技术领域technical field
本发明涉及药物载体技术领域，尤其是涉及一种磁性纳米药物载体及其制备方法和应用。The invention relates to the technical field of drug carriers, in particular to a magnetic nano drug carrier and its preparation method and application.
背景技术Background technique
近年来，磁性纳米颗粒因其独特的性质被广泛应用于生物治疗和诊断等领域，其具有的表面化学活性可以很容易的结合生物大分子，通过在其表面修饰上特异性的配体使之成为很好的靶向性载体，磁性微粒在交变磁场作用下能吸收电磁波能量转化为热能，且可使热能局限于肿瘤组织，当温度超过41℃时可导致细胞的凋亡及坏死，因而可实现对肿瘤的热疗。In recent years, magnetic nanoparticles have been widely used in the fields of biotherapy and diagnosis due to their unique properties. Their surface chemical activity can easily bind biomacromolecules, and make them It becomes a good targeting carrier. Under the action of alternating magnetic field, magnetic particles can absorb electromagnetic wave energy and convert it into heat energy, and can confine the heat energy to tumor tissue. When the temperature exceeds 41°C, it can lead to apoptosis and necrosis of cells, thus Hyperthermia for tumors can be achieved.
在临床治疗中，药物的低毒和高效一直是人们研究的重点，药物靶向传递的研究，即如何药物高效治疗的效果越来越受到研究者的重视。叶酸是小分子量维生素，相对于单分子抗体等蛋白质，具有结构稳定、价格低廉、无免疫原性等特点，而且叶酸与叶酸受体结合力强，能被高效介导进入肿瘤细胞，是一种很有应用价值的靶向物质。根据叶酸受体在绝大多数恶性肿瘤细胞膜内有大量表达而正常细胞很少有表达这一特性，有研究将叶酸通过化学键连接到药物载体以及它们制成的纳米颗粒上，致使叶酸/叶酸受体介导靶向传递成为该领域的研究热点。In clinical treatment, the low toxicity and high efficiency of drugs have always been the focus of people's research, and the research on drug targeted delivery, that is, how to effectively treat drugs with high efficiency, has attracted more and more attention from researchers. Folic acid is a small molecular weight vitamin. Compared with proteins such as monomolecular antibodies, it has the characteristics of stable structure, low price, and no immunogenicity. Moreover, folic acid has a strong binding force with folic acid receptors and can be efficiently mediated into tumor cells. A target substance with great application value. According to the characteristic that folic acid receptors are highly expressed in the cell membrane of most malignant tumors but rarely expressed in normal cells, some studies have linked folic acid to drug carriers and nanoparticles made of them through chemical bonds, causing folic acid/folic acid to be stimulated. Body-mediated targeted delivery has become a research hotspot in this field.
磁纳米颗粒研究发展至今，颗粒的尺寸、表面分子的修饰、靶向性等都成为人们所关注的重点，磁性纳米颗粒可用在多种纳米生物技术中，例如利用磁共振成像(MRI)的分子成像、疾病的跟踪和诊断、高热疗法、药物递送、磁性生物传感器和微流体系统。尤其是，磁性纳米颗粒可用作MRI的诊断探针。Since the development of magnetic nanoparticle research, particle size, surface molecular modification, and targeting have become the focus of attention. Magnetic nanoparticles can be used in a variety of nanobiotechnologies, such as molecular imaging using magnetic resonance imaging (MRI). Imaging, disease tracking and diagnosis, hyperthermia, drug delivery, magnetic biosensors and microfluidic systems. In particular, magnetic nanoparticles can be used as diagnostic probes for MRI.
然而现有文献报道的纳米药物载体存在(1)毒性大、(2)难降解、(3)活体示踪困难等缺点，使得纳米药物载体的使用受到了一定的局限性。However, the nano-drug carriers reported in the existing literature have disadvantages such as (1) high toxicity, (2) difficult to degrade, and (3) difficult to trace in vivo, which makes the use of nano-drug carriers subject to certain limitations.
发明内容Contents of the invention
本发明的目的在于克服上述现有技术的不足之处而提供一种磁性纳米药物载体及其制备方法，将SP94靶向肽和siRNA输送载体相偶联，该磁性纳米药物载体具有较佳的安全性、稳定性、转染效率和生物相容性，并且磁性纳米药物载体将实时示踪、可控的siRNA释放和基因治疗相结合，实现了副作用低的抗肿瘤治疗的最佳策略。The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a magnetic nano drug carrier and its preparation method, which couples the SP94 targeting peptide and siRNA delivery carrier, and the magnetic nano drug carrier has better safety properties, stability, transfection efficiency and biocompatibility, and the combination of real-time tracking, controllable siRNA release and gene therapy by magnetic nano-drug carriers realizes the best strategy for anti-tumor therapy with low side effects.
为实现上述目的，本发明采取的技术方案为：In order to achieve the above object, the technical scheme that the present invention takes is:
本发明的第一方面提供了一种磁性纳米药物载体，所述磁性纳米药物载体包括SP94靶向肽和siRNA输送载体的偶联产物，所述siRNA输送载体的偶联产物包括超顺磁性氧化铁纳米颗粒、季铵阳离子化直链淀粉和四苯基乙烯偶联形成的季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒，所述siRNA吸附于磁性纳米药物载体中。The first aspect of the present invention provides a magnetic nano-drug carrier, the magnetic nano-drug carrier includes a coupling product of an SP94 targeting peptide and an siRNA delivery carrier, and the coupling product of the siRNA delivery carrier includes superparamagnetic iron oxide Nanoparticles, quaternary ammonium cationized amylose and tetraphenylethylene coupling formed quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles, the siRNA is adsorbed in the magnetic nano drug carrier .
本发明RNAi技术改善了纳米药物载体的安全性、稳定性、生物相容性及转染效率。与其他载体相比，本发明的载体在跨细胞膜运输时具有更高效率，并且若载体的大小和表面涂层合适，则可以防止排泄；有机纳米颗粒包括阳离子聚合物纳米颗粒，基于脂质的体系等，可以通过化学键合或物理包埋将药物掺入有机纳米颗粒中；阳离子聚合物纳米颗粒具有诸多优点：1.毒性低，生物相容性好，安全性高；2.可生物降解，免疫原性低；3.纳米载体表面应带正电；4.高基因转染效率；5.批量生产，成本低；6.更高的热力学稳定性和动态稳定性。本发明所用的直链淀粉构成的纳米颗粒除了具有阳离子聚合物纳米颗粒的共同优点外，还具有更高的生物相容性。The RNAi technology of the invention improves the safety, stability, biocompatibility and transfection efficiency of the nano drug carrier. Compared with other carriers, the carrier of the present invention has a higher efficiency in transport across the cell membrane, and if the size and surface coating of the carrier are suitable, it can prevent excretion; organic nanoparticles include cationic polymer nanoparticles, lipid-based system, etc., drugs can be incorporated into organic nanoparticles through chemical bonding or physical embedding; cationic polymer nanoparticles have many advantages: 1. Low toxicity, good biocompatibility, and high safety; 2. Biodegradable, Low immunogenicity; 3. The surface of the nanocarrier should be positively charged; 4. High gene transfection efficiency; 5. Mass production, low cost; 6. Higher thermodynamic stability and dynamic stability. In addition to the common advantages of cationic polymer nanoparticles, the nanoparticle composed of amylose used in the present invention also has higher biocompatibility.
本发明利用具有SPOI的MRI靶向性特征对纳米颗粒进行体外监测，SPIO具有超顺磁性，这些颗粒在没有外部磁场的情况下不具有任何磁性，当放置在外部磁场中时，它们的晶体会对齐并产生很高的局部磁场梯度，从而引起水质子自旋移相，从而减少周围水磁场的T1WI和T2WI弛豫时间；因此，SPIO产生具有T2WI、T2WI*序列的低信号，并能提高图像质量和对比度；SPIO具有良好的药效和药代动力学特征，其中的铁具有生物相容性，可通过生理性铁代谢被细胞再利用或回收；SPIO表面包覆多糖、聚乙二醇(PEG)，聚吡咯(PPy)，聚乳酸(PLA)等，可以提高生物相容性、生物降解性选用SPIO这一经典的T2WI阴性对比剂，其生物相容性高、低毒，具备MRI可视化的功能；The present invention utilizes the MRI targeting characteristics of SPOI for in vitro monitoring of nanoparticles, SPIO is superparamagnetic, these particles do not have any magnetism in the absence of an external magnetic field, when placed in an external magnetic field, their crystals will Align and generate high local magnetic field gradients, which cause water proton spin phase shifts, thereby reducing the T1WI and T2WI relaxation times of the surrounding water magnetic fields; thus, SPIO produces low signal with T2WI, T2WI* sequences and improves image Quality and contrast; SPIO has good pharmacodynamic and pharmacokinetic characteristics, and the iron in it is biocompatible and can be reused or recycled by cells through physiological iron metabolism; SPIO surface is coated with polysaccharides, polyethylene glycol ( PEG), polypyrrole (PPy), polylactic acid (PLA), etc., can improve biocompatibility and biodegradability. SPIO, a classic T2WI negative contrast agent, is selected for its high biocompatibility, low toxicity, and MRI visualization. function;
SP94靶向肽具有对各种人类肝细胞癌细胞系的高特异性亲和力，其与肝细胞癌细胞的亲和力比正常肝细胞、内皮细胞、外周血单核细胞、B淋巴细胞、T淋巴细胞高1万倍，SP94靶向肽的精确靶向特异性与递送系统相结合，可增强其靶向性。SP94 targeting peptide has high specific affinity to various human hepatocellular carcinoma cell lines, and its affinity to hepatocellular carcinoma cells is higher than that of normal liver cells, endothelial cells, peripheral blood mononuclear cells, B lymphocytes, and T lymphocytes 10,000-fold, the precise targeting specificity of the SP94 targeting peptide combined with the delivery system enhances its targeting.
在合成了油胺和油酸稳定的疏水SPIO后，由于四苯基乙烯(TPE)和SPIO都具有疏水特性，TPE通过疏水相互作用通过物理包埋的方式被引入到SPIO纳米粒子的内核中。因此，季铵阳离子化直链淀粉-超顺磁性氧化铁纳米颗粒可以在内核中引入大量疏水的TPE和SPIO，同时利用季铵阳离子化直链淀粉的亲水外壳层保持水溶性。After synthesizing hydrophobic SPIO stabilized by oleylamine and oleic acid, since both tetraphenylethylene (TPE) and SPIO have hydrophobic properties, TPE was introduced into the inner core of SPIO nanoparticles by physical embedding through hydrophobic interactions. Therefore, the quaternary amylose-cationized amylose-superparamagnetic iron oxide nanoparticles can introduce a large amount of hydrophobic TPE and SPIO in the inner core, while maintaining water solubility by utilizing the hydrophilic shell layer of quaternary amylose.
