEG-ceramide)能形成胶束结构，胶束是一种表面活性剂分子在其浓度超过临界值后形成特殊聚集体的形状与分子在其中的空间排布的纳米药物。在水溶液中，分子的亲水基向外伸向水相，疏水基则以范德华力紧密排列。Although salinomycin is an effective anti-tumor stem cell drug, it still needs to be strengthened against common tumor cells, and its toxic side effects are relatively large. These two shortcomings have seriously hindered the clinical application of salinomycin. Combined administration is a way of administering multiple chemotherapy drugs in combination. Clinical treatment often requires combined administration to improve the efficacy of chemotherapy drugs and reduce the side effects of chemotherapy drugs. Ceramide is a lipid molecule composed of sphingosine and fatty acids, which has a significant chemosensitizing effect. A number of studies have shown that it can significantly enhance the therapeutic effect of chemotherapy drugs. Our previous research results show that PEG-ceramide can form a micelle structure. Nanomedicines that form special aggregate shapes and the spatial arrangement of molecules within them. In aqueous solution, the hydrophilic groups of the molecules extend outward to the water phase, and the hydrophobic groups are closely arranged by van der Waals force.
盐霉素水溶性较差，且直接用药，对机体的毒副作用较强，同时，在体内循环时间也较短，不利于肿瘤治疗。因此迫切需要一种载体能有效的包载游离药物盐霉素、高效渗透肿瘤内部并选择性地杀伤肿瘤细胞，减少非靶向器官的药物积聚，降低对机体的毒副作用，延长体内循环时间。有报道指出，将神经酰胺聚乙二醇化，能增强阿霉素诱导细胞的能力。聚乙二醇-神经酰胺是两亲性分子共聚物，具有亲水和疏水段，容易形成小粒径胶束，因此具有制备成载体包载盐霉素的可能性。而且小粒径胶束(<30nm)不仅可以逃避单核细胞吞噬作用，延长体内循环时间，增加药物在实体瘤的蓄积，还具有肿瘤组织高渗透能力，能有效地渗透入肿瘤组织内部，释放药物达到消灭肿瘤干细胞的目的。以PEG-ceramide为载体包载盐霉素制备成粒径小于30nm的胶束可以加强药物对肿瘤的靶向性与渗透性，消灭肝癌细胞和干细胞。Salinomycin is poor in water solubility and has strong toxic and side effects on the body when administered directly. At the same time, the circulation time in the body is also short, which is not conducive to tumor treatment. Therefore, there is an urgent need for a carrier that can effectively carry the free drug salinomycin, efficiently penetrate into the tumor and selectively kill tumor cells, reduce the accumulation of drugs in non-target organs, reduce the toxic and side effects on the body, and prolong the circulation time in the body. It has been reported that pegylation of ceramide can enhance the ability of doxorubicin to induce cells. Polyethylene glycol-ceramide is an amphiphilic molecular copolymer with hydrophilic and hydrophobic segments, and it is easy to form small-sized micelles, so it has the possibility of being prepared as a carrier to load salinomycin. Moreover, small-sized micelles (<30nm) can not only escape the phagocytosis of monocytes, prolong the circulation time in the body, increase the accumulation of drugs in solid tumors, but also have high permeability of tumor tissues, which can effectively penetrate into tumor tissues and release Drugs achieve the purpose of destroying tumor stem cells. Using PEG-ceramide as a carrier to load salinomycin into micelles with a particle size of less than 30nm can enhance the targeting and penetration of drugs to tumors, and eliminate liver cancer cells and stem cells.
中国专利文献CN104257628A公开一种载盐霉素胶束及其制备方法和应用，是以DSPE-PEG2000-CRGDK共聚物为载体，包载盐霉素钠的胶束。目前尚无文献报道以PEG-ceramide为载体包载盐霉素制备的胶束针对肝癌细胞和干细胞进行靶向给药。Chinese patent document CN104257628A discloses a salinomycin-loaded micelle and its preparation method and application, which use DSPE-PEG2000-CRGDK copolymer as a carrier and salinomycin sodium-loaded micelle. At present, there is no literature report that the micelles prepared with salinomycin loaded with PEG-ceramide as the carrier can carry out targeted drug delivery on liver cancer cells and stem cells.
