技术领域technical field
本发明属于生物医药技术领域，涉及一种连接有荧光示踪基团的聚赖氨酸纳米自组装微球载体的制备工艺，并进一步采用点击化学技术共价偶联被修饰药物的方法。以及利用本发明所制备的载药微球，对被修饰药物进行的整体动物和细胞水平的示踪成像分析，药物靶点定位，靶点蛋白捕获分离方面的应用。The invention belongs to the technical field of biomedicine, and relates to a preparation process of a polylysine nanometer self-assembled microsphere carrier connected with a fluorescent tracer group, and a method for covalently coupling modified drugs by click chemistry technology. And using the drug-loaded microspheres prepared by the present invention, the whole animal and cell-level tracer imaging analysis of the modified drug, drug target positioning, and application of target protein capture and separation.
背景技术Background technique
ε-聚赖氨酸（Poly-L-lysine，PL)是由25～30个赖氨酸残基通过酰胺键依次连接而成的同型单体直链状聚合物。聚赖氨酸为白色或淡黄色粉末，吸湿性强，略有苦味，不受pH值影响。ε-聚赖氨酸是一种具有抑菌功效的多肽，其具有抑菌谱广、水溶性大、热稳定性好、可生物降解且对人和环境无毒害等优点，已经成为优良的食品防腐保鲜剂。ε-聚赖氨酸能在人体内分解为赖氨酸，而赖氨酸是人体必需的氨基酸，也是世界各国允许在食品中强化的氨基酸。因此ε-聚赖氨酸是一种营养型抑菌剂，安全性高于其他化学防腐剂，其急性口服毒性为5 g/kg。ε-聚赖氨酸的抑菌谱广，对于酵母属中的尖锐假丝酵母、法红酵母、产膜毕氏酵母、玫瑰掷孢酵母；革兰氏阳性菌中的耐热脂肪芽孢杆菌、凝结芽孢杆菌、枯草芽孢杆菌；以及革兰氏阴性菌中的产气节杆菌、大肠杆菌等引起食物中毒与腐败的菌有强烈的抑制作用。分子量在3600～4300之间的ε-聚赖氨酸其抑菌活性最好，当分子量低于1300时，ε-聚赖氨酸则失去抑菌活性。ε-Poly-L-lysine (Poly-L-lysine, PL) is a linear polymer of the homotype monomer formed by sequentially linking 25 to 30 lysine residues through amide bonds. Polylysine is white or light yellow powder, strong hygroscopicity, slightly bitter taste, not affected by pH value. ε-polylysine is a polypeptide with antibacterial effect. It has the advantages of broad antibacterial spectrum, high water solubility, good thermal stability, biodegradability and non-toxicity to humans and the environment. It has become an excellent food Antiseptic preservative. ε-polylysine can be decomposed into lysine in the human body, and lysine is an essential amino acid for the human body, and it is also an amino acid that is allowed to be fortified in food in various countries around the world. Therefore, ε-polylysine is a nutritional bacteriostatic agent with higher safety than other chemical preservatives, and its acute oral toxicity is 5 g/kg. ε-polylysine has a broad antibacterial spectrum, for Candida acuminata, Rhodotorula fascia, Pichia membranosa, and Rosesporium in the genus Saccharomyces; Bacillus coagulans, Bacillus subtilis, and Gram-negative bacteria Arthrobacter aerogenes, Escherichia coli and other bacteria that cause food poisoning and spoilage have a strong inhibitory effect. The ε-polylysine with a molecular weight between 3600 and 4300 has the best antibacterial activity, and when the molecular weight is lower than 1300, the ε-polylysine loses its antibacterial activity.
此外，由于ε-聚赖氨酸带正电，能与带负电的物质发生强烈的静电吸引力，易通过生物膜且不易分解，可以有效降低药物阻力提高药物的转运效率。聚赖氨酸还具有能促进细胞生长和粘附以及促进细胞发挥正常功能等作用，因此作为药物的缓释和靶向载体ε-聚赖氨酸已被广泛用于生物医药领域。作为生物高分子材料骨架，中国专利（申请号：2014100723354）公开了一种可溶解凝血酶纳米颗粒的制备方法，该纳米颗粒以凝血酶为核心，外壳是由水溶性直链淀粉与聚赖氨酸交联形成的纳米粒子；中国专利（申请号：2013107289643，2013106763740）分别公开了ε-聚赖氨酸水凝胶的制备方法，以及由该发明衍生的伤口组织愈合材料；中国专利（申请号：2012101329801）提供了一种两亲性三嵌段共聚物的纳米载体制剂，该共聚物由包含聚乙二醇衍生物、聚赖氨酸和聚亮氨酸的线性高分子化合物组成，在水溶液中可以自组装形成具有三层结构的纳米载体，并能有效负载小分子疏水性药物、基因以及蛋白质或多肽。In addition, since ε-polylysine is positively charged, it can have a strong electrostatic attraction with negatively charged substances, and it is easy to pass through biomembranes and is not easy to decompose, which can effectively reduce drug resistance and improve drug transport efficiency. Polylysine can also promote cell growth and adhesion, and promote normal cell function. Therefore, as a slow-release and targeting carrier of drugs, ε-polylysine has been widely used in the field of biomedicine. As the skeleton of biopolymer materials, Chinese patent (application number: 2014100723354) discloses a preparation method of soluble thrombin nanoparticles. Nanoparticles formed by acid cross-linking; Chinese patent (application number: 2013107289643, 2013106763740) respectively discloses the preparation method of ε-polylysine hydrogel, and the wound tissue healing material derived from the invention; Chinese patent (application number : 2012101329801) provided a nanocarrier preparation of an amphiphilic triblock copolymer composed of a linear polymer compound containing polyethylene glycol derivatives, polylysine and polyleucine, in aqueous solution It can self-assemble to form a nanocarrier with a three-layer structure, and can effectively load small molecule hydrophobic drugs, genes, and proteins or polypeptides.
