技术领域Technical Field
本发明属于生物医药技术领域，具体涉及多功能基因载体的制备及在递送miRNA中的应用。The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation of a multifunctional gene vector and its application in delivering miRNA.
背景技术Background technique
区别于传统的化疗药物和生物药物(抗体和细胞药物)，核酸药物(例如siRNA和miRNA)能够以化学药物或抗体药物无法靶向的分子作为作用靶点，有望对传统药物疗效不佳的疾病产生突破性的进展，特别是难以治疗的遗传疾病、癌症等。但是核酸类药物的输送面临着很大的问题。核酸药物进入细胞面临两大挑战，一个是RNA暴露在血液中容易被血浆和组织中的RNase降解，而且会引发不必要的免疫反应；另一个是带负电的RNA难以跨膜进入胞内，所以需要一个递送系统将它们传送到细胞中行使功能。递送平台的开发是整个核酸疗法产业链条的重点，也是核酸疗法获得成功的必要条件。Different from traditional chemotherapy drugs and biological drugs (antibodies and cell drugs), nucleic acid drugs (such as siRNA and miRNA) can target molecules that chemical drugs or antibody drugs cannot target, and are expected to produce breakthrough progress in diseases that are not well treated by traditional drugs, especially difficult-to-treat genetic diseases and cancers. However, the delivery of nucleic acid drugs faces great problems. Nucleic acid drugs face two major challenges in entering cells. One is that RNA exposed to the blood is easily degraded by RNase in plasma and tissues, and it will trigger unnecessary immune responses; the other is that negatively charged RNA is difficult to cross the membrane and enter the cell, so a delivery system is needed to deliver them into the cell to perform their functions. The development of the delivery platform is the focus of the entire nucleic acid therapy industry chain and a necessary condition for the success of nucleic acid therapy.
随着纳米科技的发展，纳米材料成为递送miRNA的新选择。例如Hosseinpour等将miRNA-26a-5p封装在介孔二氧化硅纳米粒(MSN-CC-PEI)中，并将其包裹在聚乙烯亚胺(MSN-CC-PEI)上，作为向大鼠骨髓间充质细胞促进其成骨分化的系统，该递送体系在体内表现出稳定性和高效性。With the development of nanotechnology, nanomaterials have become a new choice for delivering miRNA. For example, Hosseinpour et al. encapsulated miRNA-26a-5p in mesoporous silica nanoparticles (MSN-CC-PEI) and wrapped it on polyethyleneimine (MSN-CC-PEI) as a system to promote osteogenic differentiation of rat bone marrow mesenchymal cells. The delivery system showed stability and high efficiency in vivo.
岩藻多糖是一种海洋来源的硫酸多糖，已经被纳入纳米医学应用领域中。岩藻多糖可用于纳米递药体系，不仅可以稳定纳米载体，还可增加增强化疗药物的抗癌活性。公布号CN 108451931 A的专利公布了一种由聚丙烯胺盐酸盐和岩藻聚糖通过聚电解质复合法制成的纳米颗粒，可以增加化疗药物甲氨蝶呤的抗癌活性。我们设计制备岩藻多糖多功能基因载体递送miRNA，不仅能够能够实现miRNA的靶向递送，其作为免疫激活剂，还可改善免疫抑制的肿瘤微环境。这种设计将为癌症的治疗提供新的治疗策略。Fucoidan is a sulfated polysaccharide of marine origin that has been incorporated into the field of nanomedicine applications. Fucoidan can be used in nano drug delivery systems, which can not only stabilize nanocarriers, but also increase the anti-cancer activity of chemotherapeutic drugs. Patent publication number CN 108451931 A discloses a nanoparticle made of polyacrylamine hydrochloride and fucoidan by a polyelectrolyte composite method, which can increase the anti-cancer activity of the chemotherapeutic drug methotrexate. We designed and prepared a fucoidan multifunctional gene carrier to deliver miRNA, which can not only achieve targeted delivery of miRNA, but also improve the immunosuppressive tumor microenvironment as an immune activator. This design will provide a new therapeutic strategy for the treatment of cancer.
发明内容Summary of the invention
本发明的目的在于提供一种多功能基因载体负载抗肿瘤活性的miRNA，以实现miRNA在体内的靶向运输及miRNA和岩藻多糖的协同抗癌作用。The purpose of the present invention is to provide a multifunctional gene carrier loaded with miRNA with anti-tumor activity, so as to achieve the targeted transport of miRNA in vivo and the synergistic anti-cancer effect of miRNA and fucoidan.
本发明的具体技术方案如下：The specific technical solutions of the present invention are as follows:
一种多功能基因载体。该纳米颗粒包括聚乙烯亚胺(Polyethyleneimine，PEI)修饰的Fe3O4纳米磁核和组装在外层的岩藻多糖。岩藻多糖通过静电吸附自组装的方式结合至磁核外层。该载体的的平均粒径为50～150nm。A multifunctional gene carrier. The nanoparticles include a Fe3 O4 nanomagnetic core modified by polyethyleneimine (PEI) and fucoidan assembled on the outer layer. The fucoidan is bound to the outer layer of the magnetic core by electrostatic adsorption self-assembly. The average particle size of the carrier is 50 to 150 nm.