发明内容Contents of the invention
本发明的目的在于提供一种新的载盐霉素具有高靶向性及渗透能力的小粒径胶束及其制备方法。本发明的另一个目的是提供该胶束在制备治疗肝癌药物中的应用。The purpose of the present invention is to provide a new salinomycin-loaded small particle size micelle with high targeting and penetrating ability and a preparation method thereof. Another object of the present invention is to provide the application of the micelles in the preparation of drugs for treating liver cancer.
本发明选择新型抗肿瘤药物盐霉素，将其用PEG-ceramide包载用于肝癌的靶向药物治疗。In the present invention, a novel antitumor drug salinomycin is selected, and PEG-ceramide is used to carry it for targeted drug treatment of liver cancer.
本发明的技术方案包括：Technical scheme of the present invention comprises:
一、新型载盐霉素的聚乙二醇-神经酰胺胶束的制备；1. Preparation of novel salinomycin-loaded polyethylene glycol-ceramide micelles;
二、新型载药胶束体外穿透肝癌干细胞能力的检测；2. Detection of the ability of the new drug-loaded micelles to penetrate liver cancer stem cells in vitro;
三、载药胶束体外抗肝癌干细胞活性。3. Anti-liver cancer stem cell activity of drug-loaded micelles in vitro.
本发明的第一方面，提供一种载盐霉素的聚乙二醇-神经酰胺胶束，该胶束为：以聚乙二醇-神经酰胺为载体，包载盐霉素，粒径为11±3nm(最优为11nm)的胶束，所述的盐霉素与载体摩尔比为1:4。The first aspect of the present invention provides a polyethylene glycol-ceramide micelle loaded with salinomycin. 11±3nm (optimally 11nm) micelles, the molar ratio of salinomycin to carrier is 1:4.
本发明的第二方面，提供一种上述载盐霉素的聚乙二醇-神经酰胺胶束的制备方法，包括以下步骤：将聚乙二醇-神经酰胺溶解于氯仿中，另取盐霉素，溶解于甲醇中，将两种溶液混合，37℃下减压旋转蒸发除去有机溶剂，形成干燥薄膜；向旋转蒸发瓶中加入pH7.4的磷酸盐缓冲液并充满氮气，水化30min；过200nm的聚碳酸酯膜去除未包封的盐霉素。The second aspect of the present invention provides a method for preparing the above-mentioned polyethylene glycol-ceramide micelles loaded with salinomycin, comprising the following steps: dissolving polyethylene glycol-ceramide in chloroform, and taking another salinomycin Dissolve the two solutions in methanol, mix the two solutions, and remove the organic solvent by rotary evaporation under reduced pressure at 37°C to form a dry film; add pH 7.4 phosphate buffer solution to the rotary evaporation flask and fill it with nitrogen, and hydrate for 30 minutes; Unencapsulated salinomycin was removed by passing through a 200 nm polycarbonate membrane.
较优的，包括以下步骤：将5mg的聚乙二醇-神经酰胺溶解于3ml氯仿中，另取0.454mg的盐霉素，溶解于1ml甲醇中，将两种溶液混合，37℃下减压旋转蒸发0.5--2h(最优为2h)除去有机溶剂，形成干燥薄膜。向旋转蒸发瓶中加入pH 7.4的磷酸盐缓冲液并充满氮气，水化30min。过200nm的聚碳酸酯膜去除未包封的盐霉素。Preferably, the following steps are included: dissolving 5 mg of polyethylene glycol-ceramide in 3 ml of chloroform, and dissolving another 0.454 mg of salinomycin in 1 ml of methanol, mixing the two solutions, and depressurizing at 37°C Rotary evaporation for 0.5--2h (optimally 2h) to remove the organic solvent to form a dry film. Add pH 7.4 phosphate buffer solution into the rotary evaporating flask and fill it with nitrogen gas, and hydrate for 30 min. Unencapsulated salinomycin was removed by passing through a 200 nm polycarbonate membrane.