作为药物载体，中国专利（申请号：2013106705336）提供了一种作为疏水性药物的载体的双敏感响应型聚合物纳米胶束，其成分为正丁胺-聚赖氨酸(叶酸/2，3-二甲基马来酸)-b-聚半胱氨酸；中国专利（申请号：2004100680618）公开了一种短肽修饰的聚赖氨酸与聚乳酸共聚物纳米粒，该纳米粒包括药物和由精氨酰－甘氨酰－天冬氨酰序列短肽、修饰的聚赖氨酸－聚乳酸共聚物纳米粒载体组成，可用于有机药物、水溶性药物或水不溶性抗癌药物；中国专利（申请号：2004100466794）提供了一种应用“成胶现象”合成半乳糖化海藻酸钠与多聚赖氨酸纳米胶的制备方法，所得到的肝靶向性纳米基因载体系统能为原发性肝癌的靶向基因治疗提供材料；此外，中国专利（申请号：2013101802363）还公开了一种双敏感可崩解式纳米囊泡药物载体，其中双敏感两亲性嵌段共聚物由还原敏感的二硫键及pH敏感的碳氮双键桥连亲水性聚乙二醇和疏水性苄氧羰基保护的聚赖氨酸而成。该载体制剂具有疏水双分子膜及亲水内腔，疏水双分子膜负载疏水药物，亲水内腔负载亲水药物，可有效利用肿瘤细胞中的还原环境及酸性环境使得载体制剂崩解释放药物实现靶向释药。As a drug carrier, Chinese patent (application number: 2013106705336) provides a double-sensitive responsive polymer nanomicelle as a hydrophobic drug carrier, the composition of which is n-butylamine-polylysine (folate/2,3 -Dimethylmaleic acid)-b-polycysteine; Chinese patent (application number: 2004100680618) discloses a short peptide-modified polylysine and polylactic acid copolymer nanoparticle, which includes drug And it is composed of arginyl-glycyl-aspartyl sequence short peptide and modified polylysine-polylactic acid copolymer nanoparticle carrier, which can be used for organic drugs, water-soluble drugs or water-insoluble anticancer drugs; China The patent (application number: 2004100466794) provides a preparation method for synthesizing galactosylated sodium alginate and polylysine nanogel by applying the "gelation phenomenon". In addition, Chinese patent (application number: 2013101802363) also discloses a dual-sensitive disintegrable nanovesicle drug carrier, in which the dual-sensitive amphiphilic block copolymer is reduced Sensitive disulfide bonds and pH-sensitive carbon-nitrogen double bonds are bridged by hydrophilic polyethylene glycol and hydrophobic benzyloxycarbonyl-protected polylysine. The carrier preparation has a hydrophobic bimolecular membrane and a hydrophilic inner cavity, the hydrophobic bimolecular membrane is loaded with hydrophobic drugs, and the hydrophilic inner cavity is loaded with hydrophilic drugs, which can effectively utilize the reducing environment and acidic environment in tumor cells to cause the carrier preparation to disintegrate and release drugs achieve targeted drug release.
作为荧光成像纳米材料的骨架，中国专利（申请号：2012102228846）公开了一种功能性近红外荧光纳米微粒的制备方法。该纳米微粒平均粒径在15nm左右，以所装载的近红外荧光染料作为发光中心，以壳聚糖、聚赖氨酸为基本骨架，经海藻酸钠自组装包裹成壳制备而成；中国专利（申请号：201310470907X）还提供了一种信号放大型免疫荧光探针的制备方法。该探针的制备方法包括：在缩合剂的存在下，对苯二胺和均苯三甲酸经缩合反应得到多羧基大分子，多羧基大分子经活化后，依次加入抗体、聚赖氨酸进行反应，再用荧光标记物标记制得所述的探针。该探针标记有较多荧光标记物，结构稳定，可用于荧光免疫检测，具有检测灵敏度高、检测时间短、成本低等特点。此外，中国专利（申请号：2013101802363）还公开了一种两亲性三嵌段聚多肽ICG胶束，该胶束包括了分散有吲哚菁绿并由聚亮氨酸形成的内核，以及环绕内核由聚赖氨酸形成的中间层及部分穿插于所述中间层的由聚乙二醇形成的外壳。该胶束具备良好的空间稳定性以及出色的荧光性能、光热转换能力，可以实现对肿瘤细胞或组织进行光学成像和光热治疗。As the skeleton of fluorescent imaging nanomaterials, Chinese patent (application number: 2012102228846) discloses a preparation method of functional near-infrared fluorescent nanoparticles. The average particle size of the nanoparticles is about 15nm, and the loaded near-infrared fluorescent dye is used as the luminescent center, and chitosan and polylysine are used as the basic skeleton, which are self-assembled and wrapped into shells by sodium alginate; Chinese patent (Application No.: 201310470907X) also provides a method for preparing a signal-amplifying immunofluorescence probe. The preparation method of the probe comprises: in the presence of a condensing agent, p-phenylenediamine and trimesic acid are condensed to obtain a polycarboxy macromolecule, and after the polycarboxy macromolecule is activated, antibodies and polylysine are added in sequence to carry out reaction, and then labeled with a fluorescent marker to prepare the probe. The probe is marked with many fluorescent markers, has a stable structure, can be used for fluorescent immunoassay, and has the characteristics of high detection sensitivity, short detection time, low cost and the like. In addition, Chinese patent (application number: 2013101802363) also discloses an amphiphilic triblock polypeptide ICG micelle, which includes an inner core formed of polyleucine dispersed with indocyanine green, and surrounding The inner core is a middle layer formed by polylysine, and the outer shell made of polyethylene glycol is partially interspersed in the middle layer. The micelles have good steric stability, excellent fluorescence properties, and photothermal conversion capabilities, and can realize optical imaging and photothermal therapy of tumor cells or tissues.
综上所述，目前国内外均以聚赖氨酸或共混其他高分子材料为骨架进行相关材料的开发。但尚未见以聚赖氨酸自身为骨架，一方面通过酰胺键共价连接荧光分子（罗丹明），并通过自组装技术制备纳米颗粒，另一方面采用点击化学技术共价结合可释放药物分子；同时可进行被修饰药物的体内外示踪成像分析，靶点定位以及靶点蛋白捕获分离，集合上述功能用途为一体的聚赖氨酸纳米材料的报道。To sum up, at present, polylysine or blended with other polymer materials are used as the skeleton for the development of related materials at home and abroad. However, it has not been seen that polylysine itself is used as the backbone. On the one hand, fluorescent molecules (rhodamine) are covalently linked through amide bonds, and nanoparticles are prepared by self-assembly technology. On the other hand, drug molecules can be released by covalently combining with click chemistry technology. ; At the same time, in vivo and in vitro tracer imaging analysis of modified drugs, target location and target protein capture and separation can be carried out, and reports on polylysine nanomaterials that integrate the above functions and uses.
发明内容Contents of the invention
本发明的目的是利用纳米自组装技术，通过引入荧光基团和可被裂解释药的二硫键基团制备聚赖氨酸载药纳米微球，并利用聚赖氨酸的生物相容性实现被修饰药物在动物组织和细胞内的示踪，以及靶点蛋白及其相关蛋白的捕获分离，提供一种多用途聚赖氨酸荧光自组装纳米微球载体的制备方法与应用。The purpose of the present invention is to use nano self-assembly technology to prepare polylysine drug-loaded nano-microspheres by introducing fluorescent groups and disulphide bond groups that can be cracked, and utilize the biocompatibility of polylysine Realize the tracking of modified drugs in animal tissues and cells, as well as capture and separation of target proteins and related proteins, and provide a preparation method and application of a multi-purpose polylysine fluorescent self-assembled nano-microsphere carrier.