本发明制备的磁性纳米药物载体具有良好的释放Survivin siRNA的效果，5h后释放50.0％的Survivin siRNA，24h后释放79.8％。The magnetic nano drug carrier prepared by the invention has a good effect of releasing Survivin siRNA, releasing 50.0% of Survivin siRNA after 5 hours, and releasing 79.8% after 24 hours.
作为本发明所述磁性纳米药物载体的优选实施方式，所述超顺磁性氧化铁纳米颗粒的粒径为5～8nm。As a preferred embodiment of the magnetic nano drug carrier of the present invention, the particle diameter of the superparamagnetic iron oxide nanoparticles is 5-8 nm.
作为本发明所述磁性纳米药物载体的优选实施方式，所述磁性纳米药物载体的粒径为100～200nm。As a preferred embodiment of the magnetic nano-drug carrier of the present invention, the particle diameter of the magnetic nano-drug carrier is 100-200 nm.
作为本发明所述磁性纳米药物载体的优选实施方式，所述磁性纳米药物载体的表面电位为5.8mV，所述磁性纳米药物载体的聚合物分散性指数为0.25。As a preferred embodiment of the magnetic nano-drug carrier of the present invention, the surface potential of the magnetic nano-drug carrier is 5.8 mV, and the polymer dispersibility index of the magnetic nano-drug carrier is 0.25.
作为本发明所述磁性纳米药物载体的优选实施方式，所述季铵阳离子化直链淀粉的制备方法包括以下步骤：As a preferred embodiment of the magnetic nano drug carrier of the present invention, the preparation method of the quaternary ammonium cationized amylose comprises the following steps:
将直链淀粉加入蒸馏水中，用NaOH溶液调整pH＝12～14，并加热搅拌，缓慢滴加活性醚化剂的水溶液，继续搅拌反应12小时；反应结束后，用盐酸溶液调整pH到中性，将溶液通过截留分子量8000～14000Da的纤维素透析袋中，透析、过滤并冷冻干燥后得到季铵阳离子化直链淀粉。Add amylose to distilled water, use NaOH solution to adjust the pH to 12-14, heat and stir, slowly add the aqueous solution of active etherifying agent dropwise, and continue stirring for 12 hours; after the reaction, adjust the pH to neutral with hydrochloric acid solution The solution is passed through a cellulose dialysis bag with a molecular weight cut-off of 8000-14000 Da, dialyzed, filtered and freeze-dried to obtain quaternary ammonium cationized amylose.
本发明药物载体的骨架采用直链淀粉，是一种生物相容性好和降解性好的天然大分子，负载的MRI对比剂为超顺磁性氧化铁纳米颗粒(SPIO)，用来修饰载体的叶酸是小分子量维生素，结构稳定且无免疫原性。The skeleton of the drug carrier of the present invention adopts amylose, which is a natural macromolecule with good biocompatibility and good degradability, and the loaded MRI contrast agent is superparamagnetic iron oxide nanoparticles (SPIO), which is used to modify the carrier. Folic acid is a small molecular weight vitamin with stable structure and non-immunogenicity.
本发明在纳米药物载体的制作材料上选用安全、低毒的物质，采用“一锅法”合成，具有不经中间体的分离提纯和操作简化的优点，以保证合成载体的纯度。In the present invention, safe and low-toxic substances are selected as the production materials of the nano-medicine carrier, and the "one-pot method" is used for synthesis, which has the advantages of no separation and purification of intermediates and simplified operation, so as to ensure the purity of the synthesized carrier.
本发明的第二方面提供了上述磁性纳米药物载体的制备方法，包括以下步骤：A second aspect of the present invention provides a method for preparing the above-mentioned magnetic nano drug carrier, comprising the following steps:
S1、将季铵阳离子化直链淀粉加入蒸馏水中溶解形成季铵阳离子化直链淀粉水溶液，称量FeCl3·6H2O和FeCl2·4H2O溶于蒸馏水，然后加入至季铵阳离子化直链淀粉水溶液中形成混合物I，将混合物水浴加热，加入氨水，反应完后降温，将混合物透析、离心，得上清液，得季铵阳离子化直链淀粉-超顺磁性氧化铁纳米颗粒水溶液；S1. Add quaternary ammonium cationized amylose to dissolve in distilled water to form quaternary ammonium cationized amylose aqueous solution, weigh FeCl3 6H2 O and FeCl2 4H2 O and dissolve them in distilled water, then add to quaternary ammonium cationization Mixture I is formed in the amylose aqueous solution, the mixture is heated in a water bath, ammonia water is added, the temperature is lowered after the reaction is completed, the mixture is dialyzed and centrifuged to obtain a supernatant, and a quaternary ammonium cationized amylose-superparamagnetic iron oxide nanoparticle aqueous solution is obtained ;
S2、取四苯基乙烯溶于CH2Cl2中，加入步骤S1获得的季铵阳离子化直链淀粉-超顺磁性氧化铁纳米颗粒水溶液，混合均匀形成混合物II，在超声中将混合物II滴入至纯水中，然后透析、冷冻干燥，收集固体产物得季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒；S2. Dissolve tetraphenylethylene in CH2 Cl2 , add the quaternary ammonium cationized amylose-superparamagnetic iron oxide nanoparticle aqueous solution obtained in step S1, mix well to form mixture II, and drop the mixture II in ultrasonic into pure water, then dialyzed, freeze-dried, and collect the solid product to obtain quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles;
S3、将SP94靶向肽溶解于DMSO中，加入EDC·HCl和NHS，避光搅拌，得到SP94靶向肽活性酯的DMSO溶液，在步骤S2中季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒的水溶液加入SP94靶向肽活性酯的DMSO溶液形成混合物III，避光反应，透析，离心取上清液，得SP94靶向肽靶向修饰季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒的水溶液；S3. Dissolve the SP94 targeting peptide in DMSO, add EDC·HCl and NHS, and stir in the dark to obtain a DMSO solution of the SP94 targeting peptide active ester. In step S2, quaternized ammonium cationized amylose-tetraphenylethylene -The aqueous solution of superparamagnetic iron oxide nanoparticles is added to the DMSO solution of the SP94 targeting peptide active ester to form a mixture III, which is reacted in the dark, dialyzed, centrifuged to take the supernatant, and the SP94 targeting peptide is targeted to modify the quaternary ammonium cationized straight chain Aqueous solution of starch-tetraphenylethylene-superparamagnetic iron oxide nanoparticles;
S4、将上述步骤S3中的SP94靶向肽靶向修饰季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒的水溶液稀释，加入siRNA水溶液混合，形成磁性纳米药物载体。S4. Dilute the aqueous solution of SP94 targeting peptide targeting modified quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles in the above step S3, add siRNA aqueous solution and mix to form a magnetic nano drug carrier.
作为本发明所述磁性纳米药物载体的制备方法，所述步骤S1中氨水的浓度为质量浓度为25％。As the preparation method of the magnetic nano drug carrier of the present invention, the concentration of ammonia water in the step S1 is 25% by mass.
作为本发明所述磁性纳米药物载体的制备方法，所述步骤S1中透析截留混合物I的分子量为8000～14000Da；所述步骤S2中透析截留混合物II的分子量为2000Da；所述步骤S3中透析截留混合物III的分子量为8000～14000Da。As the preparation method of the magnetic nano drug carrier of the present invention, the molecular weight of the dialysis cut-off mixture I in the step S1 is 8000-14000Da; the molecular weight of the dialysis cut-off mixture II in the step S2 is 2000Da; the dialysis cut-off mixture II in the step S3 is The molecular weight of mixture III is 8000-14000 Da.
作为本发明所述磁性纳米药物载体的制备方法，所述步骤S4中SP94靶向肽靶向修饰季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒的水溶液中的氮与siRNA水溶液中的磷的摩尔比为5:1。As the preparation method of the magnetic nano drug carrier of the present invention, in the step S4, the SP94 targeting peptide targets to modify the nitrogen in the aqueous solution of quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles The molar ratio to phosphorus in the siRNA aqueous solution was 5:1.
本发明的第三方面提供了上述磁性纳米药物载体在制备癌症基因治疗药物中的应用。The third aspect of the present invention provides the application of the above-mentioned magnetic nano drug carrier in the preparation of cancer gene therapy drugs.
本发明还利用了活体荧光成像与磁共振成像相结合的双模态成像对药物治疗效果进行体外评估。与传统的成像技术相比，荧光成像具有成本低，灵敏度高和使用便利等优点，可用于实时监测分子过程和生物结构的时空分布，传统荧光团通常以低浓度在溶液中使用，因为由于聚集引起的ACQ效应，它们在高浓度下猝灭，从而限制了它们在超灵敏分析和长期监测中的进一步应用；具有AIE特性的荧光团溶解在溶液中时发射微弱，但在聚集态时显示荧光，此功能使它们可以在高浓度下发挥作用，使其具有出色的灵敏度和光稳定性。装有相应配体的AIE发光剂特别适用于具有高信噪比的癌细胞的光成像，荧光团在水溶液中几乎不发光，但在通过内吞作用被内化到癌细胞中后变为发射状态，因此可方便地以免洗方式进行癌细胞成像。MRI是一种非侵入性成像方式，无电离辐射，T2WI MRI序列可显示炎症和水肿；虽然传统的MRI只能基于任意单位的信号强度分析进行定性图像分析，但T2mapping基于体素方式评估弛豫时间可以实现组织成分的非侵入性可视化和量化，由于定量的T2值反映了组织成分，尤其是自由水含量，因此T2 mapping对组织的水合作用或水肿敏感，不需要对比剂，因此它有成为“非侵入性”活检的潜力。本发明使用经典T2 mapping方法量化分析T2WI信号变化，获得了同荧光图像相似的信号变化，证明可以通过定量分析MRI图像更好地分析纳米颗粒影响肿瘤信号的变化。采用多模态成像可以结合多种成像手段，实现各影像学方法优势互补，扩大各影像学方法的适用范围，集成MRI和光学成像将提供高分辨率、选择性和灵敏度。The present invention also utilizes the dual-mode imaging combining living body fluorescence imaging and magnetic resonance imaging to evaluate the drug treatment effect in vitro. Compared with traditional imaging techniques, fluorescence imaging has the advantages of low cost, high sensitivity and convenient use, and can be used to monitor the temporal and spatial distribution of molecular processes and biological structures in real time. Traditional fluorophores are usually used in solution at low concentrations because due to aggregation induced ACQ effect, they are quenched at high concentrations, thus limiting their further application in ultrasensitive analysis and long-term monitoring; fluorophores with AIE properties emit weakly when dissolved in solution, but show fluorescence when aggregated , this feature allows them to function at high concentrations, giving them excellent sensitivity and photostability. AIE luminescent agents loaded with corresponding ligands are particularly suitable for optical imaging of cancer cells with a high signal-to-noise ratio, the fluorophore hardly emits light in aqueous solution but becomes emitting after being internalized into cancer cells by endocytosis state, thus facilitating cancer cell imaging in a wash-free manner. MRI is a non-invasive imaging modality without ionizing radiation, and T2WI MRI sequences can show inflammation and edema; while traditional MRI can only perform qualitative image analysis based on signal intensity analysis in arbitrary units, T2mapping assesses relaxation based on a voxel-wise manner Time can achieve non-invasive visualization and quantification of tissue composition. Since the quantitative T2 value reflects tissue composition, especially free water content, T2 mapping is sensitive to tissue hydration or edema and does not require contrast agents, so it has Potential to become a "non-invasive" biopsy. The present invention uses the classic T2 mapping method to quantitatively analyze T2WI signal changes, and obtains signal changes similar to those of fluorescence images, which proves that quantitative analysis of MRI images can better analyze the changes of nanoparticles affecting tumor signals. The use of multimodal imaging can combine multiple imaging methods to realize the complementary advantages of each imaging method and expand the scope of application of each imaging method. The integration of MRI and optical imaging will provide high resolution, selectivity and sensitivity.