为了寻找最佳盐霉素包封率，将盐霉素与PEG-ceramide载体质量比值进行了优化。优化方法为：制备不同摩尔比例(如1:2、1:3、1:4)的载盐霉素的聚乙二醇-神经酰胺胶束，测其包封率，得出盐霉素与载体摩尔比为1:4时，制备的新型载药胶束盐霉素包封率最佳。In order to find the best encapsulation efficiency of salinomycin, the mass ratio of salinomycin to PEG-ceramide carrier was optimized. The optimization method is: prepare polyethylene glycol-ceramide micelles loaded with salinomycin in different molar ratios (such as 1:2, 1:3, 1:4), measure the encapsulation efficiency, and obtain the salinomycin and salinomycin When the carrier molar ratio was 1:4, the encapsulation efficiency of the new drug-loaded micelles salinomycin was the best.
另外，为将聚乙二醇-神经酰胺和盐霉素分别选择了不同的溶剂，聚乙二醇-神经酰胺是溶解于氯仿中，盐霉素是溶解于甲醇，优点是将两种有机溶剂氯仿和甲醇以3：1的比例混合，能使盐霉素和载体材料形成最佳的分散薄膜，后期用PBS水化后得到的载药胶束包封率较高，粒径分散更均匀。若用其他试剂如乙腈溶解盐霉素也能形成薄膜，但用甲醇溶解结果更佳。水化过程充满氮气，可防止药物氧化；选择pH 7.4的磷酸盐缓冲液，制备所得胶束粒径均一，而且符合动物和人体体内环境。In addition, different solvents were selected for polyethylene glycol-ceramide and salinomycin. Polyethylene glycol-ceramide was dissolved in chloroform, and salinomycin was dissolved in methanol. The advantage is that the two organic solvents Mixing chloroform and methanol at a ratio of 3:1 can make salinomycin and the carrier material form an optimal dispersion film, and the drug-loaded micelles obtained after hydration with PBS in the later stage have a higher encapsulation efficiency and more uniform particle size dispersion. If other reagents such as acetonitrile are used to dissolve salinomycin, a thin film can also be formed, but the result is better when dissolved in methanol. The hydration process is filled with nitrogen to prevent drug oxidation; the phosphate buffer solution with pH 7.4 is selected, and the prepared micelles have a uniform particle size, which is in line with the in vivo environment of animals and humans.
本发明的第三方面，提供上述制备的新型载盐霉素的聚乙二醇-神经酰胺胶束在制备治疗肝癌药物中的应用。The third aspect of the present invention provides the application of the above-mentioned novel salinomycin-loaded polyethylene glycol-ceramide micelles in the preparation of drugs for treating liver cancer.
所述的载盐霉素的聚乙二醇-神经酰胺胶束具体靶向已分化的肝癌干细胞和普通的肝癌细胞。The polyethylene glycol-ceramide micelles loaded with salinomycin specifically target differentiated liver cancer stem cells and common liver cancer cells.
(1)本发明制备所得新型胶束体外高靶向性及渗透能力的检测(1) Detection of high targeting and penetration ability of novel micelles prepared by the present invention in vitro
本发明用激光共聚焦显微镜检测肝癌细胞和肝癌干细胞中对含有荧光物质胶束的摄取能力，来评价胶束体外靶向性及渗透能力。激光共聚焦显微镜可对肝癌干细胞内荧光强弱进行定性分析。The invention uses a laser confocal microscope to detect the uptake ability of micelles containing fluorescent substances in liver cancer cells and liver cancer stem cells to evaluate the in vitro targeting and penetration capabilities of micelles. Laser confocal microscopy can be used to qualitatively analyze the fluorescence intensity in liver cancer stem cells.
(2)本发明制备所得新型载盐霉素胶束体外抗肝癌和肝癌干细胞活性(2) In vitro anti-liver cancer and liver cancer stem cell activity of the novel salinomycin-loaded micelles prepared by the present invention
本发明采用CCK-8法测定细胞在经不同药物浓度处理后细胞存活率，来评价药物和载药纳米粒的细胞毒性。细胞的生存曲线经对数模拟后，计算细胞的IC50来定量比较其细胞毒性大小。The invention adopts the CCK-8 method to measure the cell viability of cells treated with different drug concentrations to evaluate the cytotoxicity of drugs and drug-loaded nanoparticles. After the survival curve of the cells was logarithmically simulated, the IC50 of the cells was calculated to quantitatively compare their cytotoxicity.
