技术领域technical field
本发明涉及纳米结构脂质载体给药系统的用途，主要涉及纳米结构脂质载体给药系统在抗肿瘤治疗和逆转肿瘤多药耐药中的应用。The invention relates to the application of the nanostructure lipid carrier delivery system, and mainly relates to the application of the nanostructure lipid carrier delivery system in anti-tumor treatment and reversal of tumor multidrug resistance.
背景技术Background technique
肿瘤一直是直接威胁人类健康的重大疾病，肿瘤化疗由于药物本身缺乏分子靶向性，因而出现治愈率低、多药耐药性、毒副作用巨大等重大治疗问题。临床化疗失败的重要原因之一，是肿瘤细胞对化疗药物产生耐药性。多数肿瘤病人的死因，与肿瘤细胞对抗肿瘤药物的耐药直接或间接相关。因此，寻找并研究能够逆转耐药肿瘤细胞耐药性的药物给药系统，是肿瘤化疗研究与发展的重要策略与方向之一。Tumor has always been a major disease that directly threatens human health. Due to the lack of molecular targeting of drugs themselves, tumor chemotherapy has major treatment problems such as low cure rate, multidrug resistance, and huge toxic and side effects. One of the important reasons for the failure of clinical chemotherapy is the resistance of tumor cells to chemotherapy drugs. The cause of death of most cancer patients is directly or indirectly related to the resistance of tumor cells to anti-tumor drugs. Therefore, finding and studying drug delivery systems that can reverse the drug resistance of drug-resistant tumor cells is one of the important strategies and directions for the research and development of cancer chemotherapy.
研究发现，有多种原因导致了肿瘤多药耐药性的产生，并且与P-糖蛋白(P-gp)、谷胱甘肽S转移酶(GST-л)，以及拓扑异构酶(TOPO-II)的表达异常有关。P-糖蛋白(P-gp)主要分布于细胞膜，是依赖ATP酶的药物输出泵，也是一种Ca2+与Cl-的通道。P-gp能够与进入细胞浆的抗肿瘤药物结合，借助ATP水解释放出的能量，直接或间接将药物泵出细胞；或借P-gp本身的通道作用，将药物从细胞内泵出细胞外，导致肿瘤细胞内药物浓度降低、细胞毒作用减弱或丧失，进而使肿瘤产生耐药。Studies have found that there are many reasons that lead to the generation of multidrug resistance in tumors, and it is related to P-glycoprotein (P-gp), glutathione S transferase (GST-л), and topoisomerase (TOPO -II) abnormal expression. P-glycoprotein (P-gp) is mainly distributed in the cell membrane, is a drug export pump dependent on ATPase, and is also a channel for Ca2+ and Cl- . P-gp can combine with anti-tumor drugs that enter the cytoplasm, and use the energy released by ATP hydrolysis to directly or indirectly pump the drug out of the cell; or use the channel function of P-gp itself to pump the drug out of the cell. , leading to a decrease in drug concentration in tumor cells, weakening or loss of cytotoxicity, and thus drug resistance in tumors.
研究显示，聚合物载体可在一定程度上缓解肿瘤的多药耐药性。非离子型表面活性剂聚环氧乙烯-聚苯醚-聚环氧乙烯三聚体(PEO-PPO-PEO)，包裹抗肿瘤药物后，当聚合物浓度在临界胶团浓度附近或以下时，可以发挥最佳的抗肿瘤效果，体现逆转多药耐药性倾向。聚合物载体可有效减少耐药细胞的ATP量，却不会引起敏感细胞的ATP量的变化。耐药细胞中ATP量的减少，直接减少了被耐药细胞泵出的肿瘤药物量，表现为对肿瘤耐药性的逆转。又如，聚左旋组氨酸(PolyHis)，通过咪唑基团的“质子海绵体”机制，可实现核内体膜裂解活性。如果所设计的聚合物胶团，能够在早期的核内体中发生触发性释放并内在化，随后使成熟的核内体膜破裂，则可以使药物逃逸，并在细胞浆和细胞核(潜在的药物作用位点)中表现出更高的药物浓度，这样的给药系统将成为有效的克服多药耐药性的药物传递模型。因此，带有聚左旋组氨酸核心的聚合物胶团，也有望成为有效的肿瘤细胞多药耐药性的逆转剂。Studies have shown that polymer carriers can alleviate the multidrug resistance of tumors to a certain extent. Nonionic surfactant polyethylene oxide-polyphenylene ether-polyethylene oxide terpolymer (PEO-PPO-PEO), after wrapping anti-tumor drugs, when the polymer concentration is near or below the critical micelle concentration, It can exert the best anti-tumor effect and reflect the tendency of reversing multi-drug resistance. The polymer carrier can effectively reduce the amount of ATP in drug-resistant cells, but it will not cause changes in the amount of ATP in sensitive cells. The reduction of the amount of ATP in drug-resistant cells directly reduces the amount of tumor drugs pumped out by drug-resistant cells, which is manifested as the reversal of tumor drug resistance. As another example, poly-L-histidine (PolyHis), through the "proton sponge" mechanism of imidazole groups, can achieve endosomal membrane lysis activity. If the polymer micelles are designed to trigger release and internalization in early endosomes, followed by rupture of mature endosomal membranes, drugs can escape and be released in the cytoplasm and nucleus (potentially Such a drug delivery system will be an effective drug delivery model to overcome multidrug resistance. Therefore, polymer micelles with a poly-L-histidine core are also expected to be effective reversal agents of multidrug resistance in tumor cells.