本发明可解决现有技术难以实现的药物示踪和靶点捕获一体化的问题。并以含有羟基或羧基的药物为例，通过丙炔酸酯化或丙炔胺的酰胺化修饰引入炔基，并通过和微球上修饰的叠氮基团的点击化学反应将药物连接到微球载体上，运用同一纳米载药微球，分别实现了药物体内代谢示踪、细胞内定位、以及靶点蛋白捕获分离等不同应用。The invention can solve the problem of integration of drug tracing and target capture which is difficult to realize in the prior art. And taking drugs containing hydroxyl or carboxyl groups as an example, the alkynyl group is introduced by propynoesterification or amidation of propynylamine, and the drug is linked to the microsphere through a click chemical reaction with the modified azide group on the microsphere. On the spherical carrier, the same nano drug-loaded microspheres are used to realize different applications such as drug metabolism tracking in vivo, intracellular localization, and target protein capture and separation.
本发明提供了一种多用途聚赖氨酸荧光自组装纳米微球载体，该载体是在骨架材料上共价连接有荧光标记基团和药物分子制成；所述的骨架材料是由含有25～30个赖氨酸残基通过酰胺键依次连接而成的同型单体聚合物；所述骨架材料上的赖氨酸残基同时修饰了带有二硫键的叠氮基团侧链以及荧光标记物，能够通过自组装制备纳米微球，并能用于药物分子的连接。The invention provides a multi-purpose polylysine fluorescent self-assembled nano-microsphere carrier, which is made of a fluorescent labeling group and a drug molecule covalently connected to a skeleton material; the skeleton material is composed of 25 A homopolymer of ~30 lysine residues connected sequentially through amide bonds; the lysine residues on the backbone material are simultaneously modified with disulfide-bonded azide side chains and fluorescent The marker can prepare nano-microspheres through self-assembly and can be used for the connection of drug molecules.
所述的荧光标记基团是含有罗丹明的标记分子。具体是通过市售的罗丹明活化酯（NHS-罗丹明），与聚赖氨酸的氨基以酰胺键的形式和纳米微球载体相连接。The fluorescent labeling group is a labeling molecule containing rhodamine. Specifically, the commercially available rhodamine activated ester (NHS-rhodamine) is connected to the amino group of polylysine in the form of an amide bond to the nano-microsphere carrier.
所述的带有二硫键的叠氮基团侧链，具体是通过市售的叠氮化试剂（Sulfo-SADP），其一端是活化酯，通过聚赖氨酸的氨基以酰胺键的形式与纳米微球载体相连接。The azide side chain with a disulfide bond is specifically passed through a commercially available azide reagent (Sulfo-SADP), one end of which is an activated ester, through the amino group of polylysine in the form of an amide bond Connected with the nanosphere carrier.
所述的药物分子，是炔基修饰的药物衍生物，通过丙炔酸与含有羟基的药物进行酯化反应引入炔基，或通过丙炔胺与含有羧酸的药物进行酰胺化反应引入炔基，并进一步通过点击化学的方法将其炔基修饰的药物衍生物与含有叠氮侧链纳米微球载体相连接。The drug molecule is an alkynyl-modified drug derivative, and an alkynyl group is introduced through an esterification reaction between propiolic acid and a drug containing a hydroxyl group, or an alkynyl group is introduced through an amidation reaction between a propynylamine and a drug containing a carboxylic acid , and further link its alkyne-modified drug derivatives to nanosphere carriers containing azide side chains by click chemistry.
具体制备方法如下：The specific preparation method is as follows:
第1、向含有1%至5% Span-80的由汽油和四氯化碳组成的混合有机相（汽油和四氯化碳的体积比可为1:3至3:1）中加入NHS-罗丹明溶解，并再加入溶解有0.2%-2%的聚赖氨酸的水相溶液。其中有机相和水相的比例为1:1至5:1，NHS-罗丹明的用量为聚赖氨酸的0.5%至10%。10,000至20,000转高速搅拌后得到乳化液。1. Add NHS- The rhodamine is dissolved, and the aqueous phase solution in which 0.2%-2% polylysine is dissolved is added. The ratio of the organic phase to the aqueous phase is 1:1 to 5:1, and the amount of NHS-rhodamine is 0.5% to 10% of polylysine. After stirring at 10,000 to 20,000 rpm at high speed, an emulsion is obtained.
第2、将上述乳化液转移至分液漏斗中，分别加入上述体积比1:1至5:1的石油醚以及水溶液，混合均匀后静置分层，收集水相，离心弃去沉淀，上清液即为自组装的聚赖氨酸荧光纳米微球；2. Transfer the above-mentioned emulsion to a separatory funnel, add petroleum ether and an aqueous solution with a volume ratio of 1:1 to 5:1, mix evenly, let stand to separate layers, collect the aqueous phase, centrifuge to discard the precipitate, and The serum is self-assembled polylysine fluorescent nanospheres;
第3、向第2步得到的纳米微球溶液中加入叠氮化试剂 Sulfo-SADP，室温搅拌反应0.5小时至5小时，得叠氮修饰的纳米微球，其中Sulfo-SADP的用量为聚赖氨酸用量的10%至100%；3. Add the azide reagent Sulfo-SADP to the nanosphere solution obtained in step 2, and stir and react at room temperature for 0.5 hours to 5 hours to obtain azide-modified nanospheres, wherein the amount of Sulfo-SADP is polylysine 10% to 100% of the amount of amino acid;
第4、进一步将炔基修饰的药物缓慢滴入第3步得到的纳米微球溶液中，搅拌条件下加入点击化学反应的催化剂，室温反应0.5小时至5小时，反应液经超滤浓缩，即为连接有药物的荧光纳米微球。其中炔基修饰药物的用量为聚赖氨酸用量的10%至100%；所述的炔基修饰的药物，是采用丙炔酸与含有羟基的药物通过酯化的方式制备的丙炔酸酯，或采用丙炔胺与含有羧基的药物通过酰胺化的方式制备的丙炔酰胺衍生物。4. Slowly drop the alkyne-modified drug into the nanosphere solution obtained in step 3, add a click chemical reaction catalyst under stirring conditions, and react at room temperature for 0.5 hours to 5 hours. The reaction solution is concentrated by ultrafiltration, that is Fluorescent nanospheres connected with drugs. Wherein the dosage of the alkynyl-modified drug is 10% to 100% of the dosage of polylysine; the alkynyl-modified drug is a propiolate prepared by esterification of propiolic acid and a drug containing a hydroxyl group , or propynylamide derivatives prepared by amidation of propynylamine and carboxyl-containing drugs.
此外，本发明还提供了上述聚赖氨酸荧光自组装纳米微球在药物示踪与靶点蛋白捕获方面的应用。其中包括被修饰药物牛蒡子苷元的动物以及细胞水平的成像分析；以及利用甘草次酸修饰的纳米微球捕获甘草次酸靶点蛋白的过程，包括通过纳滤分级截留分离，裂解二硫键释放出靶点蛋白的具体步骤。In addition, the present invention also provides the application of the above-mentioned polylysine fluorescent self-assembled nanometer microspheres in drug tracing and target protein capture. These include animal and cellular imaging analysis of the modified drug arctigenin; and the process of using glycyrrhetinic acid-modified nanospheres to capture glycyrrhetinic acid target proteins, including fractional cut-off separation by nanofiltration, and cleavage of disulfide bonds Specific steps for releasing the target protein.