本发明提供了上述多功能基因载体的制备方法，包括以下步骤：The present invention provides a method for preparing the multifunctional gene vector, comprising the following steps:
将10～15mmol无水氯化铁，优选为15mmol，3～5mmol氢氧化钠，优选为5mmol，45～55mL乙二醇，优选为50mL和3.6mL无菌水混合均匀后转移到三口瓶中，加热至沸腾，随后保持沸腾回流8h～12h，停止反应，冷却至室温，经磁性分离、洗涤得到Fe3O4纳米颗粒，备用；10-15 mmol of anhydrous ferric chloride, preferably 15 mmol, 3-5 mmol of sodium hydroxide, preferably 5 mmol, 45-55 mL of ethylene glycol, preferably 50 mL, and 3.6 mL of sterile water are mixed evenly and transferred to a three-necked flask, heated to boiling, then kept boiling and refluxed for 8 h to 12 h, the reaction is stopped, cooled to room temperature, magnetically separated, washed to obtain Fe3 O4 nanoparticles, and set aside;
(2)三聚磷酸钠的交联和聚乙烯亚胺(PEI)修饰(2) Cross-linking of sodium tripolyphosphate and modification with polyethyleneimine (PEI)
将步骤(1)得到的四氧化三铁纳米颗粒分散于无菌水中，加入三聚磷酸钠，Fe3O4纳米颗粒与三聚磷酸钠的质量比为1：1至1：1.5，优选为1：1.2，混合均匀，室温反应30-50min，得到四氧化三铁磷酸盐纳米颗粒；将上述反应后的四氧化三铁磷酸盐纳米颗粒磁性分离后重新分散于50-80mL超纯水中；将该溶液缓慢加入到50-80mL浓度为80～100mg/L聚乙烯亚胺(PEI)水溶液中，搅拌20-30min混合均匀，经磁性分离(磁性分离为强力磁铁分离)，重悬至100-120mL的无菌水得到聚乙烯亚胺(PEI)包覆磁性氧化铁纳米颗粒溶液；The ferroferric oxide nanoparticles obtained in step (1) are dispersed in sterile water, and sodium tripolyphosphate is added, the mass ratio of Fe3 O4 nanoparticles to sodium tripolyphosphate is 1:1 to 1:1.5, preferably 1:1.2, mixed evenly, and reacted at room temperature for 30-50 minutes to obtain ferroferric oxide phosphate nanoparticles; the ferroferric oxide phosphate nanoparticles after the above reaction are magnetically separated and then redispersed in 50-80 mL ultrapure water; the solution is slowly added into 50-80 mL of 80-100 mg/L polyethyleneimine (PEI) aqueous solution, stirred for 20-30 minutes to mix evenly, subjected to magnetic separation (magnetic separation is separation with a strong magnet), and resuspended in 100-120 mL of sterile water to obtain polyethyleneimine (PEI) coated magnetic iron oxide nanoparticle solution;
(3)岩藻多糖的自组装(3) Self-assembly of fucoidan
将步骤(1)得到的纳米颗粒溶液与岩藻多糖溶液室温下混合吸附30min，7000～8000rpm离心10min，弃掉上清，加入无菌水重悬，制得结合岩藻多糖的磁性纳米颗粒(Fuc-NPs)。The nanoparticle solution obtained in step (1) was mixed with the fucoidan solution for adsorption at room temperature for 30 minutes, centrifuged at 7000-8000 rpm for 10 minutes, the supernatant was discarded, and sterile water was added for resuspending to obtain magnetic nanoparticles (Fuc-NPs) bound to fucoidan.
其中，岩藻多糖的分子量范围为5kDa～130kDa，优选为80～130kDa。The molecular weight of fucoidan ranges from 5 kDa to 130 kDa, preferably from 80 to 130 kDa.
其中，纳米颗粒溶液与岩藻多糖的质量比为3：1～1：1，其中最优为2：1。Among them, the mass ratio of nanoparticle solution to fucoidan is 3:1 to 1:1, and the optimal ratio is 2:1.
本发明还提供了多功能基因载体在递送抗肿瘤活性的miRNA中的用途。其中，miRNA与磁性纳米颗粒的结合方法为：The present invention also provides the use of a multifunctional gene carrier in delivering miRNA with anti-tumor activity. The method for combining miRNA with magnetic nanoparticles is as follows:
Fuc-NPs溶液与无菌水(无菌超纯水)DEPC溶解的miRNA(20μM)混合，两者在室温吸附30min，使miRNA组装至磁核，离心7000～8000rpm，10min，弃上清，用无菌水重悬，制得负载miRNA磁性纳米颗粒(Fuc-5-FU-miRNA NPs)。The Fuc-NPs solution was mixed with miRNA (20 μM) dissolved in DEPC in sterile water (sterile ultrapure water), and the two were adsorbed at room temperature for 30 min to assemble the miRNA into the magnetic core. The mixture was centrifuged at 7000-8000 rpm for 10 min, the supernatant was discarded, and the solution was resuspended in sterile water to obtain miRNA-loaded magnetic nanoparticles (Fuc-5-FU-miRNA NPs).
其中，纳米颗粒溶液与miRNA的体积比为10：1至10：3，其中最优为10：2。The volume ratio of the nanoparticle solution to miRNA is 10:1 to 10:3, with the optimal ratio being 10:2.
本发明还提供了一种多功能基因载体负载抗肿瘤活性的miRNA分子得到的复合物。The present invention also provides a complex obtained by loading a multifunctional gene carrier with miRNA molecules having anti-tumor activity.