绝大多数抗肿瘤药物的分子作用靶标位于细胞内。现有的肿瘤化疗技术，基本沿用了药物的非靶向性给药模式，通过合适的载体技术，将药物直接靶向病变组织(器官)、细胞及亚细胞器，是解决癌症化疗治愈率低和毒副作用的重要手段之一。目前，国内外科学家通过纳米载体技术，在抗肿瘤药物的组织(器官)和细胞靶向上取得了一定的进展，但并没有取得突破性的疗效。其基本原因之一，在于绝大多数抗肿瘤药物的分子作用靶点位于细胞内。因此，针对肿瘤细胞内药物分子作用靶点(亚细胞器)的靶向性纳米载体材料技术的研究开发，是突破肿瘤化疗瓶颈技术的关键。The molecular targets of most anticancer drugs are located in cells. The existing cancer chemotherapy technology basically follows the non-targeted drug delivery mode. Through appropriate carrier technology, the drug is directly targeted to the diseased tissue (organ), cell and subcellular organelle, which is a solution to the low cure rate of cancer chemotherapy and the One of the important means of toxic side effects. At present, scientists at home and abroad have made some progress in the tissue (organ) and cell targeting of anti-tumor drugs through nanocarrier technology, but have not achieved breakthrough efficacy. One of the basic reasons is that the molecular targets of most antineoplastic drugs are located in cells. Therefore, the research and development of targeted nano-carrier material technology for drug molecular targets (subcellular organelles) in tumor cells is the key to breaking through the bottleneck technology of tumor chemotherapy.
固体脂质纳米粒给药系统，是20世纪90年代初发展起来的新型胶体给药系统，它是继乳剂、脂质体、聚合物纳米粒之后，极具发展潜力的靶向控释给药系统。固体脂质纳米粒给药系统，采用天然或合成的类脂材料，如硬脂酸、卵磷脂、单甘酯等为载体，将药物包裹于类脂核中制成固体胶粒给药系统。它既具备聚合物给药系统控释、避免药物泄漏等优点，又兼具了乳剂、脂质体的毒性低、生物相容性好、生物利用度高的优点。但是，固体脂质纳米粒给药系统也存在一些潜在的局限性，如有限的载药能力、储存过程的药物排挤现象等问题。The solid lipid nanoparticle drug delivery system is a new type of colloid drug delivery system developed in the early 1990s. It is a targeted controlled-release drug delivery system with great development potential after emulsions, liposomes, and polymer nanoparticles. system. The solid lipid nanoparticle drug delivery system uses natural or synthetic lipid materials, such as stearic acid, lecithin, monoglyceride, etc., as carriers, and encapsulates the drug in the lipid core to form a solid micelle drug delivery system. It not only has the advantages of controlled release of polymer drug delivery system and avoiding drug leakage, but also has the advantages of low toxicity, good biocompatibility and high bioavailability of emulsion and liposome. However, the solid lipid nanoparticle drug delivery system also has some potential limitations, such as limited drug loading capacity, drug exclusion during storage and other issues.
在固体脂质纳米粒中，加入形态相异的液体脂质作为混合类脂基质，可制备得到新型纳米结构脂质载体(nanostructured lipid carrier，NLC)。液体脂质的加入，可扰乱固体脂质规则的晶格结构，增加纳米粒结构中不规则晶型的比例，可使包封药物的空间容量增加，进而提高载体的载药能力。通过控制液体脂质比例，还可使NLC在体温下保持固体骨架结构，实现NLC药物的控制释放。In solid lipid nanoparticles, liquid lipids with different shapes are added as a mixed lipid matrix to prepare a new nanostructured lipid carrier (NLC). The addition of liquid lipids can disturb the regular lattice structure of solid lipids, increase the proportion of irregular crystal forms in the nanoparticle structure, increase the space capacity of encapsulated drugs, and then improve the drug-loading capacity of the carrier. By controlling the ratio of liquid lipids, NLC can also maintain a solid skeleton structure at body temperature to achieve controlled release of NLC drugs.
发明内容Contents of the invention
本发明的第一个目的是提供一种纳米结构脂质载体给药系统在抗肿瘤治疗药物中的应用。The first object of the present invention is to provide the application of a nanostructured lipid carrier drug delivery system in antitumor therapeutic drugs.
本发明的另一个目的是提供一种纳米结构脂质载体给药系统在逆转耐药肿瘤细胞多药耐药性中的应用。Another object of the present invention is to provide the application of a nanostructured lipid carrier delivery system in reversing the multidrug resistance of drug-resistant tumor cells.
本发明的纳米结构脂质载体给药系统由固态脂质材料、液态脂质材料和抗肿瘤药物组成。固态脂质为单甘酯，液态脂质为油酸，抗肿瘤药物可为紫杉醇和阿霉素。油酸的比例可为0-30％，抗肿瘤药物的比例为1-5％。纳米结构脂质载体给药系统采用水性溶剂扩散法制备。本发明中的纳米结构脂质载体的组成与制备方法已为专利《一种具有高效抗肿瘤活性的纳米结构脂质载体》(专利申请号200610155605.3)所涵盖。The nanostructure lipid carrier drug delivery system of the present invention is composed of solid lipid material, liquid lipid material and antitumor drug. The solid lipid is monoglyceride, the liquid lipid is oleic acid, and the antitumor drugs can be paclitaxel and doxorubicin. The proportion of oleic acid can be 0-30%, and the proportion of antitumor drugs can be 1-5%. The nanostructured lipid carrier drug delivery system is prepared by the aqueous solvent diffusion method. The composition and preparation method of the nanostructured lipid carrier in the present invention have been covered by the patent "A Nanostructured Lipid Carrier with Efficient Anti-tumor Activity" (Patent Application No. 200610155605.3).
本发明是利用具有高效细胞摄取和细胞浆浓集功能的纳米结构脂质载体，包封紫杉醇、阿霉素等分子靶标位于细胞内的抗肿瘤药物，在提高该类药物抗肿瘤疗效的同时，逆转耐药肿瘤细胞的耐药和多药耐药性。The present invention utilizes nanostructured lipid carriers with efficient cell uptake and cytoplasmic concentration functions to encapsulate paclitaxel, doxorubicin and other antitumor drugs whose molecular targets are located in cells, while improving the antitumor efficacy of such drugs, Reversal of drug resistance and multidrug resistance in resistant tumor cells.