本发明提供的纳米微球的应用之一：在药物示踪方面的应用。One of the applications of the nano-microspheres provided by the present invention is application in drug tracing.
本发明首先制备了载有牛蒡子苷元的聚赖氨酸荧光自组装纳米微球，并通过激光粒度仪、透射电子显微镜分析了上述微球的粒径和形状；采用荧光光谱仪考察了其荧光特性；并进一步通过激光共聚焦显微镜以及小动物活体成像技术评价了其在体和细胞水平的分布情况。The present invention firstly prepares polylysine fluorescent self-assembled nano-microspheres loaded with arctigenin, and analyzes the particle size and shape of the above-mentioned microspheres through a laser particle size analyzer and a transmission electron microscope; uses a fluorescence spectrometer to investigate its fluorescence properties; and further evaluated its distribution at the in vivo and cellular levels by laser confocal microscopy and small animal live imaging techniques.
本发明提供的纳米微球的应用之二：靶点蛋白捕获方面的应用。The second application of the nano-microspheres provided by the present invention is the application in the capture of target proteins.
本发明中所述的靶点蛋白捕获方面的应用，利用超滤分级截留分离捕获靶点蛋白，包括以下步骤：孵育细胞、细胞裂解、离心、超滤除杂、洗涤、还原释放蛋白、超滤洗脱等步骤（附图1）。具体操作如下：The application of target protein capture described in the present invention uses ultrafiltration fractional cut-off to separate and capture target protein, including the following steps: incubating cells, cell lysis, centrifugation, ultrafiltration to remove impurities, washing, reduction to release proteins, ultrafiltration Steps such as elution (Figure 1). The specific operation is as follows:
向人肺上皮BEAS-2B细胞中加入连接甘草次酸的聚赖氨酸荧光自组装纳米微球，培养48 小时后，经胰酶消化，超声破碎，离心，得到结合有靶标蛋白的聚赖氨酸纳米微球悬浊液。将上述含有纳米微球的溶液加入到截留分子量为3 kD的超滤管中，离心弃去小分子，将截留液加入到截留分子量为100 kD的超滤管中，离心弃去未结合的大分子蛋白，进一步向截留液中加入二硫苏糖醇（DTT）溶液还原二硫键，再次加入到截留分子量为100 kD的超滤管中，离心，收集滤液得到与甘草次酸结合的靶点蛋白以及相关蛋白。Add polylysine fluorescent self-assembled nanospheres linked to glycyrrhetinic acid to human lung epithelial BEAS-2B cells, culture for 48 hours, trypsinize, sonicate, and centrifuge to obtain polylysine bound to the target protein Suspension of acid nanospheres. Add the above-mentioned solution containing nanospheres into an ultrafiltration tube with a molecular weight cut-off of 3 kD, centrifuge to discard small molecules, add the retentate to an ultrafiltration tube with a molecular weight cut-off of 100 kD, and discard unbound large molecules by centrifugation. Molecular protein, further add dithiothreitol (DTT) solution to the retentate to reduce the disulfide bond, add it again to an ultrafiltration tube with a molecular weight cut-off of 100 kD, centrifuge, collect the filtrate to obtain the target that binds to glycyrrhetinic acid protein and related proteins.
综上所述，本发明提供了一种研究药物分布以及作用靶点所需要的简便快捷的载体工具和方法，该聚赖氨酸载药荧光纳米微球，集靶点定位，靶点蛋白捕获与分离功能于一体，可用于药物作用机制的研究。In summary, the present invention provides a simple and quick carrier tool and method for studying drug distribution and target sites. The polylysine drug-loaded fluorescent nanospheres can be used for target positioning and target protein capture. Integrating with the separation function, it can be used for the study of the mechanism of drug action.
本发明的优点和积极效果：Advantage and positive effect of the present invention:
本发明提供了一种制备多用途聚赖氨酸荧光纳米自组装载药微球的新方法，并创新性的同时引入罗丹明荧光标记和可被还原裂解的二硫键的叠氮基团，通过点击化学反应连接炔基修饰药物。所制备的纳米自组装载药微球呈球形，粒度分散均匀，平均粒径为38nm，具有良好的胶体性质，最大发射波长为600 nm，可以有效避免生物样本自身的干扰，被荧光检测和示踪。同时该纳米微球生物相容性好，有利于穿过细胞膜，可以顺利地指示和捕获靶点蛋白，并通过裂解二硫键可释放出靶点蛋白及其关联蛋白。该方法制备工艺简便易行，所制备的载药纳米微球集靶点示踪定位，靶点蛋白捕获及分离功能于一体，可用于被修饰药物的动物和细胞水平的成像分析，以及靶点蛋白分离鉴定，在药物作用机制的研究方面有很好的应用前景。The present invention provides a new method for preparing multi-purpose polylysine fluorescent nanometer self-assembled drug-loaded microspheres, and innovatively introduces rhodamine fluorescent label and azide group of disulfide bond that can be reductively cleaved at the same time, Linking alkyne-modified drugs via click chemistry. The prepared nano-self-assembled drug-loaded microspheres are spherical, uniformly dispersed in particle size, with an average particle size of 38nm, good colloidal properties, and a maximum emission wavelength of 600 nm, which can effectively avoid the interference of biological samples themselves, and can be detected and displayed by fluorescence. trace. At the same time, the nanosphere has good biocompatibility, is conducive to passing through the cell membrane, can smoothly indicate and capture the target protein, and can release the target protein and its associated protein by cleaving the disulfide bond. The preparation process of this method is simple and easy, and the prepared drug-loaded nano-microspheres integrate target tracking and positioning, target protein capture and separation functions, and can be used for imaging analysis of modified drugs at the animal and cell levels, as well as target Protein separation and identification have a good application prospect in the study of the mechanism of drug action.