本发明优点在于：The present invention has the advantage that:
本发明的新型载盐霉素聚乙二醇-神经酰胺胶束在能够提高化疗药物盐霉素疗效，降低药物毒副作用的同时可达到靶向肝癌干细胞和肝癌细胞的作用，这是一种肝癌治疗的新策略。它利用小粒径胶束载体的优势，制备纳米药物靶向治疗肿瘤，具有广阔的应用前景。The new salinomycin-loaded polyethylene glycol-ceramide micelles of the present invention can improve the curative effect of the chemotherapy drug salinomycin and reduce the toxic and side effects of the drug, and at the same time can achieve the effect of targeting liver cancer stem cells and liver cancer cells. This is a liver cancer New strategies for treatment. It utilizes the advantages of the small particle size micelle carrier to prepare nano-medicine for targeted therapy of tumors, and has broad application prospects.
附图说明Description of drawings
图1.薄膜分散法制备新型的载药PEG-ceramide胶束。Figure 1. Preparation of novel drug-loaded PEG-ceramide micelles by thin-film dispersion method.
图2.载盐霉素的聚乙二醇-神经酰胺胶束的表征，其中，A为胶束的水化粒径分布图；B为胶束的水化电位分布图；C为胶束的透射电镜图片。Figure 2. Characterization of the polyethylene glycol-ceramide micelles loaded with salinomycin, wherein, A is the hydration particle size distribution diagram of the micelles; B is the hydration potential distribution diagram of the micelles; C is the hydration potential distribution diagram of the micelles TEM image.
图3.盐霉素和聚乙二醇-神经酰胺协同比例的筛选。Figure 3. Screening for the synergistic ratio of salinomycin and polyethylene glycol-ceramide.
图4.肝癌细胞HepG2及干细胞摄取PECF的荧光值强度。Figure 4. Fluorescence intensity of PECF uptake by liver cancer cells HepG2 and stem cells.
图5.激光共聚焦检测肝癌和肝癌干细胞对胶束的摄取能力，其中，A为肝癌细胞摄取新型载荧光物质胶束；B为肝癌干细胞摄取新型载荧光物质胶束；C为肝癌干细胞单细胞悬液摄取新型载荧光物质胶束。Figure 5. Laser confocal detection of micelles uptake ability of liver cancer and liver cancer stem cells, in which, A is the uptake of new fluorescent substance-loaded micelles by liver cancer cells; B is the uptake of new fluorescent substance-loaded micelles by liver cancer stem cells; C is single cells of liver cancer stem cells Suspension uptake of novel fluorescent substance-loaded micelles.
图6.载盐霉素胶束及游离盐霉素对肝癌细胞和肝癌干细胞的半数抑制浓度(IC50)，其中，A为载盐霉素胶束及游离盐霉素对肝癌细胞的半数抑制浓度；B为载盐霉素胶束及游离盐霉素对肝癌干细胞的半数抑制浓度。Figure 6. The half-inhibitory concentration (IC50) of salinomycin-loaded micelles and free salinomycin on liver cancer cells and liver cancer stem cells, where A is the half-inhibitory concentration of salinomycin-loaded micelles and free salinomycin on liver cancer cells ; B is the half inhibitory concentration of salinomycin-loaded micelles and free salinomycin to liver cancer stem cells.
具体实施方式Detailed ways
下面结合实施例对本发明提供的具体实施方式作详细说明。The specific implementation modes provided by the present invention will be described in detail below in conjunction with the examples.