本发明的有益之处，是通过具有高效细胞摄取和细胞浆浓集功能的纳米结构脂质载体，包封分子靶标位于细胞内的抗肿瘤药物，可显著增加肿瘤细胞对抗肿瘤药物的摄取，提高药物分子靶标部位的药物浓度，增强抗肿瘤药物的疗效。通过纳米结构脂质载体包封药物，可避免耐药细胞细胞浆中P-糖蛋白对抗肿瘤药物的识别，减少药物从细胞内的外排，在提高抗肿瘤药物疗效的同时，逆转耐药肿瘤细胞的耐药和多药耐药性。The benefit of the present invention is that the anti-tumor drug whose molecular target is located in the cell is encapsulated by the nano-structured lipid carrier with high-efficiency cell uptake and cytoplasmic concentration functions, which can significantly increase the uptake of anti-tumor drugs by tumor cells and improve The drug concentration at the target site of the drug molecule enhances the efficacy of anti-tumor drugs. Encapsulating drugs with nanostructured lipid carriers can avoid the recognition of anti-tumor drugs by P-glycoprotein in the cytoplasm of drug-resistant cells, reduce the efflux of drugs from cells, and reverse drug-resistant tumors while improving the efficacy of anti-tumor drugs Cellular resistance and multidrug resistance.
附图说明Description of drawings
图1为荧光标记单甘酯纳米结构脂质载体在MCF-7细胞和MCF-7-adr细胞摄取荧光照片。Figure 1 is a fluorescent photo of the uptake of fluorescently labeled monoglyceride nanostructured lipid carriers in MCF-7 cells and MCF-7-adr cells.
图2为荧光标记单甘酯纳米结构脂质载体在SKOV3细胞和SKOV3-adr细胞摄取荧光照片。Fig. 2 is a fluorescent photo of the uptake of fluorescently labeled monoglyceride nanostructured lipid carriers in SKOV3 cells and SKOV3-adr cells.
具体实施方式Detailed ways
实施例1：阿霉素单甘脂纳米结构载体给药系统的制备与应用Example 1: Preparation and Application of Doxorubicin Monoglyceride Nanostructured Carrier Delivery System
1)阿霉素单甘脂纳米结构载体给药系统制备1) Preparation of Adriamycin Monoglyceride Nanostructured Carrier Delivery System
分别取单甘酯60mg和阿霉素3mg，精密称定，加入6mL无水乙醇，水浴70℃溶解，形成有机相。以蒸馏水为分散相，置70℃水浴中，在400r·min-1机械搅拌条件下，将有机相注入到60mL分散相中，搅拌5min，制备得到阿霉素单甘酯纳米结构脂质载体给药系统分散液，分散液用3mol·L-1HCl溶液调节pH至1.2，以20000r·min-1离心10min，沉淀加0.1％泊洛沙姆(Poloxamer)(w/v)再分散后，用1.0mol·L-1NaOH溶液调节pH至7.0，得到阿霉素单甘酯纳米结构脂质载体给药系统。Take 60 mg of monoglyceride and 3 mg of doxorubicin respectively, weigh them accurately, add 6 mL of absolute ethanol, dissolve in a water bath at 70°C, and form an organic phase. Using distilled water as the dispersed phase, put it in a 70°C water bath, and under the condition of 400r·min-1 mechanical stirring, inject the organic phase into 60mL of the dispersed phase, and stir for 5min to prepare the doxorubicin monoglyceride nanostructured lipid carrier. Drug system dispersion liquid, the dispersion liquid was adjusted to pH 1.2 with 3mol·L-1 HCl solution, centrifuged at 20000r·min-1 for 10min, and the precipitate was re-dispersed with 0.1% Poloxamer (w/v). 1.0mol·L-1 NaOH solution was used to adjust the pH to 7.0 to obtain a doxorubicin monoglyceride nanostructured lipid carrier delivery system.
阿霉素单甘酯纳米结构脂质载体给药系统的粒径、表面电位和药物包封率见表1。The particle size, surface potential and drug encapsulation efficiency of the doxorubicin monoglyceride nanostructured lipid carrier delivery system are shown in Table 1.
表1阿霉素单甘酯纳米结构脂质载体给药系统的粒径、表面电位和药物包封率Table 1 Particle size, surface potential and drug encapsulation efficiency of doxorubicin monoglyceride nanostructure lipid carrier delivery system
2)单甘酯纳米结构脂质载体给药系统的抗肿瘤疗效及逆转耐药肿瘤细胞的耐药性2) Antitumor efficacy of monoglyceride nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells
分别以乳腺癌细胞MCF-7细胞和耐阿霉素乳腺癌细胞MCF-7-adr细胞为模型细胞，阿霉素单甘酯纳米结构脂质载体给药系统的抗肿瘤疗效，以及对耐药肿瘤细胞耐药性的逆转效率，通过给药系统与细胞共孵育后的IC50值(细胞的半数致死率)来评价。细胞存活率试验采用四唑盐比色法(MTT Assay)测定。于24孔板预培养24h，细胞贴壁生长后，分别加入不同浓度的空白单甘酯纳米结构脂质载体混悬液、阿霉素溶液和阿霉素单甘酯纳米结构脂质载体给药系统混悬液。实验设对照孔，每组重复3次。孵育48小时后，每孔加入60μL的MTT溶液，孵育4小时后弃去上清液，PBS溶液冲洗2次，每孔加入500μL的DMSO溶液，终止反应。将培养板水平振荡10min，用酶联检测仪在570nm处，测定吸收度，按(1)式计算细胞存活率：Taking breast cancer cell MCF-7 cells and doxorubicin-resistant breast cancer cell MCF-7-adr cells as model cells, the antitumor efficacy of doxorubicin monoglyceride nanostructured lipid carrier delivery system, and the effect on drug resistance The reversal efficiency of tumor cell drug resistance is evaluated by the IC50 value (half lethal rate of cells) after co-incubation of the drug delivery system and cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultivate in 24-well plate for 24 hours, after the cells adhere to the wall, add different concentrations of blank monoglyceride nanostructured lipid carrier suspension, doxorubicin solution and doxorubicin monoglyceride nanostructured lipid carrier for administration system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, add 60 μL of MTT solution to each well, discard the supernatant after incubation for 4 hours, wash with PBS solution twice, and add 500 μL of DMSO solution to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1):
细胞存活率(％)＝A570(样品)/A570(对照)×100％ (1)Cell viability (%)=A570 (sample)/A570 (control)×100% (1)
其中A570(样品)为加入混悬液后的细胞的吸收度，A570(对照)为空白对照的细胞的吸收度。Wherein A570 (sample) is the absorbance of cells added to the suspension, and A570 (control) is the absorbance of blank control cells.