附图说明Description of drawings
图1是实施例1，2和3，叠氮修饰的聚赖氨酸荧光纳米微球的合成路线图；Fig. 1 is embodiment 1, 2 and 3, the synthesis roadmap of the polylysine fluorescent nanosphere of azide modification;
图2是实施例4，药物分子牛蒡子苷元的炔基化修饰以及与聚赖氨酸荧光纳米微球连接路线示意图；Fig. 2 is a schematic diagram of embodiment 4, the alkynylation modification of the drug molecule arctigenin and the connection route with polylysine fluorescent nanospheres;
图3是实施例5，聚赖氨酸荧光纳米自组装载药纳米微球的形态学和荧光特性考察。其中，A为纳米微球溶液中分散状况照片； B为纳米微球的胶体特性示意图；C为纳米微球粒径分布图；D为纳米微球TEM透射电镜的整体照片；E为纳米微球TEM透射电镜的放大照片；F 为罗丹明溶液和聚赖氨酸荧光纳米自组装微球胶体溶液的荧光发射光谱图；Fig. 3 is Example 5, investigation of the morphology and fluorescence characteristics of polylysine fluorescent nanometer self-assembled drug-loaded nanospheres. Among them, A is a photo of the dispersion state of nanospheres in solution; B is a schematic diagram of the colloidal characteristics of nanospheres; C is a particle size distribution diagram of nanospheres; D is an overall photo of nanospheres by TEM transmission electron microscope; E is a nanosphere Enlarged photo of TEM transmission electron microscope; F is the fluorescence emission spectrum of rhodamine solution and polylysine fluorescent nanometer self-assembled microsphere colloidal solution;
图4是实施例6，药物分子甘草次酸的炔基化修饰以及与聚赖氨酸荧光纳米微球连接路线示意图；Figure 4 is a schematic diagram of embodiment 6, the alkynylation modification of the drug molecule glycyrrhetinic acid and the connection route with polylysine fluorescent nanospheres;
图5是实施例7，牛蒡子苷元载药的聚赖氨酸荧光纳米微球的细胞成像图和小鼠活体成像示踪图片。 A组为细胞加入实施例1所制备的聚赖氨酸荧光空白纳米微球的阴性对照，其中，A1为细胞DAPI染色图片，A2为细胞荧光照片，A3为A1和A2的叠加照片；B组为细胞加入实施例2所制备的牛蒡苷元修饰的聚赖氨酸荧光纳米载药微球的照片，其中，B1为细胞DAPI染色图片，B2为细胞荧光照片，B3为B1和B2的叠加照片；C组为实施例2所制备的聚赖氨酸荧光纳米载药微球的小鼠活体示踪图片；其中，C1为给药前0分钟，C2为给药后60分钟的活体成像图片；Fig. 5 is the cell imaging image and mouse in vivo imaging tracing image of arctigenin-loaded polylysine fluorescent nanospheres in Example 7. Group A is the negative control of cells adding the polylysine fluorescent blank nanospheres prepared in Example 1, wherein A1 is a picture of DAPI staining of cells, A2 is a photo of cell fluorescence, and A3 is a superimposed photo of A1 and A2; Group B The photos of the arctigenin-modified polylysine fluorescent nanometer drug-loaded microspheres prepared in Example 2 are added to the cells, wherein B1 is a picture of DAPI staining of cells, B2 is a photo of cell fluorescence, and B3 is a superimposed photo of B1 and B2 Group C is the mouse in vivo tracing picture of the polylysine fluorescent nano drug-loaded microspheres prepared in Example 2; wherein, C1 is 0 minutes before administration, and C2 is the in vivo imaging picture of 60 minutes after administration;
图6是实施例8，聚赖氨酸纳米载药微球捕获靶点蛋白的操作示意图；Fig. 6 is embodiment 8, the schematic diagram of the operation of polylysine nano drug-loaded microspheres capturing target protein;
图7是实施例8，实施例5所制备的甘草次酸载药纳米微球捕获靶点蛋白的SDS-PAGE电泳检测图。其中，第1泳道为蛋白Marker；第2泳道为细胞裂解蛋白样品；第3泳道为经过3 kD超滤管超滤的滤过液；第4泳道为滤液中截留蛋白；第5泳道为DTT还原后，经过100kD超滤管超滤的截留液；第6泳道为DTT还原后，经过100 kD的超滤管超滤后的滤过液。Fig. 7 is the SDS-PAGE electrophoresis detection diagram of the target protein captured by glycyrrhetinic acid drug-loaded nano-microspheres prepared in Example 8 and Example 5. Among them, the first lane is the protein marker; the second lane is the cell lysed protein sample; the third lane is the filtrate filtered by 3 kD ultrafiltration tube; the fourth lane is the retained protein in the filtrate; the fifth lane is DTT reduction After that, the retentate was ultrafiltered through a 100 kD ultrafiltration tube; the sixth lane was the filtrate after ultrafiltration through a 100 kD ultrafiltration tube after DTT reduction.
具体实施方式detailed description
实施例1、聚赖氨酸高度荧光化修饰纳米微球的制备与叠氮化修饰Example 1. Preparation and azide modification of highly fluorescent polylysine modified nanospheres
取5 g聚赖氨酸盐酸盐溶于 500 mL纯水，上样于活化的001×7阳离子交换树脂（2×80cm）脱盐，上样速度为1 mL/分钟。经1500 mL纯水平衡至流出液为中性后，用5%氨水洗脱，收集洗脱液，减压蒸馏，浓缩干燥，得白色至淡黄色固体1.9 g。Dissolve 5 g of polylysine hydrochloride in 500 mL of pure water and load it on activated 001×7 cation exchange resin (2×80 cm) for desalting at a rate of 1 mL/min. Equilibrate with 1500 mL of pure water until the effluent is neutral, elute with 5% ammonia water, collect the eluate, distill under reduced pressure, concentrate and dry to obtain 1.9 g of white to light yellow solid.
取上述脱盐后的聚赖氨酸200 mg于10 mL纯水中，搅拌使其完全溶解得水相溶液；同时分别将13 mL汽油、12 mL四氯化碳以及1.25mL Span-80混合，搅拌均匀获得有机相。向有机相中加入20 mg NHS-罗丹明（NHS-Rhodamine，Pierce 46406），搅拌混匀，放置备用。将上述水相迅速倒入有机相中，12,000转搅拌30秒，重复3次，得到均匀的乳化液，转移至分液漏斗中。分别加入50 mL石油醚，室温搅拌5分钟，再加入10 mL 纯水，继续搅拌5分钟，静置分层，收集水相，得到荧光纳米微球粗品。将粗品12,000 转离心10 分钟，弃去沉淀，上清液即为聚赖氨酸荧光化修饰的纳米自组装微球。Take 200 mg of the above-mentioned desalted polylysine in 10 mL of pure water, stir to dissolve completely to obtain an aqueous phase solution; at the same time, mix 13 mL of gasoline, 12 mL of carbon tetrachloride and 1.25 mL of Span-80, and stir The organic phase is obtained homogeneously. Add 20 mg of NHS-Rhodamine (NHS-Rhodamine, Pierce 46406) to the organic phase, mix well, and set aside. Quickly pour the above water phase into the organic phase, stir at 12,000 rpm for 30 seconds, repeat 3 times to obtain a uniform emulsion, and transfer it to a separatory funnel. Add 50 mL of petroleum ether respectively, stir at room temperature for 5 minutes, then add 10 mL of pure water, continue stirring for 5 minutes, let stand to separate layers, collect the aqueous phase, and obtain crude fluorescent nanospheres. The crude product was centrifuged at 12,000 rpm for 10 minutes, the precipitate was discarded, and the supernatant was poly-lysine fluorescently modified nanometer self-assembled microspheres.
取上述2 mL聚赖氨酸荧光纳米微球溶液，加入叠氮化试剂10 mg Sulfo-SADP（Pierce 21553）(0.02 mmoL)，室温搅拌0.5 小时，得带有二硫键并叠氮修饰的纳米自组装聚赖氨酸纳米微球，4 ℃保存备用。合成路线如图1所示。Take the above 2 mL polylysine fluorescent nanosphere solution, add azide reagent 10 mg Sulfo-SADP (Pierce 21553) (0.02 mmoL), and stir at room temperature for 0.5 h to obtain disulfide bonded and azide-modified nanoparticles Self-assembled polylysine nanospheres were stored at 4°C for later use. The synthetic route is shown in Figure 1.