并且本发明还对荷瘤裸鼠模型注射不同剂量的纳米药物载体进行MR扫描及活体荧光成像，分析MR信号与SPIO含量的关系，以及探究TPE荧光纳米颗粒在活体中的肿瘤/器官分布和身体清除速率。对肿瘤选取ROI，提取图像的定量纹理参数，结合病理玻片，分别选取肿瘤整体、肿瘤生长活跃区及坏死区，比较不同病理改变的ROI纹理参数及TPE荧光强度的差异，分析MR纹理参数及TPE荧光强度与肿瘤病理改变及survivin表达的关系，揭示隐含的基因表达变化，以提供评价预后敏感性及特异性更高的影像指标。Moreover, the present invention also performs MR scanning and in vivo fluorescence imaging on tumor-bearing nude mouse models injected with different doses of nano drug carriers, analyzes the relationship between MR signals and SPIO content, and explores the tumor/organ distribution and body distribution of TPE fluorescent nanoparticles in vivo. Clearance rate. Select the ROI of the tumor, extract the quantitative texture parameters of the image, combine the pathological slides, respectively select the whole tumor, the active tumor growth area and the necrotic area, compare the ROI texture parameters and TPE fluorescence intensity differences of different pathological changes, and analyze the MR texture parameters and The relationship between TPE fluorescence intensity and tumor pathological changes and survivin expression reveals hidden gene expression changes to provide imaging indicators with higher sensitivity and specificity for evaluating prognosis.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明提供了一种磁性纳米药物载体及其制备方法，通过细胞毒性实验证明该纳米药物载体具有良好的生物相容性，同时具有很好的稳定性、靶向性和药物释放性，并且该磁性纳米药物载体可应用于制备癌症基因治疗药物。The invention provides a magnetic nano-medicine carrier and a preparation method thereof. The nano-medicine carrier has good biocompatibility, good stability, targeting and drug release through cytotoxicity experiments, and the The magnetic nano drug carrier can be applied to prepare cancer gene therapy drugs.
附图说明Description of drawings
图1为CA-SPIO-TPE-SP94-siRNA纳米药物载体的结构图；Fig. 1 is the structural diagram of CA-SPIO-TPE-SP94-siRNA nano drug carrier;
图2为SPIO、CA-SPIO和CA-SPIO-TPE-SP94-siRNA的高分辨率透射电镜图(TEM)(a～c)，其中(a)为SPIO纳米颗粒，(b)为CA-SPIO纳米颗粒，(c)CA-SPIO-TPE-SP94-siRNA纳米颗粒；(d)为SPIO纳米颗粒的粒径分布柱形图，(e)为CA-SPIO纳米颗粒的粒径分布柱形图，(f)为CA-SPIO-TPE-SP94-siRNA纳米颗粒的粒径分布柱形图；Figure 2 is the high-resolution transmission electron microscope (TEM) (a-c) of SPIO, CA-SPIO and CA-SPIO-TPE-SP94-siRNA, where (a) is SPIO nanoparticles, (b) is CA-SPIO Nanoparticles, (c) CA-SPIO-TPE-SP94-siRNA nanoparticles; (d) is the histogram of particle size distribution of SPIO nanoparticles, (e) is the histogram of particle size distribution of CA-SPIO nanoparticles, (f) is a histogram of particle size distribution of CA-SPIO-TPE-SP94-siRNA nanoparticles;
图3(a)为SPIO纳米颗粒的红外光谱图，图3(b)为CA-SPIO-TPE纳米颗粒的光致发光光谱，图3(c)为CA-SPIO-TPE纳米颗粒的热重曲线，图3(d)为SPIO和CA-SPIO-TPE纳米颗粒的磁化率曲线，图3(e)为SPIO的X射线衍射图，图3(f)为CA-SPIO-TPE-SP94-siRNA的Survivin siRNA释放示意图；Figure 3(a) is the infrared spectrum of SPIO nanoparticles, Figure 3(b) is the photoluminescence spectrum of CA-SPIO-TPE nanoparticles, and Figure 3(c) is the thermogravimetric curve of CA-SPIO-TPE nanoparticles , Figure 3(d) is the magnetic susceptibility curve of SPIO and CA-SPIO-TPE nanoparticles, Figure 3(e) is the X-ray diffraction pattern of SPIO, Figure 3(f) is the curve of CA-SPIO-TPE-SP94-siRNA Schematic diagram of Survivin siRNA release;
图4(a)为不同浓度CA-SPIO-TPE的水溶液体外T2加权MR图像；图4(b)为不同浓度CA-SPIO-TPE的水溶液的横向弛豫率(R2)；Figure 4(a) is the in vitro T2-weighted MR image of aqueous solutions of different concentrations of CA-SPIO-TPE; Figure 4(b) is the transverse relaxation rate (R2) of aqueous solutions of different concentrations of CA-SPIO-TPE;
图5(a)为CA-SPIO和CA-SPIO-TPE对Huh-7细胞的CCK-8结果图；图5(b)为不同浓度的CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA的水溶液对Huh-7细胞的CCK-8结果图；Fig. 5 (a) is the CCK-8 result figure of CA-SPIO and CA-SPIO-TPE to Huh-7 cell; Fig. 5 (b) is the different concentrations of CA-SPIO-TPE-siRNA and CA-SPIO-TPE- CCK-8 results of Huh-7 cells in aqueous solution of SP94-siRNA;
图6(a)为CA-SPIO-TPE、CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA颗粒对死/活细胞染色结果图；图6(b)为CA-SPIO-TPE、CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA颗粒在Huh-7细胞上的WB染色结果图；Figure 6(a) is the staining results of dead/live cells by CA-SPIO-TPE, CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA particles; Figure 6(b) is CA-SPIO-TPE , WB staining results of CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA particles on Huh-7 cells;
图7(a)、图7(b)为CA-SPIO-TPE、CA-SPIO-TPE-SP94颗粒加入Huh-7细胞中培养4h和12h后用共聚焦显微镜检测细胞内的荧光图；图7(c)、图7(d)为CA-SPIO-TPE、CA-SPIO-TPE-SP94颗粒加入Huh-7细胞中培养4h和12h后用流式细胞仪检测细胞内的荧光图；图7(e)为CA-SPIO-TPE、CA-SPIO-TPE-SP94颗粒加入Huh-7细胞中培养4h和12h平均荧光图；Figure 7(a) and Figure 7(b) are the fluorescent images in the cells detected by confocal microscope after CA-SPIO-TPE and CA-SPIO-TPE-SP94 particles were added to Huh-7 cells and cultured for 4h and 12h; Figure 7 (c) and Figure 7(d) are the fluorescent images in the cells detected by flow cytometry after adding CA-SPIO-TPE and CA-SPIO-TPE-SP94 particles to Huh-7 cells for 4h and 12h; Figure 7( e) The average fluorescence graph of adding CA-SPIO-TPE and CA-SPIO-TPE-SP94 particles to Huh-7 cells for 4h and 12h;
图8(a)为静脉注射游离CA-SPIO-TPE和CA-SPIO-TPE-SP94裸鼠移植瘤后24小时内裸鼠荧光图像；图8(b)为静脉注射游离CA-SPIO-TPE和CA-SPIO-TPE-SP94裸鼠移植瘤后24小时，裸鼠主要器官CA-SPIO-TPE和CA-SPIO-TPE-SP94移植瘤的荧光成像图像；图8(c)为24小时内定量显示裸鼠肿瘤部位的总荧光强度图；图8(d)为CA-SPIO-TPE-SP94和CA-SPIO-TPE在荷Huh-7荷瘤小鼠静脉注射前后不同时间点的磁共振成像图；图8(e)为肿瘤T2降低的定量分析图；(*：p＜0.05；**：p＜0.01)；Figure 8 (a) is the fluorescence image of nude mice within 24 hours after intravenous injection of free CA-SPIO-TPE and CA-SPIO-TPE-SP94 nude mice; Figure 8 (b) is the intravenous injection of free CA-SPIO-TPE and 24 hours after CA-SPIO-TPE-SP94 tumor transplantation in nude mice, the fluorescence imaging images of CA-SPIO-TPE and CA-SPIO-TPE-SP94 transplanted tumors in the main organs of nude mice; Figure 8(c) shows the quantitative display within 24 hours Figure 8 (d) is the magnetic resonance imaging images of CA-SPIO-TPE-SP94 and CA-SPIO-TPE at different time points before and after intravenous injection of Huh-7 tumor-bearing mice; Figure 8(e) is a quantitative analysis chart of tumor T2 reduction; (*: p<0.05; **: p<0.01);
图9(a)为各组荷瘤小鼠的肿瘤生长曲线图；图9(b)为各组荷瘤小鼠体重曲线图；图9(c)为各组荷瘤小鼠的存活率曲线图；图9(d)为第14天肿瘤显像图；图9(e)为各组荷瘤小鼠肿瘤及内脏器官的HE染色图。Fig. 9 (a) is the tumor growth curve of each group of tumor-bearing mice; Fig. 9 (b) is the body weight curve of each group of tumor-bearing mice; Fig. 9 (c) is the survival rate curve of each group of tumor-bearing mice Fig. 9(d) is the imaging image of the tumor on the 14th day; Fig. 9(e) is the HE staining image of the tumor and internal organs of the tumor-bearing mice in each group.
具体实施方式Detailed ways
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.
实施例1、季铵阳离子化直链淀粉(CA)的制备Embodiment 1, the preparation of quaternary ammonium cationized amylose (CA)
称量0.50g直链淀粉加入25mL蒸馏水中，用4mol/L NaOH溶液调整pH＝12～14，并加热搅拌使其分散；缓慢滴加含0.25g活性醚化剂GTA的水溶液5mL，在50℃下继续搅拌反应12小时；反应结束后，用盐酸溶液调整pH到中性，将溶液全部转移到截留分子量8000～14000Da的纤维素透析袋中，用去离子水透析3天，透析液过滤并冷冻干燥后得到季铵阳离子化直链淀粉，记作CA。Weigh 0.50g of amylose and add it to 25mL of distilled water, adjust the pH to 12-14 with 4mol/L NaOH solution, heat and stir to disperse; slowly add 5mL of aqueous solution containing 0.25g of active etherification agent GTA, at 50℃ Continue to stir and react for 12 hours; after the reaction, adjust the pH to neutral with hydrochloric acid solution, transfer all the solution to a cellulose dialysis bag with a molecular weight cut-off of 8,000-14,000 Da, dialyze with deionized water for 3 days, filter the dialysate and freeze After drying, quaternary ammonium cationized amylose was obtained, denoted as CA.