所测定的空白单甘酯纳米结构脂质载体、阿霉素溶液和阿霉素单甘酯纳米结构脂质载体给药系统的IC50值结果，见表2。See Table 2 for the measured IC50 values of the blank monoglyceride nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride nanostructured lipid carrier delivery system.
表2单甘酯纳米结构脂质载体、阿霉素溶液和阿霉素单甘酯纳米结构脂质载体给药系统的IC50值Table 2IC50 values of monoglyceride nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride nanostructured lipid carrier delivery system
研究结果表明，单甘酯纳米结构脂质载体为低毒性载体材料，阿霉素经单甘酯纳米结构脂质载体包封后，在乳腺癌敏感细胞和耐药细胞上，可分别提高抗肿瘤疗效0.5倍和5.3倍。乳腺癌耐药细胞的耐药倍数约为35.2倍，阿霉素经单甘酯纳米结构脂质载体包封后，可部分逆转乳腺癌耐药细胞的耐药性。The research results show that the monoglyceride nanostructured lipid carrier is a low-toxicity carrier material. After doxorubicin is encapsulated by the monoglyceride nanostructured lipid carrier, it can improve the anti-tumor effect on breast cancer sensitive cells and drug-resistant cells, respectively. The curative effect is 0.5 times and 5.3 times. The drug-resistant multiple of breast cancer drug-resistant cells is about 35.2 times, and doxorubicin can partially reverse the drug resistance of breast cancer drug-resistant cells after being encapsulated by the monoglyceride nanostructured lipid carrier.
实施例2：阿霉素单甘脂/油酸纳米结构载体给药系统的制备与应用Example 2: Preparation and Application of Doxorubicin Monoglyceride/Oleic Acid Nanostructured Carrier Delivery System
1)阿霉素单甘脂/油酸纳米结构载体给药系统制备1) Preparation of doxorubicin monoglyceride/oleic acid nanostructure carrier drug delivery system
分别取单甘酯60mg、油酸6mg和阿霉素3mg，精密称定。加入6mL无水乙醇，水浴70℃溶解。以蒸馏水为分散相，置70℃水浴中，在400r·min-1机械搅拌条件下，将有机相注入到60mL分散相中，搅拌5min，得到阿霉素单甘酯/油酸纳米结构脂质载体给药系统分散液，分散液用3mol·L-1HCl溶液调节pH至1.2，以20000r·min-1离心10min，沉淀加0.1％泊洛沙姆(Poloxamer)(w/v)再分散后，用1mol·L-1NaOH溶液调节pH至7.0，得到阿霉素单甘酯/油酸纳米结构脂质载体给药系统。Take 60 mg of monoglyceride, 6 mg of oleic acid and 3 mg of doxorubicin, respectively, and weigh them accurately. Add 6 mL of absolute ethanol and dissolve in a water bath at 70°C. Using distilled water as the dispersed phase, put it in a water bath at 70°C, and inject the organic phase into 60 mL of the dispersed phase under the condition of 400 r·min-1 mechanical stirring, and stir for 5 min to obtain the doxorubicin monoglyceride/oleic acid nanostructured lipid Carrier drug delivery system dispersion, the dispersion is adjusted to pH 1.2 with 3mol L-1 HCl solution, centrifuged at 20000r min-1 for 10min, and redispersed after adding 0.1% Poloxamer (w/v) to the precipitate , adjust the pH to 7.0 with 1mol·L-1 NaOH solution, and obtain the doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system.
阿霉素单甘酯/油酸纳米结构脂质载体给药系统的粒径、表面电位和药物包封率见表3。The particle size, surface potential and drug encapsulation efficiency of the doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system are shown in Table 3.
表3阿霉素单甘酯纳米结构脂质载体给药系统的粒径、表面电位和药物包封率Table 3 Particle size, surface potential and drug encapsulation efficiency of doxorubicin monoglyceride nanostructure lipid carrier delivery system
2)单甘酯/油酸纳米结构脂质载体给药系统的抗肿瘤疗效及逆转耐药肿瘤细胞的耐药性2) Antitumor efficacy of monoglyceride/oleic acid nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells
分别以乳腺癌细胞MCF-7细胞和耐阿霉素乳腺癌细胞MCF-7-adr细胞为模型细胞，阿霉素单甘酯/油酸纳米结构脂质载体给药系统的抗肿瘤疗效，以及对耐药肿瘤细胞耐药性的逆转效率，通过给药系统与细胞共孵育后的IC50值(细胞的半数致死率)来评价。细胞存活率试验采用四唑盐比色法(MTT Assay)测定。于24孔板预培养24h，细胞贴壁生长后，分别加入不同浓度的空白单甘酯/油酸纳米结构脂质载体的混悬液、阿霉素溶液和阿霉素单甘酯/油酸纳米结构脂质载体给药系统混悬液。实验设对照孔，每组重复3次。孵育48小时后，每孔加入60μL的MTT溶液，孵育4小时后，弃去上清液，PBS溶液冲洗2次，每孔加入500μL的DMSO溶液，终止反应。将培养板水平振荡10min，用酶联检测仪在570nm处，测定吸收度，按(1)式计算细胞存活率。Taking breast cancer cell MCF-7 cells and doxorubicin-resistant breast cancer cell MCF-7-adr cells as model cells, the antitumor efficacy of doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system, and The reversal efficiency of drug resistance to drug-resistant tumor cells is evaluated by the IC50 value (half lethal rate of cells) after co-incubation of the drug delivery system and cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultivate in 24-well plate for 24 hours, after the cells adhere to the wall, add different concentrations of blank monoglyceride/oleic acid nanostructured lipid carrier suspension, doxorubicin solution and doxorubicin monoglyceride/oleic acid Nanostructured lipid carrier drug delivery system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, 60 μL of MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, washed twice with PBS solution, and 500 μL of DMSO solution was added to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1).