本发明中的递送的miRNA有如下特点：The delivered miRNA in the present invention has the following characteristics:
miRNA mimics的正义链中尿嘧啶和/或反义链中尿嘧啶全部被氟尿嘧啶替代，即5-FU-miRNA mimics。The uracil in the sense strand and/or the uracil in the antisense strand of miRNA mimics are all replaced by fluorouracil, namely 5-FU-miRNA mimics.
进一步的，本发明还提供了一种上述复合物在制备预防或治疗肿瘤疾病的药物和制剂中的用途。Furthermore, the present invention also provides a use of the above-mentioned complex in the preparation of drugs and preparations for preventing or treating tumor diseases.
本发明的优点为：The advantages of the present invention are:
本发明提供的多功能基因载体。该纳米颗粒有如下优点：The multifunctional gene carrier provided by the present invention has the following advantages:
(1)制备工艺简单：岩藻多糖和miRNA通过层层自组装的方式结合至纳米磁核，制备方法简单，条件温和，易于操作和重复。(1) Simple preparation process: Fucoidan and miRNA are combined to the nanomagnetic core through layer-by-layer self-assembly. The preparation method is simple, the conditions are mild, and it is easy to operate and repeat.
(2)双靶向作用：磁靶向和岩藻多糖对高表达于肿瘤中的p-选择素的靶向作用，均能高效的将miRNA递送到肿瘤内部，减少对其他器官组织的毒副作用，增加肿瘤部位的miRNA有效剂量。(2) Dual targeting: Magnetic targeting and fucoidan targeting p-selectin, which is highly expressed in tumors, can both efficiently deliver miRNA into the tumor, reduce toxic side effects on other organs and tissues, and increase the effective dose of miRNA in the tumor site.
(3)三重给药系统：该纳米颗粒负载的岩藻多糖作为效应分子能够激活免疫系统，改善免疫抑制的肿瘤微环境，与5-FU-miRNA mimics产生协同抗肿瘤作用；其携载的miRNA能靶向癌基因抑制肿瘤生长，释放的游离5-FU又能直接杀伤肿瘤细胞和肿瘤间质细胞，从而实现肿瘤的系统化治疗。本项目设计的“一箭三雕”的纳米给药系统为肿瘤靶向纳米释药体系提供了新的协同设计策略。(3) Triple drug delivery system: The fucoidan loaded on the nanoparticles can activate the immune system as an effector molecule, improve the immunosuppressive tumor microenvironment, and produce a synergistic anti-tumor effect with 5-FU-miRNA mimics; the miRNA it carries can target oncogenes to inhibit tumor growth, and the released free 5-FU can directly kill tumor cells and tumor stromal cells, thereby achieving systematic treatment of tumors. The "three birds with one stone" nano drug delivery system designed in this project provides a new synergistic design strategy for tumor-targeted nano drug release systems.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1.不同分子量的岩藻多糖免疫免疫调节活性比较Figure 1. Comparison of immunomodulatory activity of fucoidan with different molecular weights
图2.Fuc-5-FU-miRNA NPs的形态及表征Figure 2. Morphology and characterization of Fuc-5-FU-miRNA NPs
图3.Fuc-5-FU-miRNA NPs的平均粒径及Zeta电位Figure 3. Average particle size and Zeta potential of Fuc-5-FU-miRNA NPs
图4.Fuc-5-FU-miRNA NPs的摄取Figure 4. Uptake of Fuc-5-FU-miRNA NPs
图5.激光共聚焦显微镜表征Fuc-5-FU-miRNA NPs在细胞内的摄取Figure 5. Confocal laser scanning microscopy characterization of the intracellular uptake of Fuc-5-FU-miRNA NPs
图6.Fuc-5-FU-miRNA NPs对靶基因表达的影响Figure 6. Effect of Fuc-5-FU-miRNA NPs on target gene expression
图7.Fuc-5-FU-miRNA NPs的细胞毒性作用Figure 7. Cytotoxic effect of Fuc-5-FU-miRNA NPs
图8.Transwell共培养体系表征Fuc-5-FU-miRNA NPs和5-FU-miRNA NPs对细胞活力的影响Figure 8. Transwell co-culture system characterization of the effects of Fuc-5-FU-miRNA NPs and 5-FU-miRNA NPs on cell viability
图9.Fuc-5-FU-miRNA NPs的体内抗肿瘤活性。(a)肿瘤组织拍照图像；Figure 9. In vivo antitumor activity of Fuc-5-FU-miRNA NPs. (a) Photograph of tumor tissue;
(b)肿瘤的体积和重量；(c)肿瘤组织Ki67、CD68、CD206免疫组化代表性图像。所有图像的比例尺为50μm。(b) Tumor volume and weight; (c) Representative images of Ki67, CD68, and CD206 immunohistochemistry of tumor tissues. The scale bar of all images is 50 μm.
具体实施方式Detailed ways
通过以下具体实施例对本发明方案作进一步的具体说明，但本发明要求保护的范围并不局限于实例表述的范围。The present invention is further described in detail by the following specific examples, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.
实施例1：岩藻多糖和低分子量岩藻多糖免疫调节活性比较Example 1: Comparison of immunomodulatory activity of fucoidan and low molecular weight fucoidan
本发明比较了低分子量岩藻多糖LF2(7.2kDa)和岩藻多糖FPS1M(130kDa)的免疫调节活性。The present invention compares the immunomodulatory activities of low molecular weight fucoidan LF2 (7.2 kDa) and fucoidan FPS1M (130 kDa).