材料和仪器Materials and Instruments
激光粒度测定仪，英国马尔文仪器有限公司；Laser particle size analyzer, British Malvern Instruments Co., Ltd.;
R系列旋转蒸发器，上海申生科技有限公司；R series rotary evaporator, Shanghai Shensheng Technology Co., Ltd.;
CRGDK线肽，吉尔生化有限公司；CRGDK line peptide, Gill Biochemical Co., Ltd.;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt)(DSPE-mPEG2000)，Avanti PolarLipids；1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt)(DSPE-mPEG2000), Avanti Polar Lipids;
PEG-ceramide，上海炎怡生物技术有限公司；PEG-ceramide, Shanghai Yanyi Biotechnology Co., Ltd.;
盐霉素，美国Sigma公司；Salinomycin, American Sigma Company;
HPLC色谱纯度>93.8％；HPLC chromatographic purity>93.8%;
透析袋(截留分子量1000)，上海绿鸟生物发展有限公司；Dialysis bag (MWCO 1000), Shanghai Green Bird Biological Development Co., Ltd.;
磷酸盐缓冲液(PBS)，美国Hyclone公司；Phosphate buffer saline (PBS), American Hyclone Company;
乙腈及四氢呋喃为色谱纯，其他试剂均为分析纯；Acetonitrile and tetrahydrofuran are chromatographically pure, and other reagents are analytically pure;
碱性成纤细胞生长因子(bFGF)，以色列prospec公司；Basic fibroblast growth factor (bFGF), Israeli Prospec Company;
内皮生长因子(EGF)、胰岛素，以色列prospec公司；Endothelial growth factor (EGF), insulin, Israeli Prospec Company;
胎牛血清、胰酶、青链双抗、B27添加物，美国Gibico公司；Fetal bovine serum, trypsin, blue chain double antibody, B27 supplement, American Gibico company;
其他化学试剂均购自上海国药集团；Other chemical reagents were purchased from Shanghai Sinopharm Group;
杜氏磷酸缓冲液(D-PBS)，美国Thermo公司；Duchenne's phosphate buffered solution (D-PBS), American Thermo company;
万分之一电子天平AL104，梅特勒托利多公司；1/10,000 electronic balance AL104, Mettler Toledo;
十万分之一电子天平MS205DU，梅特勒托利多公司；1/100,000 electronic balance MS205DU, Mettler Toledo;
透射电子显微镜JEM2100F，日本JEOL公司；Transmission electron microscope JEM2100F, Japan JEOL company;
Zetasizer nano ZS激光粒度分析仪，英国马尔文公司；Zetasizer nano ZS laser particle size analyzer, Malvern, UK;
岛津高效液相色谱仪LC-20A，日本岛津公司。Shimadzu high performance liquid chromatograph LC-20A, Shimadzu Corporation, Japan.
实施例1:载盐霉素的聚乙二醇-神经酰胺胶束的制备Embodiment 1: the preparation of the polyethylene glycol-ceramide micelles of loading salinomycin
将5mg PEG-ceramide溶解于3ml氯仿中，另取0.454mg盐霉素溶解于1ml甲醇中，将上述两种溶液混合，37℃下减压旋转蒸发2h除去有机溶剂，形成干燥薄膜。向瓶中加入2mLpH 7.4的磷酸盐缓冲液并充满氮气，水化30min。过200nm聚碳酸酯膜去除未包封的盐霉素(图1)。Dissolve 5 mg of PEG-ceramide in 3 ml of chloroform, and another 0.454 mg of salinomycin in 1 ml of methanol, mix the above two solutions, and remove the organic solvent by rotary evaporation under reduced pressure at 37 °C for 2 h to form a dry film. Add 2 mL of pH 7.4 phosphate buffer to the bottle and fill it with nitrogen gas, and hydrate for 30 min. Unencapsulated salinomycin was removed by passing through a 200 nm polycarbonate membrane (Figure 1).
通过薄膜分散法制备的胶束粒径在11nm(图2A)，透射电镜(图2C),表明本发明制备的新型载药胶束为球形，且具备典型的核-壳结构。通过该实验表明本发明制备的新型PEG-ceramide自组装胶束具备明确的微观结构。The particle size of the micelles prepared by the film dispersion method was 11nm (Figure 2A), and the transmission electron microscope (Figure 2C) showed that the novel drug-loaded micelles prepared by the present invention were spherical and had a typical core-shell structure. This experiment shows that the novel PEG-ceramide self-assembled micelles prepared by the present invention have a definite microstructure.