所测定的空白单甘酯/油酸纳米结构脂质载体、阿霉素溶液和阿霉素单甘酯/油酸纳米结构脂质载体给药系统的IC50值，见表4。See Table 4 for the measured IC50 values of the blank monoglyceride/oleic acid nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system.
表4单甘酯/油酸纳米结构脂质载体、阿霉素溶液和阿霉素单甘酯/油酸纳米结构脂质载体给药系统的IC50值Table 4IC50 values of monoglyceride/oleic acid nanostructured lipid carrier, doxorubicin solution and doxorubicin monoglyceride/oleic acid nanostructured lipid carrier delivery system
研究结果表明，单甘酯/油酸纳米结构脂质载体为低毒性载体材料，阿霉素经单甘酯/油酸纳米结构脂质载体包封后，在乳腺癌敏感细胞和耐药细胞上可分别提高抗肿瘤疗效5％和7.7倍。乳腺癌耐药细胞的耐药倍数约为35.2倍，阿霉素经单甘酯/油酸纳米结构脂质载体包封后，可部分逆转乳腺癌耐药细胞的耐药性。The research results show that the monoglyceride/oleic acid nanostructured lipid carrier is a low-toxicity carrier material, and after doxorubicin is encapsulated by the monoglyceride/oleic acid nanostructured lipid carrier, it can inhibit breast cancer sensitive cells and drug-resistant cells. The anti-tumor efficacy can be increased by 5% and 7.7 times respectively. The drug-resistant multiple of breast cancer drug-resistant cells is about 35.2 times, and doxorubicin can partially reverse the drug resistance of breast cancer drug-resistant cells after being encapsulated by monoglyceride/oleic acid nanostructured lipid carrier.
实施例3：紫杉醇单甘脂纳米结构载体给药系统的制备与应用Example 3: Preparation and Application of Paclitaxel Monoglyceride Nanostructured Carrier Delivery System
1)紫杉醇单甘脂纳米结构载体给药系统制备1) Preparation of paclitaxel monoglyceride nanostructure carrier drug delivery system
分别取单甘酯60mg和紫杉醇3mg，精密称定，加入6mL无水乙醇，水浴70℃溶解。以蒸馏水为分散相，置70℃水浴中，在400r·min-1机械搅拌条件下，将有机相注入60mL分散相中，搅拌5min，得到紫杉醇单甘酯纳米结构脂质载体给药系统分散液，分散液用3mol·L-1HCl溶液调节pH至1.2，以20000r·min-1离心10min，沉淀加0.1％泊洛沙姆(Poloxamer)(w/v)再分散后，用1mol·L-1NaOH溶液调节pH至7.0，得到紫杉醇单甘酯纳米结构脂质载体给药系统。Take 60 mg of monoglyceride and 3 mg of paclitaxel respectively, weigh them accurately, add 6 mL of absolute ethanol, and dissolve in a water bath at 70°C. Using distilled water as the dispersed phase, put it in a water bath at 70°C, and under the condition of 400r·min-1 mechanical stirring, inject the organic phase into 60mL of the dispersed phase, and stir for 5min to obtain the dispersion liquid of paclitaxel monoglyceride nanostructured lipid carrier drug delivery system , the dispersion was adjusted to pH 1.2 with 3mol L-1 HCl solution, centrifuged at 20000r min-1 for 10 min, and the precipitate was re-dispersed by adding 0.1% Poloxamer (w/v), then washed with 1mol L-1 1 NaOH solution to adjust the pH to 7.0 to obtain the paclitaxel monoglyceride nanostructured lipid carrier drug delivery system.
紫杉醇单甘酯纳米结构脂质载体给药系统的粒径、表面电位和药物包封率见表5。The particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride nanostructured lipid carrier delivery system are shown in Table 5.
表5紫杉醇单甘酯纳米结构脂质载体给药系统的粒径、表面电位和药物包封率Table 5 Particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride nanostructure lipid carrier delivery system
2)单甘酯纳米结构脂质载体给药系统的抗肿瘤疗效及逆转耐药肿瘤细胞的耐药性2) Antitumor efficacy of monoglyceride nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells
分别以乳腺癌细胞MCF-7细胞和耐阿霉素乳腺癌细胞MCF-7-adr细胞，卵巢癌细胞SKOV3细胞耐紫杉醇卵巢癌细胞SKOV3-adr细胞为模型细胞，紫杉醇单甘酯纳米结构脂质载体给药系统的抗肿瘤疗效，以及对耐药肿瘤细胞耐药性的逆转效率，通过给药系统与细胞共孵育后的IC50值(细胞的半数致死率)来评价。细胞存活率试验采用四唑盐比色法(MTT Assay)测定。于24孔板预培养24h，细胞贴壁生长后，分别加入不同浓度的空白单甘酯纳米结构脂质载体的混悬液、紫杉醇溶液(溶剂为Cremophor∶无水乙醇混合溶剂(1∶1，v/v))和紫杉醇单甘酯纳米结构脂质载体(油酸含量为0)给药系统混悬液。实验设对照孔，每组重复3次。孵育48小时后，每孔加入60μL的MTT溶液，孵育4小时后，弃去上清液，PBS溶液冲洗2次，每孔加入500μL的DMSO溶液，终止反应。将培养板水平振荡10min，用酶联检测仪在570nm处，测定吸收度，按(1)式计算细胞存活率。Breast cancer cell MCF-7 cells and doxorubicin-resistant breast cancer cell MCF-7-adr cells, ovarian cancer cell SKOV3 cells resistant to paclitaxel ovarian cancer cell SKOV3-adr cells were used as model cells, paclitaxel monoglyceride nanostructured lipids The anti-tumor efficacy of the carrier delivery system and the reversal efficiency of drug resistance to drug-resistant tumor cells are evaluated by the IC50 value (half lethality of the cells) after the delivery system is co-incubated with the cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultured in a 24-well plate for 24 h, after the cells adhered to the wall, different concentrations of blank monoglyceride nanostructured lipid carrier suspension and paclitaxel solution (solvent: Cremophor: absolute ethanol mixed solvent (1:1, v/v)) and paclitaxel monoglyceride nanostructure lipid carrier (oleic acid content is 0) delivery system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, 60 μL of MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, washed twice with PBS solution, and 500 μL of DMSO solution was added to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1).