Raw264.7以7000细胞/孔接种于96孔板。用未诱导的Raw264.7细胞作为M0巨噬细胞。20ng/mL IFN-γ(PEPROTECH)和100ng/mL LPS(源叶生物)孵育24h后，M0巨噬细胞极化为M1巨噬细胞。20ng/mL IL-4(PEPROTECH)和20ng/mL IL-13(PEPROTECH)孵育M0巨噬细胞24h，得到M2型巨噬细胞。继续培养细胞12h，收集培养基上清。同时，FPS1M(200μg/mL)和LF2(200μg/mL)分别孵育M0巨噬细胞36h，收集细胞的上清测定NO和细胞因子(IL-6和TNF-α)的水平。使用ELISA(博士德)检测Raw264.7细胞培养液上清中IL-6和TNF-α的含量。一氧化氮(NO)用一氧化氮测定试剂盒按说明书(碧云天)检测。Raw264.7 was seeded in a 96-well plate at 7000 cells/well. Uninduced Raw264.7 cells were used as M0 macrophages. After incubation with 20ng/mL IFN-γ (PEPROTECH) and 100ng/mL LPS (Yuanye Bio) for 24h, M0 macrophages were polarized into M1 macrophages. M0 macrophages were incubated with 20ng/mL IL-4 (PEPROTECH) and 20ng/mL IL-13 (PEPROTECH) for 24h to obtain M2 macrophages. The cells were cultured for 12h and the culture supernatant was collected. At the same time, FPS1M (200μg/mL) and LF2 (200μg/mL) were used to incubate M0 macrophages for 36h, and the supernatant of the cells was collected to determine the levels of NO and cytokines (IL-6 and TNF-α). ELISA (Boster) was used to detect the content of IL-6 and TNF-α in the culture supernatant of Raw264.7 cells. Nitric oxide (NO) was detected using a nitric oxide assay kit according to the instructions (Biyuntian).
图1显示，FPS1M和LF2均能促进巨噬细胞释放促炎因子(NO,IL-6和TNFα)。其中，FPS1M较LF2发挥更好的促进巨噬细胞M1分化为M1表型的作用。这表明高分子量的岩藻多糖较低分子量的岩藻多糖发挥更好的免疫刺激活性。因此，Fuc-5-FU-miR-15a NPs的制备优选高分子量岩藻多糖(80～130kDa)。Figure 1 shows that both FPS1M and LF2 can promote the release of pro-inflammatory factors (NO, IL-6 and TNFα) from macrophages. Among them, FPS1M plays a better role in promoting the differentiation of macrophage M1 into M1 phenotype than LF2. This indicates that high molecular weight fucoidan has better immunostimulatory activity than low molecular weight fucoidan. Therefore, high molecular weight fucoidan (80-130 kDa) is preferred for the preparation of Fuc-5-FU-miR-15a NPs.
实施例2：负载miRNA的多功能基因载体的制备和表征Example 2: Preparation and characterization of multifunctional gene vector loaded with miRNA
2.1负载miR-15a的多功能基因载体(Fuc-5-FU-miR-15a NPs)的制备2.1 Preparation of multifunctional gene carriers loaded with miR-15a (Fuc-5-FU-miR-15a NPs)
将15mmol无水氯化铁，5mmol氢氧化钠，50mL乙二醇以及3.6ml去离子水混合均匀后转移到三口瓶中，加热至沸腾，随后保持沸腾回流8h，停止反应，冷却至室温，经分离、洗涤得到Fe3O4纳米颗粒。15mmol anhydrous ferric chloride, 5mmol sodium hydroxide, 50mL ethylene glycol and 3.6ml deionized water were mixed evenly and transferred to a three-necked flask, heated to boiling, then kept boiling and refluxed for 8h, the reaction was stopped, cooled to room temperature, andFe3O4nanoparticles were obtained by separation and washing.
将0.2g上述所得到的四氧化三铁纳米颗粒分散于100mL去离子水中，加入200mg三聚磷酸钠，混合均匀，磁性分离后重新分散于100mL水中；将该溶液缓慢加入到50mL浓度为100mg/L聚乙烯亚胺水溶液中，混合均匀，经磁性分离或离心分离，得到PEI包覆磁性氧化铁纳米颗粒。0.2 g of the above-obtained ferrosoferric oxide nanoparticles was dispersed in 100 mL of deionized water, 200 mg of sodium tripolyphosphate was added, mixed evenly, and redispersed in 100 mL of water after magnetic separation; the solution was slowly added into 50 mL of a 100 mg/L polyethyleneimine aqueous solution, mixed evenly, and subjected to magnetic separation or centrifugal separation to obtain PEI-coated magnetic iron oxide nanoparticles.
纳米颗粒溶液100μL与岩藻多糖溶液10μL(1mg/mL)室温下吸附30min，使岩藻多糖自组装至磁核外层。上述溶液离心7000rpm，10min，弃掉上清，加入无菌水重悬，制得结合岩藻多糖的磁性纳米颗粒(Fuc-NPs)。100 μL of the nanoparticle solution and 10 μL of the fucoidan solution (1 mg/mL) were adsorbed at room temperature for 30 min to allow the fucoidan to self-assemble on the outer layer of the magnetic core. The above solution was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and sterile water was added to resuspend it to obtain magnetic nanoparticles (Fuc-NPs) bound to fucoidan.