实施例2、季铵阳离子化直链淀粉和超顺磁性氧化铁纳米颗粒复合物的水溶液Embodiment 2, the aqueous solution of quaternary ammonium cationized amylose and superparamagnetic iron oxide nanoparticle composite(CA-SPIO)的制备Preparation of (CA-SPIO)
称量0.20g季铵阳离子化直链淀粉于50mL烧瓶中，加入20mL蒸馏水并搅拌使其溶解；称量0.20g FeCl3·6H2O和0.10g FeCl2·4H2O溶于5mL蒸馏水中，并加入到上述体系中，通氮气搅拌30分钟。将烧瓶置于80℃水浴，在剧烈搅拌下用注射器加入2.5mL 25％的氨水，反应1小时。反应完后降至室温，将全部溶液转移到截留分子量8000～14000Da的纤维素透析袋中透析2天。将透析液离心除去不溶物后，上层液体在4℃下保存，得到季铵阳离子化直链淀粉与超顺磁性纳米颗粒(Superparamagnetic iron oxide，SPIO)复合物的水溶液，记作CA-SPIO。Weigh 0.20g quaternary ammonium cationized amylose in a 50mL flask, add 20mL distilled water and stir to dissolve; weigh 0.20g FeCl3 6H2 O and 0.10g FeCl2 4H2 O and dissolve in 5mL distilled water, and added into the above system, and stirred under nitrogen gas for 30 minutes. The flask was placed in a water bath at 80°C, and 2.5 mL of 25% ammonia water was added with a syringe under vigorous stirring, and reacted for 1 hour. After the reaction was completed, the temperature was lowered to room temperature, and the entire solution was transferred to a cellulose dialysis bag with a molecular weight cut-off of 8000-14000 Da for dialysis for 2 days. After the dialysate was centrifuged to remove insoluble matter, the upper layer was stored at 4°C to obtain an aqueous solution of a complex of quaternary ammonium cationized amylose and superparamagnetic iron oxide (SPIO), which was designated as CA-SPIO.
实施例3、季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒(CA-Embodiment 3, quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles (CA-SPIO-TPE)的制备Preparation of SPIO-TPE)
称取4mg TPE溶于4mL CH2Cl2中，然后加入20mg季铵阳离子化直链淀粉和超顺磁性纳米颗粒复合物的水溶液(CA-SPIO)，室温混合均匀，然后边超声边滴入40mL纯水中，超声1小时后，产物移入到截留分子量2000Da的透析袋中，透析3天，每天换纯水2次，将透析液冷冻干燥后，收集固体产物于4℃下保存备用，得到季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒，记作CA-SPIO-TPE。Weigh 4mg TPE and dissolve it in 4mL CH2 Cl2 , then add 20mg aqueous solution (CA-SPIO) of quaternary ammonium cationized amylose and superparamagnetic nanoparticle composite, mix well at room temperature, then drop into 40mL After ultrasonication for 1 hour in pure water, the product was transferred to a dialysis bag with a molecular weight cut-off of 2000 Da, and was dialyzed for 3 days, with pure water changed twice a day. Ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles, denoted as CA-SPIO-TPE.
实施例4、SP94靶向肽靶向修饰季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧Example 4, SP94 targeting peptide targeting modification quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic oxygen化铁纳米颗粒的水溶液(CA-SPIO-TPE-SP94)的制备Preparation of Aqueous Solution of Iron Fe Nanoparticles (CA-SPIO-TPE-SP94)
称量20mg SP94短肽于4mL DMSO中，搅拌约1小时使其溶解；随即加入40mgEDC.HCl和20mg NHS，室温下避光搅拌4小时，得到SP94靶向肽活性酯的DMSO溶液。取实施例3制备得到的CA-SPIO-TPE的水溶液(含0.10g CA)于25mL圆底烧瓶，滴加冰醋酸调整pH至5左右，再加入上述SP94靶向肽活性酯的DMSO溶液，在室温下避光反应48小时。反应完后，把反应液全部移到截留分子量8000～14000Da的纤维素透析袋中透析3天。将透析液离心除去不溶物后，上层液体在4℃下避光保存，得到SP94靶向肽靶向修饰季铵阳离子化直链淀粉-四苯基乙烯-超顺磁性氧化铁纳米颗粒的水溶液，记作CA-SPIO-TPE-SP94。Weigh 20 mg of SP94 short peptide in 4 mL of DMSO, stir for about 1 hour to dissolve; then add 40 mg of EDC.HCl and 20 mg of NHS, and stir for 4 hours at room temperature in the dark to obtain a DMSO solution of the active ester of the SP94 targeting peptide. Get the aqueous solution (containing 0.10g CA) of the CA-SPIO-TPE prepared in Example 3 in a 25mL round bottom flask, add dropwise glacial acetic acid to adjust the pH to about 5, then add the DMSO solution of the above-mentioned SP94 targeting peptide active ester, in The reaction was protected from light at room temperature for 48 hours. After the reaction, all the reaction solution was transferred to a cellulose dialysis bag with a molecular weight cut-off of 8000-14000 Da and dialyzed for 3 days. After the dialysate was centrifuged to remove insoluble matter, the upper liquid was stored in the dark at 4°C to obtain an aqueous solution of SP94 targeting peptide targeting modified quaternary ammonium cationized amylose-tetraphenylethylene-superparamagnetic iron oxide nanoparticles, Denoted as CA-SPIO-TPE-SP94.
实施例5、CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA纳米药物载体的制备Example 5, Preparation of CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA Nano Drug Carriers
按照N/P(其中N来源于CA-SPIO-TPE，P来源于siRNA)摩尔比为5：1加入蒸馏水稀释，然后加入等量的siRNA溶液(2pmol/μL)。将混合物旋转5秒，然后在室温下放置20分钟，形成CA-SPIO-TPE-siRNA纳米药物载体。Add distilled water to dilute according to the molar ratio of N/P (where N comes from CA-SPIO-TPE, P comes from siRNA) at a molar ratio of 5:1, and then add an equal amount of siRNA solution (2pmol/μL). The mixture was spun for 5 seconds and then left at room temperature for 20 minutes to form the CA-SPIO-TPE-siRNA nano-drug carrier.
按照N/P(其中N来源于CA-SPIO-TPE-SP94，P来源于siRNA)摩尔比为5：1加入蒸馏水稀释，然后加入等量的siRNA溶液(2pmol/μL)。将混合物旋转5秒，然后在室温下放置20分钟，形成CA-SPIO-TPE-SP94-siRNA纳米药物载体。其结构图如图1所示。Add distilled water to dilute according to the molar ratio of N/P (where N comes from CA-SPIO-TPE-SP94, P comes from siRNA) at a molar ratio of 5:1, and then add an equal amount of siRNA solution (2pmol/μL). The mixture was spun for 5 seconds and then left at room temperature for 20 minutes to form the CA-SPIO-TPE-SP94-siRNA nano-drug carrier. Its structural diagram is shown in Figure 1.
实施例6、细胞实验及动物实验Embodiment 6, cell experiment and animal experiment
1、细胞培养及动物培养1. Cell culture and animal culture
1.1、细胞培养1.1. Cell culture
配置完全培养基，将10％的FBS、1％的双抗混合于DMEM培养基内。将Huh-7人肝细胞癌细胞放入含有完全培养基的培养皿中，并于37℃、5％CO2饱和湿度培养箱中培养，经过2～3天传代，取对数生长期细胞进行实验。Prepare complete medium, mix 10% FBS and 1% double antibody in DMEM medium. Put Huh-7 human hepatocellular carcinoma cells into a culture dish containing complete medium, and culture them in a 37°C, 5% CO2 saturated humidity incubator. experiment.
1.2、动物培养1.2. Animal culture
采用BALB/c裸鼠，雄性，年龄大小约4周(28～34天)，平均体重约15～18g，将裸鼠置于温度约22～26℃、湿度约40～60％的SPF环境中，自由摄食与饮水，食用SPF级小鼠饲料、蒸馏水，笼具等经过高压灭菌处理，定期更换垫料，保持干燥清洁，适应性饲养7天后用于实验。本实验遵守实验动物福利与伦理准则，经过中山大学实验动物管理与使用委员批准，中山大学动物实验伦理审查同意书批准编号SYSU-IACUC-2020-000048。BALB/c nude mice, male, aged about 4 weeks (28-34 days), with an average body weight of about 15-18g, were placed in an SPF environment with a temperature of about 22-26°C and a humidity of about 40-60%. , free to eat and drink, eat SPF-grade mouse feed, distilled water, cages, etc., after autoclaving, replace the litter regularly, keep dry and clean, and use them for experiments after adaptive feeding for 7 days. This experiment complied with the experimental animal welfare and ethical guidelines, and was approved by the Committee on the Management and Use of Experimental Animals of Sun Yat-sen University.
1.3、肿瘤建模1.3. Tumor modeling
取处对数生长期的Huh-7人肝细胞癌细胞，向贴壁细胞加入胰蛋白酶液消化重悬形成细胞悬液，将细胞悬液进行1000转/分钟离心5分钟，然后丢弃上层液体，将下层细胞重悬于PBS中，调整细胞浓度约1×107个/mL。取裸鼠，用10％水合氯醛腹腔注射麻醉，将细胞悬液缓慢皮下注射种植于右侧腹股沟，接种液体体积约0.2mL，定期观察裸鼠，约5天后可见裸鼠腹股沟有小肿瘤生成(肿瘤最大经线＜10mm)，即为造模成功，可用于进一步实验。Huh-7 human hepatocellular carcinoma cells in the logarithmic growth phase were taken, and trypsin solution was added to the adherent cells to digest and resuspend to form a cell suspension. The cell suspension was centrifuged at 1000 rpm for 5 minutes, and then the upper layer was discarded. Resuspend the lower layer of cells in PBS and adjust the cell concentration to about 1×107 cells/mL. Take nude mice and anesthetize them by intraperitoneal injection of 10% chloral hydrate. Slowly inject the cell suspension subcutaneously into the right groin. The volume of the inoculated liquid is about 0.2 mL. Observe the nude mice regularly. After about 5 days, small tumors can be seen in the groin of the nude mice. (the largest meridian of the tumor <10mm), it means that the modeling is successful and can be used for further experiments.