所测定的空白单甘酯纳米结构脂质载体、紫杉醇溶液和紫杉醇单甘酯纳米结构脂质载体给药系统的IC50值，见表6。See Table 6 for the measured IC50 values of the blank monoglyceride nanostructured lipid carrier, paclitaxel solution and paclitaxel monoglyceride nanostructured lipid carrier delivery system.
表6单甘酯纳米结构脂质载体、紫杉醇溶液和紫杉醇单甘酯纳米结构脂质载体给药系统的IC50值TheIC50 value of table 6 monoglyceride nanostructure lipid carrier, paclitaxel solution and paclitaxel monoglyceride nanostructure lipid carrier delivery system
研究结果表明，单甘酯纳米结构脂质载体为低毒性载体材料，紫杉醇经单甘酯纳米结构脂质载体(不含油酸)包封后，在乳腺癌敏感细胞和耐药细胞上，可分别提高抗肿瘤疗效2.2倍和125.5倍。乳腺癌耐药细胞对紫杉醇的多药耐药倍数约为29.7倍，紫杉醇经单甘酯纳米结构脂质载体包封后，可完全逆转乳腺癌耐药细胞的多药耐药性。紫杉醇经单甘酯纳米结构脂质载体(不含油酸)包封后，在卵巢癌敏感细胞和耐药细胞上，可分别提高抗肿瘤疗效0.8倍和5.9倍，可部分逆转卵巢癌耐药细胞的耐药性。The research results show that the monoglyceride nanostructured lipid carrier is a low-toxicity carrier material. After paclitaxel is encapsulated by the monoglyceride nanostructured lipid carrier (without oleic acid), it can be expressed on breast cancer sensitive cells and drug-resistant cells, respectively. Improve the anti-tumor efficacy by 2.2 times and 125.5 times. The multiple drug resistance of breast cancer drug-resistant cells to paclitaxel is about 29.7 times. After paclitaxel is encapsulated by monoglyceride nanostructured lipid carrier, it can completely reverse the multi-drug resistance of breast cancer drug-resistant cells. After paclitaxel is encapsulated by a monoglyceride nanostructured lipid carrier (without oleic acid), the anti-tumor efficacy can be increased by 0.8 times and 5.9 times on ovarian cancer sensitive cells and drug-resistant cells, respectively, and it can partially reverse ovarian cancer drug-resistant cells drug resistance.
实施例4：紫杉醇单甘脂/油酸纳米结构载体给药系统的制备与应用Example 4: Preparation and Application of Paclitaxel Monoglyceride/Oleic Acid Nanostructured Carrier Delivery System
1)紫杉醇单甘脂/油酸纳米结构载体给药系统制备1) Preparation of paclitaxel monoglyceride/oleic acid nanostructure carrier drug delivery system
分别取单甘酯60mg、油酸6或18mg和紫杉醇3mg，精密称定，加入6mL无水乙醇，水浴70℃溶解。以蒸馏水为分散相，置70℃水浴中，在400r·min-1机械搅拌条件下，将有机相注入到60mL分散相中，搅拌5min，得到紫杉醇单甘酯/油酸纳米结构脂质载体给药系统分散液，分散液用3mol·L-1HCl溶液调节pH至1.2，以20000r·min-1离心10min，沉淀加0.1％泊洛沙姆(Poloxamer)(w/v)再分散后，用1mol·L-1NaOH溶液调节pH至7.0，得到紫杉醇单甘酯/油酸纳米结构脂质载体给药系统。Take 60 mg of monoglyceride, 6 or 18 mg of oleic acid, and 3 mg of paclitaxel, weigh them accurately, add 6 mL of absolute ethanol, and dissolve in a water bath at 70°C. Using distilled water as the dispersed phase, put it in a water bath at 70°C, and under the condition of 400r·min-1 mechanical stirring, inject the organic phase into 60mL of the dispersed phase, and stir for 5min to obtain paclitaxel monoglyceride/oleic acid nanostructured lipid carrier. Drug system dispersion liquid, the dispersion liquid was adjusted to pH 1.2 with 3mol·L-1 HCl solution, centrifuged at 20000r·min-1 for 10min, and the precipitate was re-dispersed with 0.1% Poloxamer (w/v). 1mol·L-1 NaOH solution was used to adjust the pH to 7.0 to obtain the paclitaxel monoglyceride/oleic acid nanostructured lipid carrier drug delivery system.
紫杉醇的单甘酯/油酸纳米结构脂质载体给药系统的粒径、表面电位和药物包封率见表7。The particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride/oleic acid nanostructured lipid carrier delivery system are shown in Table 7.