Fuc-NPs溶液100μL与20μL无菌水溶解的5-FU-miR-15a mimics(20μM)混合，两者在室温吸附30min，使miRNA吸附组装至磁核，离心7000rpm，10min，弃上清，用无菌水重悬，100 μL of Fuc-NPs solution was mixed with 20 μL of 5-FU-miR-15a mimics (20 μM) dissolved in sterile water. The two were adsorbed at room temperature for 30 min to allow miRNA to be adsorbed and assembled onto the magnetic core. The mixture was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the mixture was resuspended in sterile water.
图2显示了Fuc-5-FU-miR-15a NPs的模拟结合方式，最终得到的Fuc-5-FU-miR-15a NPs为稳定的磁性纳米颗粒体系。FIG2 shows the simulated binding mode of Fuc-5-FU-miR-15a NPs, and the final obtained Fuc-5-FU-miR-15a NPs is a stable magnetic nanoparticle system.
2.2Fuc-5-FU-miR-15a NPs的表征2.2 Characterization of Fuc-5-FU-miR-15a NPs
采用Malvern Zetasizer纳米ZS设备(Malvern,UK)，在散射光探测角分别为90和15时，利用动态光散射(DLS)分析了25℃下胶束的尺寸分布和Zeta电位。The size distribution and zeta potential of the micelles were analyzed at 25 °C by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS instrument (Malvern, UK) at scattered light detection angles of 90 and 15, respectively.
用透射电子显微镜(TEM)分析了纳米颗粒的形态。样品溶液(10μL；1mg/mL)滴到镀有无定形碳的铜格栅上，并在干燥器中自然干燥。将1wt％的醋酸铀酰水溶液滴于铜格栅上，使样品染色1min。用滤纸吸干后，样品在干燥器中彻底干燥后，再进行TEM观察。样品最终在Philips CM120透射电子显微镜(Philips,Netherlands)上观察。The morphology of the nanoparticles was analyzed by transmission electron microscopy (TEM). The sample solution (10 μL; 1 mg/mL) was dropped onto a copper grid coated with amorphous carbon and dried naturally in a desiccator. A 1 wt % aqueous solution of uranyl acetate was dropped onto the copper grid to stain the sample for 1 min. After blotting with filter paper, the sample was thoroughly dried in a desiccator before TEM observation. The sample was finally observed on a Philips CM120 transmission electron microscope (Philips, Netherlands).
透射电镜显示该多功能基因载体为均匀分散的圆形颗粒(图2)。Transmission electron microscopy showed that the multifunctional gene carrier was uniformly dispersed round particles (Figure 2).
图3显示纳米载体平均粒径为58nm，Zeta电位为+38.6mV。将PEI-磁性氧化铁纳米颗粒与岩藻多糖溶液混合后通过自组装形成Fuc-NPs,此时纳米复合物的粒径约为72.3nm，Zeta电位为-17.7mV。最后再与5-FU-miR-15a mimics复合形成Fuc-5-FU-miR-15a NPs，此时纳米复合物粒径为143.6nm，Zeta电位为-33.1mV。随着逐步的结合，电荷逐渐降低，粒径逐渐变大，说明5-FU修饰的miRNA-15a mimics和岩藻多糖成功结合至纳米氧化铁磁核，成功制备了Fuc-5-FU-miR-15a-NPs磁性纳米颗粒。Figure 3 shows that the average particle size of the nanocarrier is 58nm and the Zeta potential is +38.6mV. After mixing PEI-magnetic iron oxide nanoparticles with fucoidan solution, Fuc-NPs are formed by self-assembly. At this time, the particle size of the nanocomposite is about 72.3nm and the Zeta potential is -17.7mV. Finally, it is compounded with 5-FU-miR-15a mimics to form Fuc-5-FU-miR-15a NPs. At this time, the particle size of the nanocomposite is 143.6nm and the Zeta potential is -33.1mV. With the gradual combination, the charge gradually decreases and the particle size gradually increases, indicating that the 5-FU modified miRNA-15a mimics and fucoidan are successfully combined with the nano-iron oxide magnetic core, and the Fuc-5-FU-miR-15a-NPs magnetic nanoparticles are successfully prepared.
实施例3.Fuc-5-FU-miR-15a NPs的细胞摄取及靶向作用Example 3. Cellular uptake and targeting of Fuc-5-FU-miR-15a NPs
2.1Fuc-5-FU-miR-15a NPs的细胞摄取2.1 Cellular uptake of Fuc-5-FU-miR-15a NPs
2.1.1qRT-PCR的方法检测miR-15a的释放2.1.1 Detection of miR-15a release by qRT-PCR
三株胰腺癌细胞(sw1990,ASPC-1,Paca-mia-2)以1x105/mL的密度接种于6孔板，24小时之后，用Fuc-5-FU-miR-15a NPs处理癌细胞6小时。总RNA使用Trizol法提取。PrimeScriptTM RT reagent Kit(Taraka,日本)用于合成cDNA。采用SYBR Green MasterMix和Light Cycler PCR检测系统(ABI PRISM-7900)进行定量RT-PCR。miR-15a的5’引物序列为：CGCCTAGCAGCACATAATGGTTTGTG，3‘端为试剂盒提供的mRQ3’通用序列。U6基因表达水平作为内参。根据公式计算相对miRNA水平:ΔΔCT＝ΔCT检测样品-ΔCT对照样品。使用2-ΔΔCT方法计算基因表达的变化。Three pancreatic cancer cell lines (sw1990, ASPC-1, Paca-mia-2) were seeded in 6-well plates at a density of 1x105 /mL. After 24 hours, the cancer cells were treated with Fuc-5-FU-miR-15a NPs for 6 hours. Total RNA was extracted using the Trizol method. PrimeScriptTM RT reagent Kit (Taraka, Japan) was used to synthesize cDNA. Quantitative RT-PCR was performed using SYBR Green MasterMix and Light Cycler PCR Detection System (ABI PRISM-7900). The 5' primer sequence of miR-15a was: CGCCTAGCAGCACATAATGGTTTGTG, and the 3' end was the mRQ3' universal sequence provided by the kit. The U6 gene expression level was used as an internal reference. The relative miRNA level was calculated according to the formula: ΔΔCT = ΔCTtest sample - ΔCTcontrol sample . The changes in gene expression were calculated using the 2-ΔΔCT method.