实施例2、聚赖氨酸荧光纳米微球的制备与叠氮化修饰Example 2, Preparation and Azide Modification of Polylysine Fluorescent Nanospheres
取上述实施例1脱盐后的聚赖氨酸50 mg溶于10 mL纯水中，得水相溶液；同时分别将5mL汽油、15mL四氯化碳以及0.5 mL Span-80混合，搅拌均匀获得有机相。向有机相中加入0.1 mg NHS-罗丹明（NHS-Rhodamine，Pierce 46406），搅拌溶解，放置备用。将上述水相迅速倒入有机相中，20,000 转高速搅拌15秒，重复3次，得到均匀的乳化液，转移至分液漏斗中。分别加入20 mL 石油醚，搅拌5 分钟，再加入10 mL 纯水，继续搅拌5 分钟，静置分层，收集水相，得到荧光纳米微球粗品。将粗品12,000转离心10 分钟，弃去沉淀，上清液即为聚赖氨酸荧光纳米自组装微球。取上述聚赖氨酸荧光纳米微球溶液2 mL，加入叠氮化试剂50 mg Sulfo-SADP（Pierce 21553）(0. 1 mmoL)，室温搅拌5 小时，得到叠氮化修饰的纳米自组装聚赖氨酸纳米微球，4 ℃备用。合成路线同图1。Dissolve 50 mg of the desalted polylysine in Example 1 above in 10 mL of pure water to obtain an aqueous phase solution; simultaneously mix 5 mL of gasoline, 15 mL of carbon tetrachloride and 0.5 mL of Span-80, and stir evenly to obtain an organic Mutually. Add 0.1 mg of NHS-Rhodamine (NHS-Rhodamine, Pierce 46406) to the organic phase, stir to dissolve, and set aside for later use. Pour the above water phase into the organic phase quickly, stir at 20,000 rpm for 15 seconds at high speed, repeat 3 times to obtain a uniform emulsion, and transfer it to a separatory funnel. Add 20 mL of petroleum ether, stir for 5 minutes, then add 10 mL of pure water, continue stirring for 5 minutes, let stand to separate layers, collect the aqueous phase, and obtain crude fluorescent nanospheres. The crude product was centrifuged at 12,000 rpm for 10 minutes, the precipitate was discarded, and the supernatant was polylysine fluorescent nanometer self-assembled microspheres. Take 2 mL of the above polylysine fluorescent nanosphere solution, add 50 mg Sulfo-SADP (Pierce 21553) (0.1 mmoL) of the azide reagent, and stir at room temperature for 5 hours to obtain the azide-modified self-assembled nanospheres. Lysine nanospheres, at 4 ℃ for later use. The synthetic route is the same as Fig. 1.
实施例3、聚赖氨酸荧光纳米微球的制备与高度叠氮化修饰Example 3. Preparation and highly azide modification of polylysine fluorescent nanospheres
取上述实施例1脱盐后的聚赖氨酸20 mg溶于10 mL纯水中，得水相溶液；同时分别将15 mL汽油、5 mL四氯化碳以及0.2mL Span-80混合，搅拌均匀获得有机相。向有机相中加入2 mg NHS-罗丹明（NHS-Rhodamine，Pierce 46406）（0.002 mmoL），搅拌溶解，放置备用。将上述水相迅速倒入有机相中，10,000 转高速搅拌20秒，重复3次，得到均匀的乳化液，转移至分液漏斗中。分别加入10 mL 石油醚，搅拌5 分钟，再加入10 mL 纯水，继续搅拌5 分钟，静置分层，收集水相，得到荧光纳米微球粗品。将粗品12,000转离心10 分钟，弃去沉淀，上清液即为聚赖氨酸荧光纳米自组装微球。取上述聚赖氨酸荧光纳米微球溶液4 mL，加入叠氮化试剂200 mg Sulfo-SADP（Pierce 21553）(0.4 mmoL)，室温搅拌5 小时，可得到高度叠氮化修饰的纳米自组装聚赖氨酸纳米微球，4 ℃备用。合成路线同图1。Dissolve 20 mg of the desalted polylysine in Example 1 above in 10 mL of pure water to obtain an aqueous phase solution; at the same time, mix 15 mL of gasoline, 5 mL of carbon tetrachloride and 0.2 mL of Span-80, and stir evenly The organic phase is obtained. Add 2 mg of NHS-Rhodamine (NHS-Rhodamine, Pierce 46406) (0.002 mmoL) to the organic phase, stir to dissolve, and set aside for later use. Pour the above water phase into the organic phase quickly, stir at 10,000 rpm for 20 seconds at high speed, repeat 3 times to obtain a uniform emulsion, and transfer it to a separatory funnel. Add 10 mL of petroleum ether, stir for 5 minutes, then add 10 mL of pure water, continue stirring for 5 minutes, let stand to separate layers, collect the aqueous phase, and obtain crude fluorescent nanospheres. The crude product was centrifuged at 12,000 rpm for 10 minutes, the precipitate was discarded, and the supernatant was polylysine fluorescent nanometer self-assembled microspheres. Take 4 mL of the above poly-lysine fluorescent nanosphere solution, add azide reagent 200 mg Sulfo-SADP (Pierce 21553) (0.4 mmoL), and stir at room temperature for 5 hours to obtain highly azide-modified nano self-assembled polysaccharides. Lysine nanospheres, at 4 ℃ for later use. The synthetic route is the same as Fig. 1.
实施例4、牛蒡子苷元的炔基化修饰以及与聚赖氨酸荧光纳米微球的连接Example 4. Alkynylation modification of arctigenin and connection with polylysine fluorescent nanospheres
以含有酚羟基的药物牛蒡苷元为例，通过与丙炔酸的酯化反应进行药物的炔基化修饰，进一步通过点击化学反应与实施例1所制备的叠氮化修饰的聚赖氨酸荧光纳米微球偶联，合成路线见图2。具体实施如下：Taking the drug arctigenin containing a phenolic hydroxyl group as an example, the drug is modified by alkynylation through the esterification reaction with propiolic acid, and further click chemical reaction with the azide-modified polylysine prepared in Example 1 Fluorescent nanosphere coupling, the synthesis route is shown in Figure 2. The specific implementation is as follows:
向圆底烧瓶中分别加入0.372 g牛蒡子苷元(1 mmoL)，0.198 g EDC·HCl (2mmoL)，以及5 mL预冷的CH2Cl2溶剂，磁力搅拌30分钟，溶解后再依次加入0.116 g N-羟基琥珀酰亚胺(NHS, 2 mmoL)，0.140 g丙炔酸(2 mmoL)，保持冰浴反应4 小时。待反应结束后，向反应液中加入15 mL CH2Cl2稀释，然后依次用1M HCl，5% NaHCO3溶液，以及饱和食盐水洗涤。收集有机相，加入无水Na2SO4干燥，过滤，减压蒸馏浓缩，得到油状物即为牛蒡子苷元丙炔酸酯粗品。Add 0.372 g arctigenin (1 mmoL), 0.198 g EDC·HCl (2 mmoL), and 5 mL of pre-cooled CH2 Cl2 solvent into the round bottom flask, stir magnetically for 30 minutes, dissolve and then add 0.116 g N-hydroxysuccinimide (NHS, 2 mmoL), 0.140 g propiolic acid (2 mmoL), keep the reaction in ice bath for 4 hours. After the reaction was completed, 15 mL of CH2 Cl2 was added to the reaction solution for dilution, and then washed with 1M HCl, 5% NaHCO3 solution, and saturated brine. The organic phase was collected, dried by adding anhydrous Na2 SO4 , filtered, and concentrated by distillation under reduced pressure to obtain an oil which was the crude arctigenin propiolate.