表7紫杉醇单甘酯纳米结构脂质载体给药系统的粒径、表面电位和药物包封率Table 7 Particle size, surface potential and drug encapsulation efficiency of paclitaxel monoglyceride nanostructure lipid carrier delivery system
2)单甘酯纳米结构脂质载体的肺癌A549细胞转运2) Translocation of monoglyceride nanostructured lipid carrier to lung cancer A549 cells
本发明采用含有异硫氰基荧光素(FITC)与硬脂胺化学嫁接物，作为单甘酯纳米结构脂质载体进行肺癌A549细胞转运研究的荧光标记物。含有异硫氰基荧光素与硬脂胺化学嫁接物的单甘酯纳米结构脂质载体，通过以下方法制备：分别取单甘酯27mg和异硫氰基荧光素与硬脂胺化学嫁接物4.5mg，精密称定，加入3mL无水乙醇，水浴70℃溶解。以蒸馏水为分散相，置70℃水浴中。在400r·min-1机械搅拌条件下，将有机相注入30mL分散相中，搅拌5min，得到荧光标记单甘酯纳米结构脂质载体的分散液，分散液用3mol·L-1HCl溶液调节pH至1.2，以20000r·min-1离心10min，沉淀加0.1％泊洛沙姆(Poloxamer)(w/v)再分散后，用1.0mol·L-1NaOH溶液调节pH至7.0，分散液用于肺癌A549细胞转运研究。The invention adopts a chemical graft containing fluorescein isothiocyanate (FITC) and stearylamine as a fluorescent marker for studying the translocation of lung cancer A549 cells as a monoglyceride nanostructured lipid carrier. The monoglyceride nanostructured lipid carrier containing fluorescein isothiocyanate and stearylamine chemical graft is prepared by the following method: take 27 mg of monoglyceride and 4.5 mg of the chemical graft of fluorescein isothiocyanate and stearylamine mg, accurately weighed, add 3mL of absolute ethanol, and dissolve in a water bath at 70°C. With distilled water as the dispersed phase, put it in a 70°C water bath. Under the condition of mechanical stirring at 400r·min-1 , inject the organic phase into 30mL of the dispersed phase, and stir for 5min to obtain a dispersion liquid of fluorescently labeled monoglyceride nanostructured lipid carrier, and adjust the pH of the dispersion liquid with 3mol·L-1 HCl solution to 1.2, centrifuge at 20000r·min-1 for 10min, add 0.1% poloxamer (Poloxamer) (w/v) to the precipitate and redisperse, adjust the pH to 7.0 with 1.0mol·L-1 NaOH solution, and use the dispersion for Translocation studies in lung cancer A549 cells.
分别取乳腺癌细胞MCF-7细胞和耐阿霉素乳腺癌细胞MCF-7-adr细胞，在含有10％小牛血清的RPMI 1640培养液中培养(5％CO2，37℃孵箱)。取对数生长期细胞，PBS润洗后，加入胰酶消化并用培养液稀释，按每孔1×105个细胞的密度接种于24孔培养板内，24孔培养板中的细胞贴壁生长后，加入荧光标记的单甘酯纳米结构脂质载体(终浓度为100μg·mL-1)，孵育1，2，4，12，24h后，用PBS冲洗细胞3次，荧光倒置显微镜观察并拍照，结果参见图1。其中纳米载体与细胞共孵育12小时，A为荧光标记单甘酯纳米结构脂质载体(含30％油酸)在MCF-7细胞摄取荧光照片，B为荧光标记单甘酯纳米结构脂质载体(含30％油酸)在MCF-7-adr细胞摄取荧光照片。The breast cancer cell MCF-7 and the doxorubicin-resistant breast cancer cell MCF-7-adr were respectively cultured in RPMI 1640 medium containing 10% calf serum (5% CO2 , 37°C incubator). Take cells in the logarithmic growth phase, rinse with PBS, add trypsin to digest and dilute with culture medium, inoculate in a 24-well culture plate at a density of 1×105 cells per well, and the cells in the 24-well culture plate grow adherently Afterwards, add fluorescently labeled monoglyceride nanostructured lipid carrier (final concentration: 100 μg·mL-1 ), incubate for 1, 2, 4, 12, and 24 h, wash the cells with PBS three times, observe and take pictures with a fluorescent inverted microscope , see Figure 1 for the results. Among them, the nanocarriers were co-incubated with the cells for 12 hours. A is the fluorescent photo taken by the fluorescently labeled monoglyceride nanostructured lipid carrier (containing 30% oleic acid) in MCF-7 cells, and B is the fluorescently labeled monoglyceride nanostructured lipid carrier. (Containing 30% oleic acid) Fluorescence photographs were taken in MCF-7-adr cells.
分别取乳腺癌细胞MCF-7细胞和耐阿霉素乳腺癌细胞MCF-7-adr细胞，卵巢癌细胞SKOV3细胞耐紫杉醇卵巢癌细胞SKOV3-adr细胞，在含有10％小牛血清的RPMI 1640培养液中培养(5％CO2，37℃孵箱)。取对数生长期细胞，PBS润洗后，加入胰酶消化并用培养液稀释，按每孔1×105个细胞的密度接种于24孔培养板内，24孔培养板中的细胞贴壁生长后，加入荧光标记的单甘酯/油酸纳米结构脂质载体(终浓度为100μg·mL-1)，孵育1，2，4，12，24h后，用PBS冲洗细胞3次，荧光倒置显微镜观察并拍照，结果参见图2，其中纳米载体与细胞共孵育12小时，为荧光标记单甘酯纳米结构脂质载体(含30％油酸)在SKOV3细胞摄取荧光照片，B荧光标记单甘酯纳米结构脂质载体(含30％油酸)在SKOV3-adr细胞摄取荧光照片。Breast cancer cells MCF-7 cells, breast cancer cells resistant to doxorubicin MCF-7-adr cells, ovarian cancer cells SKOV3 cells resistant to paclitaxel ovarian cancer cells SKOV3-adr cells were cultured in RPMI 1640 containing 10% calf serum Culture in liquid (5% CO2 , 37°C incubator). Take cells in the logarithmic growth phase, rinse with PBS, add trypsin to digest and dilute with culture medium, inoculate in a 24-well culture plate at a density of 1×105 cells per well, and the cells in the 24-well culture plate grow adherently Afterwards, add fluorescently labeled monoglyceride/oleic acid nanostructured lipid carrier (final concentration: 100 μg·mL-1 ), incubate for 1, 2, 4, 12, and 24 h, wash cells with PBS three times, and fluorescent inverted microscope Observe and take photos, the results are shown in Figure 2, where the nanocarriers were co-incubated with the cells for 12 hours, which is a fluorescent photo of the fluorescently labeled monoglyceride nanostructured lipid carrier (containing 30% oleic acid) in SKOV3 cells, B fluorescently labeled monoglyceride Fluorescent photographs of nanostructured lipid carriers (containing 30% oleic acid) in SKOV3-adr cells.