2.1.2HPLC方法检测5-FU的释放2.1.2 HPLC method to detect the release of 5-FU
HPLC方法测定5-FU的释放量。三株胰腺癌细胞以1x105/mL的密度接种于6孔板，24小时之后，用Fuc-5-FU-miR-15a NPs处理癌细胞6小时。收集细胞，超声处理30min，离心之后使用CBM-L20液相色谱系统(岛津，日本)检测5-FU的含量。The release of 5-FU was determined by HPLC. Three pancreatic cancer cells were seeded in a 6-well plate at a density of 1x105 /mL. After 24 hours, the cancer cells were treated with Fuc-5-FU-miR-15a NPs for 6 hours. The cells were collected, sonicated for 30 minutes, and centrifuged to detect the 5-FU content using a CBM-L20 liquid chromatography system (Shimadzu, Japan).
具体的，使用C18反相柱，流动相，甲醇：水＝10：90，柱温30℃，进样量为20μL，检测波长为265nm。Specifically, a C18 reverse phase column was used, the mobile phase was methanol: water = 10:90, the column temperature was 30°C, the injection volume was 20 μL, and the detection wavelength was 265 nm.
2.1.3激光共聚焦检测Fuc-5-FU-miR-15a NPs@Cy3磁性纳米颗粒在细胞的摄取2.1.3 Laser confocal microscopy detection of cellular uptake of Fuc-5-FU-miR-15a NPs@Cy3 magnetic nanoparticles
在5-FU-miR-15a mimics合成时加入Cy3标记，制备Fuc-5-FU-miR-15a NPs@Cy3磁性纳米颗粒。将有荧光标记的Fuc-5-FU-miR-15a NPs@Cy3磁性纳米颗粒(5-FU-miR-15a终浓度50nM)与接种在细胞爬片上的ASPC-1细胞共孵育0和6小时，用PBS清洗3次，用CellMaskgreen stain(细胞质膜绿色染料)37℃染色10min，PBS清洗3次之后，DAPI室温染细胞核10min。封片剂封片，用激光共聚焦显微镜进行观察。Cy3 was added to the synthesis of 5-FU-miR-15a mimics to prepare Fuc-5-FU-miR-15a NPs@Cy3 magnetic nanoparticles. Fluorescently labeled Fuc-5-FU-miR-15a NPs@Cy3 magnetic nanoparticles (5-FU-miR-15a final concentration 50nM) were co-incubated with ASPC-1 cells seeded on cell slides for 0 and 6 hours, washed 3 times with PBS, stained with CellMaskgreen stain (cytoplasmic membrane green dye) at 37°C for 10 minutes, washed 3 times with PBS, and then stained with DAPI at room temperature for 10 minutes for cell nuclei. The slides were sealed with sealing agents and observed with a laser confocal microscope.
图4基因表达分析发现Fuc-5-FU-miR-15a-NPs可被细胞高效摄取，在细胞中释放出miR-15a和5-FU，发挥细胞生长抑制作用。Figure 4 Gene expression analysis found that Fuc-5-FU-miR-15a-NPs can be efficiently taken up by cells, releasing miR-15a and 5-FU in cells, exerting a cell growth inhibitory effect.
图5显示了5-FU-miR-15a@Cy3在细胞内的积累，表明Fuc-5-FU-miR-15a NPs能够成功将5-FU-miR-15a递送至细胞内，发挥抗肿瘤活性。Figure 5 shows the accumulation of 5-FU-miR-15a@Cy3 in cells, indicating that Fuc-5-FU-miR-15a NPs can successfully deliver 5-FU-miR-15a into cells and exert antitumor activity.
2.2Fuc-5-FU-miR-15a NPs靶基因的表达情况2.2 Expression of target genes of Fuc-5-FU-miR-15a NPs
使用qRT-PCR的方法验证了Fuc-5-FU-miR-15a NPs靶基因的表达情况。三株胰腺癌细胞以1x105/mL的密度接种于6孔板，24小时之后，用含Fuc-5-FU-miR-15a NPs的DMEM处理癌细胞24小时。收集细胞，提取总RNA。实验方法参考实施例2.1.1。引物的序列YAP-1，5’-primer:CAGAACCGTTTCCCAGACTACCTTG，3’-primer:GCAGACTTGGCATCAGCTCCTC；BCL-2,5’-primerTCGCCCTGTGGATGACTGAGTAC,3’-primerACAGCCAGGAGAAATCAAACAGAGG；TS，5’-primerCTTCAGCGAGAACCCAGACCTTTC，3’-primerAGTTGGATGCGGATTGTACCCTTC。The expression of target genes of Fuc-5-FU-miR-15a NPs was verified by qRT-PCR. Three pancreatic cancer cells were seeded in 6-well plates at a density of 1x105 /mL. After 24 hours, the cancer cells were treated with DMEM containing Fuc-5-FU-miR-15a NPs for 24 hours. The cells were collected and total RNA was extracted. The experimental method was as described in Example 2.1.1. The sequences of the primers YAP-1, 5'-primer: CAGAACCGTTTCCCAGACTACCTTG, 3'-primer: GCAGACTTGGCATCAGCTCCTC; BCL-2, 5'-primerTCGCCCTGTGGATGACTGAGTAC, 3'-primerACAGCCAGGAGAAATCAAACAGAGG; TS, 5'-primerCTTCAGCGAGAACCCAGACCTTTC, 3'-primerAGTTGGATGCGGATTGTACCCTTC.