2、结构表征测试2. Structural characterization test
2.1红外(FTIR)测试2.1 Infrared (FTIR) test
采用溴化钾压片法，用傅立叶红外光(Fourier transform infrared，FTIR)谱仪分别测定超顺磁性纳米颗粒SPIO纳米胶束在4000～399cm-1范围内的红外光谱。The infrared spectrum of SPIO nanomicelles of superparamagnetic nanoparticles in the range of 4000-399 cm-1 was measured by potassium bromide tablet method and Fourier transform infrared (FTIR) spectrometer.
2.2X射线衍射谱图(XRD)测试2.2 X-ray diffraction spectrum (XRD) test
将冻干后的样品铺在样品板上，用X射线衍射仪对其晶体结构进行测试。在40kV/30mA的条件下，扫描速度为10°/min，扫描角度为20°～70°，Cu靶为Kα射线()。Spread the freeze-dried sample on the sample plate, and test its crystal structure with an X-ray diffractometer. Under the condition of 40kV/30mA, the scanning speed is 10°/min, the scanning angle is 20°～70°, and the Cu target is Kα ray ( ).
2.3高分辨率透射电镜(TEM)测试2.3 High-resolution transmission electron microscope (TEM) test
溶于正己烷的CA-SPIO和CA-SPIO-TPE纳米胶束充分稀释成0.5mg/mL，然后将样品滴在表面镀有无定形碳膜的铜网上，空气中干燥后置于高分辨率透射电镜(Transmissionelectron microscopy，TEM)中观察纳米颗粒的总体形态和粒径分布。The CA-SPIO and CA-SPIO-TPE nanomicelles dissolved in n-hexane were fully diluted to 0.5mg/mL, and then the samples were dropped on the copper grid coated with amorphous carbon film, dried in the air and placed in high resolution The overall shape and particle size distribution of the nanoparticles were observed in a transmission electron microscope (TEM).
2.4粒径分布和Zeta电位测试2.4 Particle size distribution and Zeta potential test
用动态光散射(Dynamic light scattering，DLS)测量聚合物胶束的粒径分布，采用粒径和Zeta电位分析仪测定纳米颗粒的粒径、粒径分布与Zeta电位，测试温度为25℃，每个样品测量3次，取平均值。The particle size distribution of polymer micelles was measured by dynamic light scattering (DLS), and the particle size, particle size distribution and Zeta potential of nanoparticles were measured by a particle size and Zeta potential analyzer. The test temperature was 25 °C, and each The samples were measured 3 times and the average value was taken.
2.5荧光光谱测试2.5 Fluorescence spectrum test
将纳米颗粒溶解在乙腈中，配置1mg/mL的纳米胶束溶液，然后按照梯度，配置一系列乙腈/水复合胶束溶液，利用荧光分光光度计测试，扫描波长为350nm～600nm。The nanoparticles were dissolved in acetonitrile, and a 1mg/mL nanomicelle solution was prepared, and then a series of acetonitrile/water composite micelles solutions were prepared according to the gradient, and tested by a fluorescence spectrophotometer with a scanning wavelength of 350nm to 600nm.
2.6磁学性质测试2.6 Magnetic properties test
取适量冻干后的样品用磁学性质测量系统测试磁性纳米复合粒子的磁滞曲线。温度为300K，磁场强度范围为±20000Oe。An appropriate amount of freeze-dried samples was taken to test the magnetic hysteresis curve of the magnetic nanocomposite particles with a magnetic property measuring system. The temperature is 300K, and the magnetic field strength range is ±20000Oe.
2.7热失重(TGA)测试2.7 Thermogravimetric (TGA) test
取适量冻干后的样品用热重分析仪(Thermogravimetry analysis)进行测试，升温速率为20℃/min，吹扫气为40mL/min氮气，保护气为20mL/min氮气，扫描温度范围为40～700℃。Take an appropriate amount of freeze-dried samples and test them with a Thermogravimetry analysis. The heating rate is 20°C/min, the purge gas is 40mL/min nitrogen, the protective gas is 20mL/min nitrogen, and the scanning temperature range is 40~ 700°C.
2.8磁共振成像(MRI)测试2.8 Magnetic resonance imaging (MRI) test
配制材料CA-SPIO-TPE的水溶液0.5mg/mL(Fe浓度为1.43mM)，然后逐级稀释为共8个不同的浓度梯度，浓度从高到低分别为1.43、0.72、0.36、0.19、0.09、0.04、0.02、0.01mM[Fe]，用8个2mL的透明硬质塑料离心管分别装入1.5mL溶液，按顺序放在泡沫平板上，室温下用1.5T MRI扫描仪进行扫描样品。MRI扫描序列包括T2WI、T2 mapping。The aqueous solution of the prepared material CA-SPIO-TPE is 0.5mg/mL (Fe concentration is 1.43mM), and then diluted step by step to a total of 8 different concentration gradients, the concentrations from high to low are 1.43, 0.72, 0.36, 0.19, 0.09 , 0.04, 0.02, 0.01mM [Fe], use eight 2mL transparent rigid plastic centrifuge tubes to fill 1.5mL solution respectively, put them on the foam plate in order, and scan the samples with a 1.5T MRI scanner at room temperature. The MRI scan sequence includes T2WI and T2 mapping.
T2WI参数如下：重复时间(Repetitiontime，TR)/回波时间(Echo time，TE)＝2600ms/100ms；翻转角(Flip angle)＝90°；NA(Number of acquisitions)＝6；采集矩阵(Acquisition matrix)＝384×305；FOV(Field of view)＝80mm×80mm；层厚(Slicethickness)＝2mm；层间距(Slice gap)＝2mm。T2 mapping采用单层、多回波自选回波序列扫描，参数如下：TR＝1500ms，TE＝0，20，40…160ms，NA＝3，Acquisition matrix＝176×123mm，FOV＝80mm×80mm，Slice thickness＝1.5mm，Slice gap＝1.5mm。T2WI parameters are as follows: repetition time (Repetition time, TR)/echo time (Echo time, TE) = 2600ms/100ms; flip angle (Flip angle) = 90°; NA (Number of acquisitions) = 6; acquisition matrix (Acquisition matrix) )=384×305; FOV (Field of view)=80mm×80mm; slice thickness (Slice thickness)=2mm; slice gap (Slice gap)=2mm. T2 mapping adopts single-layer, multi-echo self-selected echo sequence scanning, and the parameters are as follows: TR＝1500ms, TE＝0, 20, 40...160ms, NA＝3, Acquisition matrix＝176×123mm, FOV＝80mm×80mm, Slice thickness=1.5mm, slice gap=1.5mm.
3、体外实验3. In vitro experiments
3.1细胞毒性测试3.1 Cytotoxicity test
实验选取Huh-7细胞为材料毒性评价细胞模型，采用CCK-8检测细胞活性的方法来评价材料CA-SPIO、CA-SPIO-TPE、CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA4种纳米颗粒对Huh-7细胞的毒性。In the experiment, Huh-7 cells were selected as the cell model for material toxicity evaluation, and CCK-8 was used to detect cell activity to evaluate materials CA-SPIO, CA-SPIO-TPE, CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94 - Toxicity of siRNA4 nanoparticles to Huh-7 cells.
具体操作步骤如下：首先将Huh-7细胞以5000个/孔的密度接种于96孔板中，然后将其置于二氧化碳培养箱中培养贴壁过夜。随后，吸出原有的培养基，换上新鲜的含有不同浓度的材料的完全培养基，所选的材料的浓度设置为1000μg/mL、500μg/mL、250μg/mL、100μg/mL、10μg/mL(聚合物浓度)；2000μg/mL、1000μg/mL、500μg/mL、250μg/mL、125μg/mL(胶束浓度)每个浓度有5个平行组。然后在培养箱中培养24小时，培养后用PBS将细胞洗涤一次后并向各孔中加入100μL的新鲜培养基(含有10％CCK-8)。置于培养箱中孵育一段时间，最后使用酶标仪检测并记录在450nm波长处的吸光度，通过下公式计算细胞存活率：细胞存活率(％)＝(实验组吸光度-空白组吸光度)/(阴性对照组吸光度-空白组吸光度)×100％。The specific operation steps are as follows: first, Huh-7 cells were seeded in a 96-well plate at a density of 5000 cells/well, and then placed in a carbon dioxide incubator to culture and adhere to the wall overnight. Subsequently, aspirate the original medium and replace it with fresh complete medium containing different concentrations of materials, the concentration of the selected materials is set to 1000 μg/mL, 500 μg/mL, 250 μg/mL, 100 μg/mL, 10 μg/mL (polymer concentration); 2000 μg/mL, 1000 μg/mL, 500 μg/mL, 250 μg/mL, 125 μg/mL (micelle concentration) with 5 parallel groups for each concentration. The cells were then cultured in an incubator for 24 hours. After the culture, the cells were washed once with PBS and 100 μL of fresh medium (containing 10% CCK-8) was added to each well. Place in an incubator and incubate for a period of time, and finally use a microplate reader to detect and record the absorbance at a wavelength of 450nm, and calculate the cell survival rate by the following formula: cell survival rate (%)=(experimental group absorbance-blank group absorbance)/( Absorbance of negative control group - absorbance of blank group) × 100%.
3.2激光共聚焦测试3.2 Laser confocal test
使用激光共聚焦显微镜考察细胞内纳米颗粒的分布情况。采用CA-SPIO-TPE和CA-SPIO-TPE-SP94纳米颗粒进行细胞内荧光检测。具体方法如下：将盖玻片用酒精浸泡、超声，清洗干净后烘干，121℃灭菌，再次烘干备用。将无菌盖玻片无菌条件下放入35mm培养皿中，配制细胞悬液105个/mL，每孔加入2mL，让细胞在盖玻片上爬片生长。培养24小时后，用含有CA-SPIO-TPE和CA-SPIO-TPE-SP94纳米颗粒的复合胶束溶液置换原来的全培养基。培养到规定时间(4小时和12小时)后，去掉所有培养基，加入4％多聚甲醛的PBS溶液，在培养箱中固定细胞30分钟。30分钟后，去掉多聚甲醛溶液，用PBS溶液清洗1次，加入DAP的PBS溶液(DAPI浓度2g/mL)，在培养箱中染色10分钟。10分钟后，去掉DAPI的PBS溶液，去掉所有溶液，用PBS溶液清洗两次。然后将爬满细胞的盖玻片挑出倒扣在干净的载玻片上，甘油封片，指甲油固定，最后4℃下避光保存。将制备好的细胞爬片在激光共聚焦显微镜下观察，其中DAPI的调光参数为(激发光波长/发射光波长：405nm/453nm)，其中TPE的调光参数为(激发光波长/发射光波长：488nm/523nm)。The distribution of nanoparticles in cells was examined using confocal laser microscopy. Intracellular fluorescence detection using CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles. The specific method is as follows: soak the cover glass in alcohol, ultrasonically clean it, dry it, sterilize it at 121°C, and dry it again for use. Put the sterile coverslip into a 35mm Petri dish under aseptic conditions, prepare a cell suspension of10 cells/mL, add 2mL to each well, and let the cells grow on the coverslip. After culturing for 24 hours, the original full medium was replaced with the composite micellar solution containing CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles. After culturing for the specified time (4 hours and 12 hours), remove all the medium, add 4% paraformaldehyde in PBS solution, and fix the cells in the incubator for 30 minutes. After 30 minutes, remove the paraformaldehyde solution, wash once with PBS solution, add DAP in PBS solution (DAPI concentration 2g/mL), and stain in the incubator for 10 minutes. After 10 min, remove the DAPI in PBS solution, remove all solutions, and wash twice with PBS solution. Then pick out the cover slip covered with cells and place it upside down on a clean glass slide, cover the slide with glycerol, fix with nail polish, and finally store it in the dark at 4°C. The prepared cell slides were observed under a laser confocal microscope, wherein the dimming parameter of DAPI was (excitation light wavelength/emission light wavelength: 405nm/453nm), wherein the dimming parameter of TPE was (excitation light wavelength/emission light wavelength Wavelength: 488nm/523nm).