将粗品过硅胶柱纯化，得到白色固体粉末，称重得牛蒡子苷元丙炔酸酯0.196 g,产率约为47%。Rf=0.7 (PE / EtOAc 1:1);1H NMR [CDCl3, 400 MHz] 6.98-6.94 (m,1H), 6.79-6.75 (m, 2H), 6.68-6.65 (m, 1H), 6.58-6.50 (m, 2H), 3.84(s, 3H),3.81 (s, 3H), 3.76-3.74 (m, 2H), 2.97-2.95 (m, 2H), 2.83 (s, 1H), 2.66-2.48(m, 4H)。The crude product was purified through a silica gel column to obtain a white solid powder, which was weighed to obtain 0.196 g of arctigenin propiolate, with a yield of about 47%.Rf =0.7 (PE / EtOAc 1:1);1 H NMR [CDCl3 , 400 MHz] 6.98-6.94 (m,1H), 6.79-6.75 (m, 2H), 6.68-6.65 (m, 1H), 6.58 -6.50 (m, 2H), 3.84(s, 3H), 3.81 (s, 3H), 3.76-3.74 (m, 2H), 2.97-2.95 (m, 2H), 2.83 (s, 1H), 2.66-2.48 (m, 4H).
取上述所得的5 mg 牛蒡子苷元丙炔酸酯（0.01 mmoL）溶于100 μL 二甲基亚砜（DMSO）中，并将其缓慢滴入实施例1制备的1 mL叠氮修饰的聚赖氨酸自组装纳米微球溶液中，最后加入点击化学反应的催化剂（0.2 mmol/L Tris-triazoleamine，1.0 mmol/LCuSO4和2.0 mmol/L抗坏血酸钠），室温搅拌1 小时。反应结束后加入到截留分子量为3MilliporekD的超滤管中，3000 转离心30 分钟，弃去滤液，分别用0.2 mmol/L磷酸盐缓冲液2 mL洗涤2次，收集截留液，即为连接有牛蒡子苷元的高度荧光化修饰的纳米微球。Dissolve 5 mg arctigenin propiolate (0.01 mmoL) obtained above in 100 μL dimethyl sulfoxide (DMSO), and slowly drop it into 1 mL of the azide-modified polysaccharide prepared in Example 1. In the lysine self-assembled nanosphere solution, the catalyst for the click chemical reaction (0.2 mmol/L Tris-triazoleamine, 1.0 mmol/LCuSO4 and 2.0 mmol/L sodium ascorbate) was finally added, and stirred at room temperature for 1 hour. After the reaction, add it to an ultrafiltration tube with a molecular weight cut-off of 3 MilliporekD, centrifuge at 3000 rpm for 30 minutes, discard the filtrate, wash twice with 2 mL of 0.2 mmol/L phosphate buffer solution, and collect the retentate, that is, the burdock is connected. Highly fluorescent modified nanospheres of aglycones.
实施例5、聚赖氨酸荧光纳米微球的形态学与荧光特征Example 5, Morphological and Fluorescent Characteristics of Polylysine Fluorescent Nanospheres
上述实施例3所制备的牛蒡子苷元修饰的聚赖氨酸荧光纳米载药微球在水溶液中呈现橙黄色，且分散均匀（图3A）。通过线光源照射，能够明显看到丁达尔现象，证明是胶体溶液，如图3B所示。经激光粒度分析仪分析，其平均粒径为38 nm（图3C）。采用TEM透射电子显微镜进行观查，该聚赖氨酸纳米微球的粒径大小均匀，呈规则球状（图3D），进一步放大观测显示其内部为空心状，如图3E所示。The arctigenin-modified polylysine fluorescent nanometer drug-loaded microspheres prepared in Example 3 above were orange-yellow in aqueous solution and dispersed uniformly ( FIG. 3A ). Through the irradiation of the line light source, the Tyndall phenomenon can be clearly seen, which proves that it is a colloidal solution, as shown in Figure 3B. Analyzed by a laser particle size analyzer, the average particle size was 38 nm (Fig. 3C). Observation with a TEM transmission electron microscope showed that the polylysine nanospheres were uniform in particle size and had a regular spherical shape (Fig. 3D). Further magnification and observation showed that the interior was hollow, as shown in Fig. 3E.
采用荧光光谱仪分别对相同用量的罗丹明溶液和聚赖氨酸荧光纳米微球进行了荧光光谱扫描，考察纳米自组装球的荧光特性。如图3F所示，在507nm的激发波长下，罗丹明溶液的最大发射波长为590 nm，而聚赖氨酸荧光纳米微球的最大发射波长接近600 nm。但二者的荧光强度明显不同，罗丹明溶液的荧光强度为625 a.u，而荧光纳米微球的荧光强度为1149 a.u几乎增加了一倍。这是因为聚赖氨酸将荧光染料罗丹明较为集中地组装到纳米微球的内部，微球内部的荧光量子效率提高导致荧光强度得到增强，符合近红外荧光探针的基本要求，可有效避免生物样本的自身干扰，适合药物分子的示踪分析。Fluorescence spectrum scanning of the rhodamine solution and polylysine fluorescent nanospheres with the same dosage was carried out by a fluorescence spectrometer to investigate the fluorescence characteristics of the self-assembled nanospheres. As shown in Figure 3F, at an excitation wavelength of 507 nm, the maximum emission wavelength of rhodamine solution was 590 nm, while the maximum emission wavelength of polylysine fluorescent nanospheres was close to 600 nm. However, the fluorescence intensity of the two is obviously different. The fluorescence intensity of rhodamine solution is 625 a.u, while the fluorescence intensity of fluorescent nano-microspheres is almost doubled at 1149 a.u. This is because polylysine assembles the fluorescent dye rhodamine into the interior of the nano-microspheres, and the increase in the fluorescence quantum efficiency inside the microspheres leads to an enhanced fluorescence intensity, which meets the basic requirements of near-infrared fluorescent probes and can effectively avoid The self-interference of biological samples is suitable for trace analysis of drug molecules.