3)单甘酯/油酸纳米结构脂质载体给药系统的抗肿瘤疗效及逆转耐药肿瘤细胞的耐药性3) Antitumor efficacy of monoglyceride/oleic acid nanostructured lipid carrier delivery system and reversal of drug resistance of drug-resistant tumor cells
分别以乳腺癌细胞MCF-7细胞和耐阿霉素乳腺癌细胞MCF-7-adr细胞，卵巢癌细胞SKOV3细胞耐紫杉醇卵巢癌细胞SKOV3-adr细胞为模型细胞，紫杉醇单甘酯/油酸纳米结构脂质载体给药系统的抗肿瘤疗效，以及对耐药肿瘤细胞耐药性的逆转效率，通过给药系统与细胞共孵育后的IC50值(细胞的半数致死率)来评价。细胞存活率试验采用四唑盐比色法(MTT Assay)测定。于24孔板预培养24h，细胞贴壁生长后，分别加入不同浓度的空白单甘酯/油酸纳米结构脂质载体混悬液、紫杉醇溶液(溶剂为Cremophor∶无水乙醇混合溶剂(1∶1，v/v))和紫杉醇单甘酯/油酸纳米结构脂质载体(含10和30％油酸)给药系统混悬液。实验设对照孔，每组重复3次。孵育48小时后，每孔加入60μL的MTT溶液，孵育4小时后，弃去上清液，PBS溶液冲洗2次，每孔加入500μL的DMSO溶液，终止反应。将培养板水平振荡10min，用酶联检测仪在570nm处，测定吸收度，按(1)式计算细胞存活率。Breast cancer cells MCF-7 cells, adriamycin-resistant breast cancer cells MCF-7-adr cells, ovarian cancer cells SKOV3 cells resistant to paclitaxel ovarian cancer cells SKOV3-adr cells were used as model cells, paclitaxel monoglyceride/oleic acid nano The antitumor efficacy of the structured lipid carrier delivery system, as well as the reversal efficiency of drug resistance to drug-resistant tumor cells, are evaluated by the IC50 value (half lethality of the cells) after the delivery system is co-incubated with the cells. The cell viability test was determined by tetrazolium salt colorimetric method (MTT Assay). Pre-cultured in a 24-well plate for 24 h, after the cells adhered to the wall, different concentrations of blank monoglyceride/oleic acid nanostructured lipid carrier suspension and paclitaxel solution (solvent: Cremophor: absolute ethanol mixed solvent (1: 1, v/v)) and paclitaxel monoglyceride/oleic acid nanostructured lipid carrier (containing 10 and 30% oleic acid) delivery system suspension. Control wells were set up in the experiment, and each group was repeated 3 times. After incubation for 48 hours, 60 μL of MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, washed twice with PBS solution, and 500 μL of DMSO solution was added to each well to terminate the reaction. Shake the culture plate horizontally for 10 min, measure the absorbance at 570 nm with an enzyme-linked detector, and calculate the cell survival rate according to formula (1).
所测定的空白单甘酯/油酸纳米结构脂质载体、紫杉醇溶液和紫杉醇单甘酯/油酸纳米结构脂质载体给药系统的IC50值，见表8。See Table 8 for the measured IC50 values of the blank monoglyceride/oleic acid nanostructured lipid carrier, paclitaxel solution and paclitaxel monoglyceride/oleic acid nanostructured lipid carrier delivery system.
表8单甘酯/油酸纳米结构脂质载体、紫杉醇溶液和紫杉醇单甘酯/油酸纳米结构脂质载体给药系统的IC50值IC50 values of table 8 monoglyceride/oleic acid nanostructured lipid carrier, paclitaxel solution and paclitaxel monoglyceride/oleic acid nanostructured lipid carrier delivery system
研究结果表明，单甘酯/油酸纳米结构脂质载体为低毒性载体材料，紫杉醇经单甘酯/油酸纳米结构脂质载体(含30％油酸)包封后，在乳腺癌敏感细胞和耐阿霉素的耐药细胞上，可分别提高抗肿瘤疗效3.5倍和123.8倍。乳腺癌耐药细胞对紫杉醇的多药耐药倍数为29.7倍，紫杉醇经单甘酯纳米结构脂质载体包封后，可完全逆转乳腺癌耐药细胞的多药耐药性。紫杉醇经单甘酯纳米结构脂质载体(不含油酸)包封后，在卵巢癌敏感细胞和耐药细胞上，可分别提高抗肿瘤疗效2.0倍和175.4倍，可完全逆转卵巢癌耐药细胞的耐药性。The research results show that the monoglyceride/oleic acid nanostructured lipid carrier is a low-toxicity carrier material. After paclitaxel is encapsulated by the monoglyceride/oleic acid nanostructured lipid carrier (containing 30% oleic acid), it is effective in breast cancer sensitive cells. and doxorubicin-resistant drug-resistant cells, the anti-tumor efficacy can be increased by 3.5 times and 123.8 times, respectively. The multiple drug resistance of breast cancer drug-resistant cells to paclitaxel is 29.7 times. After paclitaxel is encapsulated by monoglyceride nanostructure lipid carrier, it can completely reverse the multi-drug resistance of breast cancer drug-resistant cells. After paclitaxel is encapsulated by a monoglyceride nanostructured lipid carrier (without oleic acid), the anti-tumor efficacy can be increased by 2.0 times and 175.4 times on ovarian cancer sensitive cells and drug-resistant cells, respectively, and it can completely reverse ovarian cancer drug-resistant cells drug resistance.
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