3.3流式细胞仪测试3.3 Flow cytometry test
利用流式细胞仪定量靶向纳米胶束对细胞吞噬的促进作用。具体操作如下：将不同的细胞配制成悬液，浓度为105个/mL，接种在35mm底径的培养皿中，每皿2mL细胞悬液。培养24小时后，用含有CA-SPIO-TPE和CA-SPIO-TPE-SP94纳米颗粒的复合胶束溶液置换原来的全培养基。在培养箱中分别再培养4小时、12小时。Quantification of the promotion effect of targeted nanomicelles on cell phagocytosis by flow cytometry. The specific operation is as follows: Different cells were prepared into a suspension at a concentration of 105 cells/mL, and inoculated in a culture dish with a bottom diameter of 35 mm, 2 mL of the cell suspension per dish. After culturing for 24 hours, the original full medium was replaced with the composite micellar solution containing CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles. They were further cultured in the incubator for 4 hours and 12 hours, respectively.
4.体内实验4. In Vivo Experiments
4.1活体荧光成像测试4.1 In vivo fluorescence imaging test
以尾静脉注射的方式注入药物，并使用小动物活体成像系统对CA-SPIO-TPE和CA-SPIO-TPE-SP94纳米颗粒进行荧光成像，探究TPE荧光纳米颗粒在活体中的肿瘤/器官分布和身体清除速率。Huh-7荷瘤肿瘤的裸鼠分别注射CA-SPIO-TPE和CA-SPIO-TPE-SP94纳米颗粒，采集注射后1小时、6小时、12小时、24小时的荧光图像。Inject the drug by tail vein injection, and use the small animal in vivo imaging system to perform fluorescence imaging of CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles, and explore the tumor/organ distribution and organ distribution of TPE fluorescent nanoparticles in vivo. Body clearance rate. Huh-7 tumor-bearing nude mice were injected with CA-SPIO-TPE and CA-SPIO-TPE-SP94 nanoparticles, respectively, and fluorescence images were collected 1 hour, 6 hours, 12 hours, and 24 hours after injection.
4.2活体磁共振成像测试4.2 In vivo magnetic resonance imaging test
以尾静脉注射的方式注入药物，并使用小动物MRI扫描探究CA-SPIO-TPE-SP94-siRNA及CA-SPIO-TPE-siRNA对肿瘤信号变化的影响。荷瘤裸鼠使用异氟烷气体麻醉后，在室温在3.0T MRI扫描仪使用小动物专用线圈扫描，扫描序列包括T2 SPIR(Spectralpresaturation with inversion recovery)、T2 mapping。The drug was injected into the tail vein, and the effects of CA-SPIO-TPE-SP94-siRNA and CA-SPIO-TPE-siRNA on tumor signal changes were explored using small animal MRI scans. After the tumor-bearing nude mice were anesthetized with isoflurane gas, they were scanned in a 3.0T MRI scanner at room temperature using a coil dedicated to small animals. The scanning sequence included T2 SPIR (Spectralpresaturation with inversion recovery) and T2 mapping.
T2WI SPIR参数如下：TR/TE＝1600ms/60ms；Flip angle＝90°；NA＝3；T2WI SPIR parameters are as follows: TR/TE=1600ms/60ms; Flip angle=90°; NA=3;
Acquisition matrix＝252×254；FOV＝128mm×128mm；Slice thickness＝1mm；Slice gap＝1mm。T2 mapping采用单层、多回波自选回波序列扫描，参数如下：TR＝1400ms，TE＝25，50，75，100ms，NA＝3，Acquisition matrix＝109×108mm，FOV＝128mm×128mm，Slice thickness＝1.5mm，Slice gap＝1.5mm。Acquisition matrix=252×254; FOV=128mm×128mm; Slice thickness=1mm; Slice gap=1mm. T2 mapping adopts single-layer, multi-echo self-selected echo sequence scanning, and the parameters are as follows: TR＝1400ms, TE＝25, 50, 75, 100ms, NA＝3, Acquisition matrix＝109×108mm, FOV＝128mm×128mm, Slice thickness=1.5mm, slice gap=1.5mm.
4.3体内抑瘤实验及组织病理学检查4.3 In vivo tumor inhibition experiment and histopathological examination
将荷瘤裸鼠随机分成不同的实验组，每组3只，按照5mg/kg的剂量在第0、2、4天给药，每只裸鼠尾静脉注射药物200μL，总计共给药三次。用游标卡尺每天测量裸鼠肿瘤的体积，用电子秤称量裸鼠体重。实验结束后处死荷瘤裸鼠并取出完整肿瘤拍照。计算肿瘤体积＝0.5×长度×宽度。荷瘤裸鼠生存曲线实验每组4只。将荷瘤裸鼠随机分成不同的实验组，每组3只，按照5mg/kg的剂量在第0、2、4天给药，每只裸鼠尾静脉注射药物200μL，在第14天实验结束后处死荷瘤裸鼠，取出动物的心、肝、脾、肺、肾、种植肿瘤，放入10％甲醛溶液中进行脱水，然后进行石蜡包埋、切片，最后进行HE染色并封片，在光镜下观察各脏器及肿瘤组织的结构改变及损伤情况。The tumor-bearing nude mice were randomly divided into different experimental groups, with 3 mice in each group, administered at a dose of 5 mg/kg on days 0, 2, and 4. Each nude mouse was injected with 200 μL of the drug through the tail vein, and administered three times in total. The tumor volume of the nude mice was measured with a vernier caliper every day, and the body weight of the nude mice was weighed with an electronic scale. After the experiment, the tumor-bearing nude mice were sacrificed and the whole tumor was taken out to take pictures. Calculate tumor volume = 0.5 x length x width. Survival curve experiment of tumor-bearing nude mice There were 4 mice in each group. The tumor-bearing nude mice were randomly divided into different experimental groups, with 3 mice in each group. The dose of 5 mg/kg was administered on days 0, 2, and 4. Each nude mouse was injected with 200 μL of the drug through the tail vein, and the experiment ended on the 14th day. Afterwards, the tumor-bearing nude mice were sacrificed, and the hearts, livers, spleens, lungs, kidneys, and implanted tumors were taken out, put into 10% formaldehyde solution for dehydration, then embedded in paraffin, sectioned, and finally HE stained and sealed. The structural changes and damage of various organs and tumor tissues were observed under a light microscope.
结果：result:
1、结构表征测试结果：1. Structural characterization test results:
在合成了油胺和油酸稳定的疏水SPIO后，由于TPE和SPIO都具有疏水特性，TPE通过疏水相互作用通过物理包埋的方式被引入到SPIO纳米粒子的内核中。因此，CA-SPIO可以在内核中引入大量疏水的TPE和SPIO，同时利用CA的亲水外壳层保持水溶性。高分辨率透射电镜(TEM)照片显示SPIO纳米颗粒为平均尺寸为5-8nm的黑点，CA-SPIO和CA-SPIO-TPE-SP94-siRNA纳米颗粒均为球形，平均尺寸分别为155nm和178nm(参照图2)。CA-SPIO和CA-SPIO-TPE-SP94-siRNA纳米颗粒的PDI分别为0.18和0.25。纳米颗粒的尺寸在100nm到200nm之间是增强渗透性和保留效果的最佳选择。CA-SPIO和CA-SPIO-TPE-SP94-siRNA的表面电位分别为23.5mV和5.8mV，表明成功吸附了带负电荷的siRNA。After synthesizing hydrophobic SPIO stabilized by oleylamine and oleic acid, since both TPE and SPIO have hydrophobic properties, TPE was introduced into the inner core of SPIO nanoparticles by physical embedding through hydrophobic interactions. Therefore, CA-SPIO can introduce a large amount of hydrophobic TPE and SPIO in the inner core, while utilizing the hydrophilic outer shell of CA to maintain water solubility. High-resolution transmission electron microscopy (TEM) photos show that SPIO nanoparticles are black dots with an average size of 5-8nm, CA-SPIO and CA-SPIO-TPE-SP94-siRNA nanoparticles are spherical, with average sizes of 155nm and 178nm, respectively (Refer to Figure 2). The PDIs of CA-SPIO and CA-SPIO-TPE-SP94-siRNA nanoparticles were 0.18 and 0.25, respectively. Nanoparticles with a size between 100nm and 200nm are optimal for enhanced permeability and retention. The surface potentials of CA-SPIO and CA-SPIO-TPE-SP94-siRNA were 23.5 mV and 5.8 mV, respectively, indicating that the negatively charged siRNA was successfully adsorbed.
SPIO纳米颗粒的红外光谱如图3(a)所示，560.0cm-1处的峰是金属原子和氧原子的伸缩振动吸收峰。1570cm-1处的两个峰对应于C＝O的对称伸缩振动峰和反对称伸缩振动峰，它们是羧酸盐的特征峰。2920cm-1和2850cm-1处的两个峰对应于油酸分子中的C-H伸缩振动吸收峰。The infrared spectrum of SPIO nanoparticles is shown in Fig. 3(a), and the peak at 560.0 cm-1 is the stretching vibration absorption peak of metal atoms and oxygen atoms. The two peaks at 1570cm-1 correspond to the symmetric stretching vibration peak and the antisymmetric stretching vibration peak of C=O, which are the characteristic peaks of carboxylate. The two peaks at 2920cm-1 and 2850cm-1 correspond to the CH stretching vibration absorption peaks in the oleic acid molecule.
CA-SPIO-TPE(25-1000μg/mL)的光致发光(PL)光谱如图3(b)所示，430nm处的吸收峰表明TPE已成功地引入到CA-SPIO中。The photoluminescence (PL) spectra of CA-SPIO-TPE (25–1000 μg/mL) are shown in Fig. 3(b), and the absorption peak at 430 nm indicates that TPE has been successfully incorporated into CA-SPIO.