事实例2：优化比例的载药胶束的确定Fact example 2: Determination of drug-loaded micelles with optimal ratio
(1)细胞接种：接种肝癌HepG2细胞于96孔板；(1) Cell inoculation: inoculate liver cancer HepG2 cells in a 96-well plate;
(2)实验分组及药物制备：将实验分为1:2，1:3，1:4(盐霉素：聚乙二醇—神经酰胺摩尔比)3组，制备三种比例的载药胶束，分别依次3倍比稀释九个浓度；(2) Experimental grouping and drug preparation: divide the experiment into 3 groups: 1:2, 1:3, 1:4 (salinomycin:polyethylene glycol-ceramide molar ratio), and prepare drug-loaded gels with three ratios Bundle, sequentially 3-fold ratio dilution nine concentrations;
(3)加药：12h待细胞贴壁后，分别将各组各浓度100uL的药物加入96孔板，置于5％CO2，37℃培养箱孵育48h；(3) Dosing: 12 hours after the cells adhered to the wall, add 100 uL of drugs in each group to the 96-well plate, place in 5% CO2 , and incubate at 37°C for 48 hours;
(4)加CCK-8，置于酶标仪中测其OD值，计算细胞杀伤效率；(4) Add CCK-8, place it in a microplate reader to measure its OD value, and calculate the cell killing efficiency;
(5)绘制药物联合指数-细胞杀伤效率图谱：依托Compusyn软件，计算并绘制两种药物的联合指数(Combination Index,CI)值对细胞的杀伤效率(Fraction affected,fa)作图，筛选得到盐霉素(SAL)与聚乙二醇-神经酰胺(PEG-ceramide)对HepG2细胞的协同比例。(5) Draw the drug combination index-cell killing efficiency map: relying on Compusyn software, calculate and draw the combination index (Combination Index, CI) value of the two drugs against the cell killing efficiency (Fraction affected, fa) map, and screen to obtain the salt Synergistic ratio of SAL and PEG-ceramide on HepG2 cells.
通过上述实验，我们得到如图3的CI-fa曲线，可知在1:2和1:4的时候两药有更好的协同杀伤效果。Through the above experiments, we obtained the CI-fa curve shown in Figure 3, which shows that the two drugs have a better synergistic killing effect at the ratio of 1:2 and 1:4.
实施例3:激光共聚焦检测肝癌细胞和肝癌干细胞对包载荧光物质胶束的摄取能力Example 3: Laser confocal detection of uptake ability of liver cancer cells and liver cancer stem cells to micelles loaded with fluorescent substances
(1)含有荧光物质胶束的制备。将400μg PECF荧光物质溶于1ml甲醇中，将5mgPEG-ceramide溶解于3ml氯仿中，采用薄膜分散法制备成含荧光物质的胶束。(1) Preparation of micelles containing fluorescent substances. Dissolve 400 μg of PECF fluorescent substance in 1 ml of methanol, and 5 mg of PEG-ceramide in 3 ml of chloroform, and prepare fluorescent substance-containing micelles by film dispersion method.
(2)肝癌细胞对荧光物质胶束的摄取能力检测：铺5×105个HepG2细胞于激光共聚焦皿中，置于37℃，5％CO2的孵箱内培养过夜。待细胞贴壁后，弃除培养基，分别加入空白培养基(Control)、游离的荧光物质PECF(Free PECF)、载PECF的聚乙二醇-神经酰胺胶束溶液(PECF-PEG-CRM-M)以及载PECF的DSPE-PEG胶束溶液(PECF-DSPE-PEG-M)。药物在37℃孵育2小时后，用PBS润洗两次，加DAPI染色5min，加PBS洗涤两次，最后加入500μLPBS重悬细胞，激光共聚焦观察其荧光强度。(2) Detection of the uptake ability of liver cancer cells to fluorescent substance micelles: 5×105 HepG2 cells were placed in a laser confocal dish, and cultured overnight in an incubator at 37° C. and 5% CO2 . After the cells adhered to the wall, the medium was discarded, and blank medium (Control), free fluorescent substance PECF (Free PECF), PECF-loaded polyethylene glycol-ceramide micellar solution (PECF-PEG-CRM- M) and PECF-loaded DSPE-PEG micellar solution (PECF-DSPE-PEG-M). After the drug was incubated at 37°C for 2 hours, it was washed twice with PBS, stained with DAPI for 5 minutes, washed twice with PBS, and finally 500 μL PBS was added to resuspend the cells, and the fluorescence intensity was observed by confocal laser.
(3)肝癌干细胞对荧光物质胶束的摄取能力检测：取一定量的微球体和微球体单细胞悬液于激光共聚焦皿中，分别加入空白培养基、游离的荧光物质PECF、载PECF的聚乙二醇-神经酰胺胶束溶液以及载PECF的DSPE-PEG胶束溶液，随后置于37℃，5％CO2的孵箱内孵育2小时，用PBS离心洗涤两次，加DAPI染色5min，加PBS洗涤两次，最后加入0.5ml PBS转移到激光共聚焦皿中观察其荧光强度。(3) Detection of the uptake ability of liver cancer stem cells to fluorescent substance micelles: Take a certain amount of microspheres and microsphere single-cell suspension in a laser confocal dish, add blank medium, free fluorescent substance PECF, PECF-loaded The polyethylene glycol-ceramide micelle solution and the PECF-loaded DSPE-PEG micelle solution were then placed in an incubator at 37°C and 5% CO2 for 2 hours, washed twice with PBS, and stained with DAPI for 5 minutes. , add PBS to wash twice, and finally add 0.5ml PBS to transfer to laser confocal dish to observe its fluorescence intensity.