Fuc-5-FU-miR-15a-NPs成功将5-FU-miR-15a释放入细胞，显著的降低了靶基因Bcl-2，Yap-1和TS的表达(图6)。Fuc-5-FU-miR-15a-NPs successfully released 5-FU-miR-15a into cells and significantly reduced the expression of target genes Bcl-2, Yap-1, and TS (Figure 6).
实施例4.Fuc-5-FU-miR-15a NPs的细胞毒性作用Example 4. Cytotoxicity of Fuc-5-FU-miR-15a NPs
3.1Fuc-5-FU-miR-15a NPs的细胞毒作用3.1 Cytotoxic effect of Fuc-5-FU-miR-15a NPs
通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑(MTT)测定法(Sigma-Aldrich)测定细胞活力。将细胞接种在96孔板(7×103个细胞/孔)中，12小时后加入Fuc-5-FU-miR-15a NPs纳米颗粒，再孵育48小时后，将10μL的MTT溶液(5mg/mL)添加到每个孔中，然后将细胞在37℃下再孵育4小时。轻轻弃去培养基，每孔加入150μL DMSO溶解不溶性甲臜，用酶标仪(Tecan，Switzerland)测定570nm处的吸光度。Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Cells were seeded in 96-well plates (7×103 cells/well), and Fuc-5-FU-miR-15a NPs nanoparticles were added 12 hours later. After incubation for another 48 hours, 10 μL of MTT solution (5 mg/mL) was added to each well, and the cells were incubated at 37°C for another 4 hours. The culture medium was gently discarded, and 150 μL of DMSO was added to each well to dissolve the insoluble formazan. The plate was analyzed using a microplate reader (Tecan, Switzerland) to measure the absorbance at 570 nm.
图7表明Fuc-5-FU-miR-15a NPs显著降低了sw1990和Mia-2细胞的细胞活力。且与单独使用5-FU-miR-15a mimics相比，表现出更好的杀伤肿瘤细胞的活性。Figure 7 shows that Fuc-5-FU-miR-15a NPs significantly reduced the cell viability of sw1990 and Mia-2 cells, and showed better tumor cell killing activity compared with 5-FU-miR-15a mimics alone.
进一步，我们比较了Fuc-5-FU-miR-15a NPs与不负载岩藻多糖的纳米颗粒5-FU-miR-15a NPs的对细胞活力的作用。Furthermore, we compared the effects of Fuc-5-FU-miR-15a NPs with those of 5-FU-miR-15a NPs without fucoidan loading on cell viability.
采用Transwell体系(0.4μm)构建SW1990细胞与巨噬细胞的共培养体系。5X104个Raw264.7细胞接种于24孔板细胞培养皿底部。并按照实施例1的方法诱导巨噬细胞为不同的表型。用药组分别加Fuc-5-FU-miR-15a NPs和5-FU-miR-15a NPs处理下层细胞。将7000个SW1990细胞接种于Transwell小室的上层，与巨噬细胞共培养48h。下腔的NPs和巨噬细胞释放的`细胞因子会影响上腔中SW1990细胞的生存。将上腔的SW1990细胞用PBS冲洗3次，4％多聚甲醛固定10min,0.2％结晶紫染色15min,PBS冲洗3次。染色细胞在光学显微镜(徕卡)下拍摄，并通过Image J软件进行量化。The co-culture system of SW1990 cells and macrophages was constructed using the Transwell system (0.4 μm). 5×10 4 Raw264.7 cells were inoculated at the bottom of a 24-well cell culture dish. And the macrophages were induced to different phenotypes according to the method of Example 1. The medication groups were treated with Fuc-5-FU-miR-15a NPs and 5-FU-miR-15a NPs to treat the lower cells. 7000 SW1990 cells were inoculated in the upper layer of the Transwell chamber and co-cultured with macrophages for 48 hours. The cytokines released by the NPs and macrophages in the lower chamber affect the survival of SW1990 cells in the upper chamber. The SW1990 cells in the upper chamber were rinsed 3 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, stained with 0.2% crystal violet for 15 minutes, and rinsed 3 times with PBS. The stained cells were photographed under an optical microscope (Leica) and quantified by Image J software.
负载岩藻多糖的纳米颗粒(Fuc-5-FU-miR15a NPs)较5-FU-miR15a NPs发挥更好的抗肿瘤活性，推测这与负载的岩藻多糖促进巨噬细胞表现为促炎表型有关(图8)。Nanoparticles loaded with fucoidan (Fuc-5-FU-miR15a NPs) exerted better antitumor activity than 5-FU-miR15a NPs, which was speculated to be related to the fact that the loaded fucoidan promoted macrophages to exhibit a pro-inflammatory phenotype ( Figure 8 ).