图3(c)显示了CA-SPIO在40℃到700℃之间的热重曲线。CA-SPIO-TPE的最终质量损失为63％，即SPIO含量为37％。由于SPIO的加入，降低了CA-SPIO中可热降解组分的含量，表明SPIO已成功引入CA-SPIO中。对于磁性材料而言，磁响应是检测磁性材料的重要性能指标之一。磁滞回线通常用来表征磁性材料对外部磁场的响应。Figure 3(c) shows the TG curves of CA-SPIO between 40 °C and 700 °C. The final mass loss of CA-SPIO-TPE was 63%, ie the SPIO content was 37%. Due to the addition of SPIO, the content of thermally degradable components in CA-SPIO was reduced, indicating that SPIO had been successfully introduced into CA-SPIO. For magnetic materials, magnetic response is one of the important performance indicators for detecting magnetic materials. Hysteresis loops are often used to characterize the response of magnetic materials to external magnetic fields.
图3(d)显示了300K时SPIO和CA-SPIO的磁滞回线。在外加磁场从-20000oE到20000oE的循环扫描中，当外加磁场强度为零时，SPIO或CA-SPIO-TPE的剩余磁化强度为零。这一结果表明，SPIO和CA-SPIO-TPE都具有超顺磁性。SPIO和CA-SPIO-TPE的饱和磁化强度分别为53.8emu/g和26.3emu/g。CA-SPIO饱和磁化强度降低的原因是CA包裹在SPIO表面，导致磁化强度降低。CA-SPIO-TPE对外加磁场仍有良好的磁响应，可用于后续MRI扫描。Figure 3(d) shows the hysteresis loops of SPIO and CA-SPIO at 300K. In the cyclic scanning of the applied magnetic field from -20000oE to 20000oE, when the applied magnetic field strength is zero, the residual magnetization of SPIO or CA-SPIO-TPE is zero. This result indicates that both SPIO and CA-SPIO-TPE are superparamagnetic. The saturation magnetizations of SPIO and CA-SPIO-TPE are 53.8emu/g and 26.3emu/g, respectively. The reason for the decreased saturation magnetization of CA-SPIO is that CA wraps on the surface of SPIO, resulting in decreased magnetization. CA-SPIO-TPE still has a good magnetic response to the applied magnetic field and can be used for subsequent MRI scans.
如图3(e)所示，XRD结果证实了SPIO是超顺磁性纳米粒子。特征峰位于30.2o、35.6o、43.0o、53.4o、57.1o和62.5o，分别对应于尖晶石的(220)、(311)、(400)、(422)、(511)和(440)。As shown in Fig. 3(e), the XRD results confirmed that SPIOs are superparamagnetic nanoparticles. The characteristic peaks are located at 30.2o, 35.6o, 43.0o, 53.4o, 57.1o and 62.5o, corresponding to (220), (311), (400), (422), (511) and (440) of spinel, respectively. ).
如图3(f)所示，CA-SPIO-TPE-SP94-siRNA纳米药物载体在37℃下有良好的药物释放Survivin siRNA的效果，5h后释放50.0％的Survivin siRNA，24h后释放79.8％。As shown in Figure 3(f), the CA-SPIO-TPE-SP94-siRNA nano-drug carrier has a good drug release effect of Survivin siRNA at 37°C, releasing 50.0% of Survivin siRNA after 5h and 79.8% after 24h.
2、体外T2加权成像2. In vitro T2-weighted imaging
如图4所示，T2加权成像采用常规自旋回波SE序列扫描，脉冲重复间隔时间TR＝1500ms，回波时间TE＝96.191,167.98,293.32,487.83,752.57,1032.6,1259.3,1430.2ms，FS＝1.5.。随着Fe浓度的增加，MR图像逐渐变暗。Fe(Mm)浓度与T2弛豫时间的倒数1/T2(s-1)相对应。由数据点拟合的直线斜率为T2弛豫效率，R2＝39.1mm-1s-1。这表明CA-SPIO-TPE的水溶液具有优异的磁共振成像性能。As shown in Figure 4, T2-weighted imaging adopts conventional spin echo SE sequence scanning, pulse repetition interval TR = 1500ms, echo time TE = 96.191, 167.98, 293.32, 487.83, 752.57, 1032.6, 1259.3, 1430.2ms, FS = 1.5.. With the increase of Fe concentration, the MR images become gradually darker. The Fe(Mm) concentration corresponds to the reciprocal 1/T2(s-1) of the T2 relaxation time. The slope of the straight line fitted from the data points is the T2 relaxation efficiency, R2 = 39.1 mm−1 s−1 . This indicates that the aqueous solution of CA-SPIO-TPE has excellent magnetic resonance imaging performance.
3、体外细胞毒性测试结果3. In vitro cytotoxicity test results
1)采用CCK8比色法检测CA-SPIO、CA-SPIO-TPE、CA-SPIO-TPE-SP94-siRNA及CA-SPIO-TPE-siRNA纳米颗粒的细胞毒性。1) The cytotoxicity of CA-SPIO, CA-SPIO-TPE, CA-SPIO-TPE-SP94-siRNA and CA-SPIO-TPE-siRNA nanoparticles was detected by CCK8 colorimetry.
如图5(a)所示，在CA-SPIO水溶液的浓度从10到1000μg mL-1的范围内，细胞存活率约为85％，表明CA-SPIO和CA-SPIO-TPE纳米颗粒在HUH-7细胞中具有良好的生物相容性。As shown in Fig. 5(a), the cell viability was about 85% in the concentration range of CA-SPIO aqueous solution ranging from 10 to 1000 μg mL−1 , indicating that CA-SPIO and CA-SPIO-TPE nanoparticles were in the HUH- 7 cells have good biocompatibility.
此外，如图5(b)所示，CA-SPIO-TPE-SP94-siRNA及CA-SPIO-TPE-siRNA纳米颗粒在低浓度下对Huh-7细胞表现出良好的抑制作用。CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA纳米颗粒与Huh-7细胞孵育24h后的半数抑制浓度(IC50值)分别为78.6μg/mL和29.4μg/mL。CA-SPIO-TPE-SP94-siRNA纳米颗粒的抑制效果优于其他两组，表明靶向SP94肽能增强Survivin siRNA在细胞内的积聚。In addition, as shown in Figure 5(b), CA-SPIO-TPE-SP94-siRNA and CA-SPIO-TPE-siRNA nanoparticles showed good inhibitory effect on Huh-7 cells at low concentrations. The half maximal inhibitory concentrations (IC50 values) of CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA nanoparticles incubated with Huh-7 cells for 24 hours were 78.6μg/mL and 29.4μg/mL, respectively. The inhibitory effect of CA-SPIO-TPE-SP94-siRNA nanoparticles was better than that of the other two groups, indicating that targeting SP94 peptide can enhance the accumulation of Survivin siRNA in cells.
2)活/死染色分析：2) Live/dead staining analysis:
如图6(a)所示，本发明人发现CA-SPIO、CA-SPIO-TPE纳米颗粒在培养48h后Huh-7细胞都保持了良好的细胞活力，因为几乎所有的样品都显示出绿色荧光信号(活细胞)。同时，CA-SPIO-TPE-siRNA和CA-SPIO-TPE-SP94-siRNA纳米颗粒对Huh-7细胞有较好的抑制作用，表现出大量的红色荧光信号(死亡细胞)。这些结果与CCK-8的结果是一致的。As shown in Figure 6(a), the inventors found that CA-SPIO and CA-SPIO-TPE nanoparticles maintained good cell viability in Huh-7 cells after 48 h of culture, because almost all samples showed green fluorescence Signaling (live cells). At the same time, CA-SPIO-TPE-siRNA and CA-SPIO-TPE-SP94-siRNA nanoparticles had a better inhibitory effect on Huh-7 cells, showing a large number of red fluorescent signals (dead cells). These results are consistent with those of CCK-8.
3)为探讨Survivin siRNA对Huh-7细胞的抑制作用机制，采用Western blotting方法分别检测了Huh-7细胞中caspase-3和Survivin的蛋白表达水平。3) In order to explore the inhibitory mechanism of Survivin siRNA on Huh-7 cells, the protein expression levels of caspase-3 and Survivin in Huh-7 cells were detected by Western blotting method.
如图6(b)所示，CA-SPIO-TPE-siRNA纳米颗粒处理Huh-7细胞48h后，caspase-3的表达水平显著上调，表明Survivin siRNA成功地抑制了肿瘤细胞的生长。Caspase-3在细胞凋亡中起着不可替代的作用。Caspase-3的过表达表明，携带Survivin siRNA的CA-SPIO-TPE纳米颗粒能显著抑制Huh-7细胞的生长。同时，Survivin蛋白的下调表明CA-SPIO-TPE-siRNA纳米颗粒具有成功的RNA干扰效应。As shown in Figure 6(b), after CA-SPIO-TPE-siRNA nanoparticles treated Huh-7 cells for 48 h, the expression level of caspase-3 was significantly up-regulated, indicating that Survivin siRNA successfully inhibited the growth of tumor cells. Caspase-3 plays an irreplaceable role in apoptosis. The overexpression of Caspase-3 showed that CA-SPIO-TPE nanoparticles carrying Survivin siRNA could significantly inhibit the growth of Huh-7 cells. Meanwhile, the down-regulation of Survivin protein indicated that CA-SPIO-TPE-siRNA nanoparticles had a successful RNA interference effect.
4、体外细胞摄取研究：用激光共聚焦扫描显微镜(CLSM)检测CSP胞内破碎物在细胞内的分布。4. In vitro cell uptake study: the distribution of CSP intracellular fragments in cells was detected by confocal laser scanning microscope (CLSM).
如图7(a)所示，与胶束共同孵育4小时后，SP94肽靶向胶束与非靶向胶束共孵育的细胞胞浆中的红色强度明显高于非靶向胶束，表明靶向胶束可以通过受体介导的内吞过程优先积聚在细胞质中。As shown in Figure 7(a), after co-incubating with micelles for 4 hours, the red intensity in the cytoplasm of cells co-incubated with SP94 peptide-targeted micelles and non-targeted micelles was significantly higher than that of non-targeted micelles, indicating that Targeted micelles can preferentially accumulate in the cytoplasm through receptor-mediated endocytosis.
同时，如图7(b)所示，SP94肽靶向胶束与非靶向胶束的荧光强度差异在孵育12h后逐渐扩大，这是因为SP94肽的靶向作用随着时间的推移而增强，12h后细胞摄取更多。Meanwhile, as shown in Figure 7(b), the difference in fluorescence intensity between SP94 peptide-targeted micelles and non-targeted micelles gradually expanded after incubation for 12 h, because the targeting effect of SP94 peptide was enhanced over time , cells uptake more after 12h.