通过比较肝癌细胞及干细胞荧光值(见图4)及激光共聚焦中细胞内荧光亮度(见图5)，可看出肝癌细胞和肝癌干细胞中荧光值明显高于其对照组。且肝癌干细胞的荧光值及荧光亮度明显高于肝癌细胞，说明肝癌干细胞对胶束的摄取能力比肝癌细胞更强。By comparing the fluorescence values of liver cancer cells and stem cells (see Figure 4) and the intracellular fluorescence brightness in laser confocal (see Figure 5), it can be seen that the fluorescence values of liver cancer cells and liver cancer stem cells are significantly higher than those of the control group. Moreover, the fluorescence value and fluorescence brightness of liver cancer stem cells were significantly higher than those of liver cancer cells, indicating that liver cancer stem cells had a stronger ability to absorb micelles than liver cancer cells.
实施例4:载盐霉素胶束体外抗肝癌的活性分析Embodiment 4: the activity analysis of loading salinomycin micelles against liver cancer in vitro
(1)对于贴壁的HepG2细胞株(1) For adherent HepG2 cell lines
将对数期的HepG2细胞经胰酶消化，用含血清培养基重悬制成单细胞悬液，调整细胞密度至3×104个/mL，以3000个/孔的密度接种于96孔板中，即每孔加入100uL的单细胞悬液，接种好的细胞于37℃，5％CO2的条件下培养过夜，待细胞生长到合适密度。HepG2 cells in the logarithmic phase were trypsinized, resuspended with serum-containing medium to make a single cell suspension, adjusted the cell density to 3×104 cells/mL, and seeded in 96 wells at a density of 3000 cells/well Add 100 uL of single cell suspension to each well of the plate, and inoculate the cells at 37°C and 5% CO2 overnight, until the cells grow to a suitable density.
待细胞过夜贴壁后，吸弃每孔培养基，随后向各孔分别加入100μl用培养基稀释的不同浓度的载药胶束溶液，浓度分别为333.333，111.111，37.037，12.346，4.115，1.372，0.457，0.152，0.051μM，每个浓度3个副孔，加药后将96孔板置于二氧化碳恒温培养箱中继续培养48h。同时设不加细胞的空白对照孔和不进行任何处理的阴性对照孔。孵育48h后小心吸弃上清液，每孔加入100μl浓度为10％的CCK-8溶液再孵育2h。然后置于酶标仪于450nm测定各孔OD值。细胞生存率(％)＝(实验组OD值－空白对照组OD值)/(阴性对照组OD值－空白组对照OD值)×100。After the cells adhered to the wall overnight, the medium in each well was discarded, and then 100 μl of drug-loaded micelles solutions of different concentrations diluted with medium were added to each well, the concentrations were 333.333, 111.111, 37.037, 12.346, 4.115, 1.372, 0.457, 0.152, 0.051 μM, 3 auxiliary wells for each concentration, after adding the drug, place the 96-well plate in a carbon dioxide constant temperature incubator to continue culturing for 48 hours. At the same time, a blank control well without adding cells and a negative control well without any treatment were set up. After incubation for 48 hours, the supernatant was discarded carefully, and 100 μl of 10% CCK-8 solution was added to each well for further incubation for 2 hours. Then place it in a microplate reader to measure the OD value of each well at 450 nm. Cell survival rate (%)=(OD value of experimental group-OD value of blank control group)/(OD value of negative control group-OD value of blank control group)×100.