实施例5.Fuc-5-FU-miR-15a NPs的体内抗肿瘤活性研究Example 5. Study on the anti-tumor activity of Fuc-5-FU-miR-15a NPs in vivo
雄性BALB/C小鼠，6～8周龄，购自北京维通利华实验动物有限公司，在SPF动物中心饲养。所有动物均按照《中国科学院海洋研究所实验动物护理与使用卫生指南》的建议进行护理。取对数生长期的HCT116细胞，以1×107细胞/mL皮下注射100μL至小鼠右肢上侧。当肿瘤生长到大约100mm3时，小鼠被随机分成CK组，5-FU-miR-15a组，Fuc-5-FU-miR-15a NPs组，CK组尾静脉注射生理盐水，5-FU-miR-15a组尾静脉注射80μg的5-FU-miR-15a mimics(in vivo-jetPEI预包装)，Fuc-5-FU-miR-15a NPs组尾静脉注射Fuc-5-FU-miR-15a NPs(NPs中含80μg的5-FU-miR-15a)，并放置磁石在肿瘤部位。每3天注射一次，共注射7次。根据公式V＝W2×L×0.5计算估算的肿瘤体积(V)，其中W代表最大的肿瘤直径(厘米)，L代表第二大的肿瘤直径。麻醉后，取小鼠血清进行生化分析(青岛金德医学检验中心)。将切除的肿瘤在4％多聚甲醛中固定至少24h，然后制备4μm的石蜡切片。固定的肿瘤组织用于免疫组化研究，其余肿瘤在-80℃冷冻备用。Male BALB/C mice, 6 to 8 weeks old, were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd. and raised in the SPF Animal Center. All animals were cared for in accordance with the recommendations of the "Guidelines for the Care and Use of Laboratory Animals, Institute of Oceanology, Chinese Academy of Sciences". HCT116 cells in the logarithmic growth phase were taken and injected subcutaneously with 100 μL at 1×107 cells/mL into the upper side of the right limb of the mouse. When the tumor grew to about 100 mm3 , the mice were randomly divided into CK group, 5-FU-miR-15a group, and Fuc-5-FU-miR-15a NPs group. The CK group was injected with normal saline, the 5-FU-miR-15a group was injected with 80 μg of 5-FU-miR-15a mimics (pre-packaged in vivo-jetPEI), and the Fuc-5-FU-miR-15a NPs group was injected with Fuc-5-FU-miR-15a NPs (NPs containing 80 μg of 5-FU-miR-15a), and a magnet was placed at the tumor site. The injection was once every 3 days for a total of 7 injections. The estimated tumor volume (V) was calculated according to the formula V = W2 × L × 0.5, where W represents the largest tumor diameter (cm) and L represents the second largest tumor diameter. After anesthesia, the mouse serum was collected for biochemical analysis (Qingdao Jinde Medical Testing Center). The excised tumors were fixed in 4% paraformaldehyde for at least 24 h, and then 4 μm paraffin sections were prepared. The fixed tumor tissues were used for immunohistochemical studies, and the remaining tumors were frozen at -80°C for future use.
与空白相比Fuc-5-FU-miR-15a NPs显著的降低了肿瘤的体积。5-FU-miR-15amimics和Fuc-5-FU-miR-15a NPs均显著降低了肿瘤的重量，但是，Fuc-5-FU-miR-15a NPs的抗肿瘤活性优于JetPEI预包装的5-FU-miR-15a mimics(图9a-b)。免疫组化显示，与JetPEI预包装的5-FU-miR-15a mimics相比，Fuc-5-FU-miR-15a NPs因携载了岩藻多糖，其能显著的增加肿瘤组织中M1巨噬细胞的浸润(CD86表达增加，CD206表达减少)，改善免疫抑制的肿瘤微环境，这可能促进了Fuc-5-FU-miR-15a NPs的抗肿瘤活性(图9c)。Compared with the blank, Fuc-5-FU-miR-15a NPs significantly reduced the tumor volume. Both 5-FU-miR-15amimics and Fuc-5-FU-miR-15a NPs significantly reduced the tumor weight, but the anti-tumor activity of Fuc-5-FU-miR-15a NPs was better than that of 5-FU-miR-15a mimics pre-packaged with JetPEI (Figure 9a-b). Immunohistochemistry showed that compared with 5-FU-miR-15a mimics pre-packaged with JetPEI, Fuc-5-FU-miR-15a NPs carried fucoidan, which could significantly increase the infiltration of M1 macrophages in tumor tissue (increased CD86 expression and decreased CD206 expression), improve the immunosuppressive tumor microenvironment, which may promote the anti-tumor activity of Fuc-5-FU-miR-15a NPs (Figure 9c).
补充内容to add on
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案精神和范围。The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20130189367A1 (en)* | 2011-07-29 | 2013-07-25 | University Of Washington Through Its Center For Commercialization | Nanovectors for targeted gene silencing and cytotoxic effect in cancer cells |
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| JP6771509B2 (en)* | 2017-05-01 | 2020-10-21 | 中國醫藥大學 | Immunomagnetic composition, its preparation method, its usage method and cancer treatment kit |
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| Publication | Publication Date | Title |
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| Rana et al. | Nanocarriers for cancer nano-immunotherapy | |
| Jin et al. | Floxuridine homomeric oligonucleotides “hitchhike” with albumin in situ for cancer chemotherapy | |
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