流式细胞仪在4h(图7(c))和12h(图7(d))的荧光结果也证明了SP94肽的主动靶向作用。除了SP94可以促进受体介导的摄取外，CA-SPIO-TPE-siRNA纳米颗粒的阳离子表面通过非特异性静电相互作用使其与阴离子细胞膜紧密相连。纳米颗粒可以通过内吞作用进入哺乳动物细胞(Conner&Schmid2003)，然后形成膜结合的囊泡并包裹纳米颗粒进行内化。The fluorescence results of flow cytometry at 4h (Fig. 7(c)) and 12h (Fig. 7(d)) also demonstrated the active targeting of SP94 peptide. In addition to the fact that SP94 can facilitate receptor-mediated uptake, the cationic surface of CA-SPIO-TPE-siRNA nanoparticles makes it tightly associated with anionic cell membranes through nonspecific electrostatic interactions. Nanoparticles can enter mammalian cells by endocytosis (Conner & Schmid 2003), and then form membrane-bound vesicles that encapsulate nanoparticles for internalization.
5、活体AIE显像及生物分布5. In vivo AIE imaging and biodistribution
为探讨CA-SPIO-TPE纳米粒子是否适用于小鼠肿瘤显像，将CA-SPIO-TPE纳米粒子1mg mL-1和CA-SPIO-TPE-SP94纳米粒子分别静脉注射给荷Huh-7肿瘤的BALB/c小鼠。CA-SPIO-TPE和CA-SPIO-TPE-SP94注射后1h内均在肿瘤内蓄积。肿瘤内的荧光信号逐渐增强，在6h达到最高亮度(图8(a))，提示CA-SPIO-TPE和CA-SPIO-TPE-SP94对Huh-7肿瘤具有良好的体内显像能力。注射后24h处死小鼠，观察CA-SPIO-TPE和CA-SPIO-TPE-SP94在肿瘤及不同脏器中的分布。To investigate whether CA-SPIO-TPE nanoparticles are suitable for tumor imaging in mice, 1 mg mL-1 of CA-SPIO-TPE nanoparticles and CA-SPIO-TPE-SP94 nanoparticles were injected intravenously into mice bearing Huh-7 tumors, respectively. BALB/c mice. Both CA-SPIO-TPE and CA-SPIO-TPE-SP94 accumulated in the tumor within 1 hour after injection. The fluorescent signal in the tumor gradually increased and reached the highest brightness at 6h (Figure 8(a)), suggesting that CA-SPIO-TPE and CA-SPIO-TPE-SP94 have good in vivo imaging capabilities for Huh-7 tumors. The mice were killed 24 hours after injection, and the distribution of CA-SPIO-TPE and CA-SPIO-TPE-SP94 in tumors and different organs was observed.
如图8(b)所示，制备的纳米颗粒在小鼠的循环系统中流动，并主要积聚在肝脏和肿瘤中。这可能是由于动物的新陈代谢功能所致。肿瘤平均荧光强度结果显示，CA-SPIO-TPE组和CA-SPIO-TPE-SP94组在注射后6h内的AIE荧光强度相似。6h后发现CA-SPIO-TPE-SP94组的荧光强度强于CA-SPIO-TPE组。两组的平均荧光强度有显著性差异。这间接反映了两组TPE聚集的情况。As shown in Figure 8(b), the prepared nanoparticles flowed in the circulatory system of mice and mainly accumulated in the liver and tumor. This may be due to the metabolic function of the animal. The results of the average fluorescence intensity of the tumor showed that the AIE fluorescence intensity of the CA-SPIO-TPE group and the CA-SPIO-TPE-SP94 group within 6 hours after injection was similar. After 6h, it was found that the fluorescence intensity of the CA-SPIO-TPE-SP94 group was stronger than that of the CA-SPIO-TPE group. There was a significant difference in the average fluorescence intensity between the two groups. This indirectly reflects the situation of TPE aggregation in the two groups.
6、体内磁共振成像6. Magnetic resonance imaging in vivo
由于SPIO能在T2WI上产生低信号，因此CA-SPIO-TPE-SP94的肿瘤靶向性可通过MRI无创监测。Since SPIO can produce low signal on T2WI, the tumor targeting of CA-SPIO-TPE-SP94 can be noninvasively monitored by MRI.
本发明通过Huh-7细胞来源的皮下移植瘤小鼠模型评估了MRI在体内的成像效率。小鼠在注射CA-SPIO-TPE或CA-SPIO-TPE-SP94前进行扫描。每只小鼠注射制备的纳米药物载体后，分别在注射前、注射后6h、注射后12h和注射后24h进行扫描。同一肿瘤切片的代表性扫描图像显示在不同的时间点(图8(c))。注射CA-SPIO-TPE-SP94后，肿瘤感兴趣区的归一化MR信号强度值和T2值在注射后6h和12h明显降低。注射CA-SPIO-TPE后，Huh-7肿瘤的归一化MR信号强度值和T2值无明显变化。注射后6h/12h，CA-SPIO-TPE组和CA-SPIO-TPE-SP94组肿瘤感兴趣区T2值分别降至5％/9％和2％/34％(p<0.003)。注射后24h，CA-SPIO-TPE组和CA-SPIO-TPE-SP94组T2值分别降至16％和4％(P>0.05)(图8(d))。这些结果与SP94修饰的纳米载体对Huh-7细胞具有更高的靶向能力的发现是一致的(图7)。使用主动靶向递送系统，可以显著改善MR对比信号。与荧光成像相比，MRI扫描提供了更有价值的细节。首先，MRI显示出比荧光成像更好的空间分辨率，并且可能不受穿透深度的限制。其次，横断面MR图像提供了更多关于肿瘤中纳米载体分布和浓度的信息，而不仅仅是关于荧光成像代谢的二维信息。最后，T2加权图像上低信号强度的不均匀分布反映了肿瘤的异质性，肿瘤内片状T2低信号可能对应于更活跃的肿瘤细胞或灌注较好的区域(图8(e))。The present invention evaluates the imaging efficiency of MRI in vivo through the subcutaneous xenograft tumor mouse model derived from Huh-7 cells. Mice were scanned before injection with CA-SPIO-TPE or CA-SPIO-TPE-SP94. After each mouse was injected with the prepared nano drug carrier, scanning was performed before injection, 6h after injection, 12h after injection and 24h after injection. Representative scan images of the same tumor section are shown at different time points (Fig. 8(c)). After injection of CA-SPIO-TPE-SP94, the normalized MR signal intensity value and T2 value of the tumor region of interest decreased significantly at 6h and 12h after injection. After injection of CA-SPIO-TPE, the normalized MR signal intensity values and T2 values of Huh-7 tumors did not change significantly. 6h/12h after injection, the T2 value of the tumor ROI in CA-SPIO-TPE group and CA-SPIO-TPE-SP94 group decreased to 5%/9% and 2%/34% respectively (p<0.003). 24 hours after injection, the T2 values of CA-SPIO-TPE group and CA-SPIO-TPE-SP94 group decreased to 16% and 4% respectively (P>0.05) (Fig. 8(d)). These results are consistent with the finding that the SP94-modified nanocarriers have a higher targeting ability to Huh-7 cells (Fig. 7). Using an active targeted delivery system, the MR contrast signal can be significantly improved. MRI scans provide more valuable detail than fluorescence imaging. First, MRI exhibits better spatial resolution than fluorescence imaging and may not be limited by depth of penetration. Second, cross-sectional MR images provide more information about the distribution and concentration of nanocarriers in tumors than just two-dimensional information about metabolism with fluorescence imaging. Finally, the heterogeneous distribution of hypointensities on T2-weighted images reflects tumor heterogeneity, and patchy T2 hypointensities within tumors may correspond to more active tumor cells or better perfused areas (Fig. 8(e)).
7、体内抗瘤作用7. Anti-tumor effect in vivo
对CA-SPIO在荷人Huh-7裸鼠体内的抗肿瘤作用进行了研究。The antitumor effect of CA-SPIO in Huh-7 nude mice was studied.
如图9(a)所示，PBS、CA-SPIO和CA-SPIO-TPE治疗的肿瘤在第14天显示肿瘤快速生长，平均肿瘤大小分别为825、574和462mm3。同时，经Survivin siRNA和CA-SPIO-TPE-SP94-siRNA治疗的肿瘤在第14天被轻微抑制，平均肿瘤大小分别为272和253mm3。CA-SPIO-TPE-SP94-siRNA可明显抑制肿瘤生长，第14天肿瘤平均大小为27.5mm3。单次注射CA-SPIO-TPE-SP94-siRNA可完全抑制肿瘤生长，且无复发，说明小剂量siRNA和SP94治疗可有效释放siRNA并进行靶向基因治疗，从而达到根治肿瘤的目的。这一发现与体内荧光和磁共振成像结果一致。此外，还对体重下降进行了分析，以表明治疗引起的毒性(图9(b))。所有纳米胶片治疗组裸鼠的体重均略有增加，但与对照组(PBS组)无明显差异(P＞0.05)，各组裸鼠的体重均略有增加(P＜0.05)，但与对照组(PBS组)无明显差异。治疗中使用的siRNA剂量耐受性良好。同时，注射CA-SPIO-TPE-SP94-siRNA的裸鼠存活率最高。主要器官的H&E染色图像进一步表明，所有含有siRNA的处理都是生物相容性的，对裸鼠是安全的(图9(e))。CA-SPIO联合siRNA治疗将实时示踪、可控的siRNA释放和基因治疗相结合，实现了副作用低的抗肿瘤治疗的最佳策略。As shown in Figure 9(a), PBS, CA-SPIO and CA-SPIO-TPE treated tumors showed rapid tumor growth on day 14, with mean tumor sizes of 825, 574 and 462 mm3 , respectively. Meanwhile, tumors treated with Survivin siRNA and CA-SPIO-TPE-SP94-siRNA were slightly suppressed on day 14, with average tumor sizes of 272 and 253 mm3 , respectively. CA-SPIO-TPE-SP94-siRNA can significantly inhibit tumor growth, and the average tumor size is 27.5mm3 on the 14th day. A single injection of CA-SPIO-TPE-SP94-siRNA can completely inhibit tumor growth without recurrence, indicating that low-dose siRNA and SP94 treatment can effectively release siRNA and carry out targeted gene therapy, so as to achieve the goal of radical tumor cure. This finding is consistent with in vivo fluorescence and magnetic resonance imaging results. In addition, body weight loss was analyzed to indicate treatment-induced toxicity (Fig. 9(b)). The body weight of nude mice in all nanofilm treatment groups increased slightly, but there was no significant difference with the control group (PBS group) (P>0.05), and the body weight of nude mice in each group increased slightly (P<0.05), but compared with the control group (PBS group). group (PBS group) had no significant difference. The doses of siRNA used in the treatment were well tolerated. Meanwhile, the survival rate of nude mice injected with CA-SPIO-TPE-SP94-siRNA was the highest. H&E staining images of major organs further demonstrated that all siRNA-containing treatments were biocompatible and safe for nude mice (Fig. 9(e)). CA-SPIO combined with siRNA therapy combines real-time tracking, controllable siRNA release and gene therapy to achieve the best strategy for anti-tumor therapy with low side effects.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.
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