(2)对于悬浮的HepG2干细胞(2) For suspended HepG2 stem cells
将对数期的HepG2细胞经胰酶消化后制成单细胞悬液，用无血清培养基重悬，得单细胞悬液，以3000个/孔的密度接种于96孔板中，即每孔加入100uL的单细胞悬液，接种好的细胞于37℃，5％CO2的条件下培养24h，待细胞生长到合适密度。HepG2 cells in the logarithmic phase were digested with trypsin to make a single cell suspension, resuspended in serum-free medium to obtain a single cell suspension, and seeded in a 96-well plate at a density of 3000 cells/well, that is, every 100 uL of single cell suspension was added to the well, and the inoculated cells were incubated at 37° C. and 5% CO2 for 24 hours until the cells grew to a suitable density.
向各孔分别加入100μL培养基稀释的药物，终浓度分别为111.111，37.037，12.346，4.115，1.372，0.457，0.152，0.051，0.017μM，每孔设3个副孔，加药后返回二氧化碳培养箱中继续培养48h。同时设不加细胞的空白对照孔(新鲜培养基)和不进行任何处理的阴性对照孔。孵育48h后，向每孔加入20μlCCK-8溶液再孵育4h。然后置于酶标仪于450nm测定各孔OD值。细胞生存率(％)＝(实验组OD值－空白对照组OD值)/(阴性对照组OD值－空白对照组OD值)×100％Add 100 μL of drugs diluted in medium to each well, the final concentrations are 111.111, 37.037, 12.346, 4.115, 1.372, 0.457, 0.152, 0.051, 0.017 μM, set 3 auxiliary wells for each well, and return to the carbon dioxide incubator after adding the drugs Continue to culture for 48h. At the same time, a blank control well (fresh medium) without adding cells and a negative control well without any treatment were set up. After incubation for 48 h, 20 μl of CCK-8 solution was added to each well and incubated for another 4 h. Then place it in a microplate reader to measure the OD value of each well at 450 nm. Cell survival rate (%)=(OD value of experimental group-OD value of blank control group)/(OD value of negative control group-OD value of blank control group)×100%
图6显示了游离盐霉素、新型载盐霉素胶束和传统载药胶束对HepG2细胞及肿瘤干细胞毒性，可以得到以下结果：Figure 6 shows the toxicity of free salinomycin, new salinomycin-loaded micelles and traditional drug-loaded micelles to HepG2 cells and tumor stem cells, and the following results can be obtained:
(1)盐霉素、新型载盐霉素胶束和传统载药胶束对HepG2细胞及肿瘤干细胞的毒性具有浓度依赖性，随着盐霉素的浓度升高，细胞存活率显著下降。(1) The toxicity of salinomycin, new salinomycin-loaded micelles and traditional drug-loaded micelles to HepG2 cells and tumor stem cells was concentration-dependent. As the concentration of salinomycin increased, the cell survival rate decreased significantly.
(2)较HepG2贴壁细胞，HepG2肿瘤干细胞对盐霉素及其胶束具有更高的的敏感性，表明盐霉素对于肝癌干细胞的治疗有更佳的疗效。(2) Compared with HepG2 adherent cells, HepG2 tumor stem cells have higher sensitivity to salinomycin and its micelles, indicating that salinomycin has better curative effect on the treatment of liver cancer stem cells.
(3)新型载盐霉素胶束无论对HepG2贴壁细胞还是干细胞的抑制作用要强于游离盐霉素组，说明该新型载药胶束可增强对肝癌细胞的抑制和杀伤作用。(3) The inhibitory effect of the new salinomycin-loaded micelles on HepG2 adherent cells and stem cells was stronger than that of the free salinomycin group, indicating that the new drug-loaded micelles can enhance the inhibitory and killing effects on liver cancer cells.
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the description only illustrates the principles of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.
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| CN201510725278.XACN105250238B (en) | 2015-10-30 | 2015-10-30 | Carry polyethylene glycol-ceramide micella and the preparation method and application thereof of salinomycin |
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| CN201510725278.XACN105250238B (en) | 2015-10-30 | 2015-10-30 | Carry polyethylene glycol-ceramide micella and the preparation method and application thereof of salinomycin |
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| US7422902B1 (en)* | 1995-06-07 | 2008-09-09 | The University Of British Columbia | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
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| US7422902B1 (en)* | 1995-06-07 | 2008-09-09 | The University Of British Columbia | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
| CN104257628A (en)* | 2014-09-15 | 2015-01-07 | 中国人民解放军第二军医大学 | Salinomycin-loaded micelle as well as preparation method and application thereof |
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