实施例6、甘草次酸的炔基化修饰以及与聚赖氨酸荧光纳米微球的连接Example 6. Alkynylation modification of glycyrrhetinic acid and connection with polylysine fluorescent nanospheres
以含有羧酸的药物甘草次酸为例，通过与丙炔胺的酰胺化反应进行药物的炔基化修饰，进一步通过点击化学反应与实施例2所制备的高度叠氮化修饰的聚赖氨酸荧光纳米微球偶联，合成路线见图4。具体实施如下：Taking the drug glycyrrhetinic acid containing carboxylic acid as an example, the alkynylation modification of the drug is carried out through the amidation reaction with propargylamine, and the highly azide-modified polylysine prepared in Example 2 is further passed through the click chemical reaction. Acid fluorescent nanosphere coupling, the synthetic route is shown in Figure 4. The specific implementation is as follows:
向圆底烧瓶中分别加入0.941 g甘草次酸(2 mmoL)，0.460 g EDC·HCl (2.4mmoL)，0.324 g HOBt (2.4 mmoL)，以及5 mL预冷的CH2Cl2溶剂，磁力搅拌30 分钟，溶解后滴加三乙胺(TEA) 0.7g (约7 mmoL)，继续搅拌30 分钟，加入0.165 g丙炔胺(3 mmoL)，保持冰浴反应过夜。待反应结束后，向反应液中加入15 ml CH2Cl2稀释，然后依次用1M HCl，5%NaHCO3溶液，以及饱和食盐水洗涤。收集有机相，加入无水Na2SO4干燥，过滤，减压蒸馏浓缩，得到油状物即为甘草次酸的丙炔酰胺衍生物粗品。Add 0.941 g glycyrrhetinic acid (2 mmoL), 0.460 g EDC·HCl (2.4 mmoL), 0.324 g HOBt (2.4 mmoL), and 5 mL pre-cooled CH2 Cl2 solvent into the round bottom flask, and stir magnetically for 30 After dissolving, add 0.7 g (about 7 mmoL) of triethylamine (TEA) dropwise, continue to stir for 30 minutes, add 0.165 g of propargylamine (3 mmoL), and keep the ice bath to react overnight. After the reaction was completed, 15 ml CH2 Cl2 was added to the reaction solution for dilution, and then washed with 1M HCl, 5% NaHCO3 solution, and saturated brine. The organic phase was collected, dried by adding anhydrous Na2 SO4 , filtered, and concentrated by distillation under reduced pressure to obtain an oily product which was a crude propynamide derivative of glycyrrhetinic acid.
进一步采用中压制备液相进行纯化，得到白色固体粉末，称重得丙炔酰胺化甘草次酸产物0.482 g, 产率约为48%。1H NMR (400 MHz, CDCl3) δ 5.93 (t,J = 5.0 Hz,1H), 5.70 (s, 1H), 4.13 (ddd,J = 17.5, 5.4, 2.5 Hz, 1H), 4.03 (ddd,J =17.5, 4.9, 2.5 Hz, 1H), 3.24 (dd,J = 10.5, 5.8 Hz, 1H), 2.80 (dt,J = 13.3,3.3 Hz, 1H), 2.35 (s, 1H), 2.26 (t,J = 2.5 Hz, 1H), 2.17 (dd,J = 12.2, 5.1Hz, 1H), 2.11 – 2.00 (m, 2H), 1.96 (d,J = 10.4 Hz, 1H), 1.90 – 1.74 (m, 3H),1.72 – 1.58 (m, 4H), 1.51 – 1.43 (m, 2H), 1.42 – 1.37 (m, 6H), 1.25 – 1.18(m, 1H), 1.18 – 1.12 (m, 9H), 1.05 – 1.00 (m, 4H), 0.96 (dd,J = 12.5, 4.7Hz, 1H), 0.87 – 0.78 (m, 6H), 0.71 (d,J = 11.7 Hz, 1H)；13C NMR (101 MHz,CDCl3) δppm 200.18, 175.51, 169.09, 128.54, 79.78, 78.78, 71.67, 61.83,54.95, 48.04, 45.37, 43.54, 43.19, 41.77, 39.16, 37.35, 37.08, 32.76, 31.90,31.44, 29.31, 28.40, 28.11, 27.28, 26.43, 23.35, 18.67, 17.48, 16.37, 15.58。Further purification was carried out by medium-pressure preparative liquid phase to obtain a white solid powder, which was weighed to obtain 0.482 g of propyne amidated glycyrrhetinic acid product, with a yield of about 48%.1 H NMR (400 MHz, CDCl3 ) δ 5.93 (t,J = 5.0 Hz, 1H), 5.70 (s, 1H), 4.13 (ddd,J = 17.5, 5.4, 2.5 Hz, 1H), 4.03 (ddd,J =17.5, 4.9, 2.5 Hz, 1H), 3.24 (dd,J = 10.5, 5.8 Hz, 1H), 2.80 (dt,J = 13.3,3.3 Hz, 1H), 2.35 (s, 1H), 2.26 (t ,J = 2.5 Hz, 1H), 2.17 (dd,J = 12.2, 5.1Hz, 1H), 2.11 – 2.00 (m, 2H), 1.96 (d,J = 10.4 Hz, 1H), 1.90 – 1.74 (m, 3H),1.72 – 1.58 (m, 4H), 1.51 – 1.43 (m, 2H), 1.42 – 1.37 (m, 6H), 1.25 – 1.18(m, 1H), 1.18 – 1.12 (m, 9H), 1.05 – 1.00 (m, 4H), 0.96 (dd,J = 12.5, 4.7Hz, 1H), 0.87 – 0.78 (m, 6H), 0.71 (d,J = 11.7 Hz, 1H);13 C NMR (101 MHz, CDCl3 ) δppm 200.18, 175.51, 169.09, 128.54, 79.78, 78.78, 71.67, 61.83,54.95, 48.04, 45.37, 43.54, 43.19, 41.77, 39.16, 37.35, 37.08, 32.76, 31.90,31.44, 29.31, 28.40, 28.11, 27.28, 26.43, 23.35, 18.67, 17.48, 16.37, 15.58.
取上述所得的甘草次酸丙炔酰胺衍生物100 mg（约0.2 mmoL）溶于500 μL DMSO中，并将其缓慢滴入实施例2制备的4 mL叠氮修饰的聚赖氨酸自组装纳米微球溶液中，最后加入点击化学反应的催化剂（2 mmol/L Tris-triazoleamine，15 mmol/L CuSO4和30mmol/L抗坏血酸钠），室温搅拌2 小时。反应结束后加入到截留分子量为3 kD的Millipore超滤管中，3000 转离心30 分钟，弃去滤液，分别用0.2 mmol/L磷酸盐缓冲液5 mL洗涤3次，收集截留液，即为连接有甘草次酸药物的荧光纳米微球，可用于靶点蛋白的捕获与分离。Take 100 mg (about 0.2 mmoL) of the glycyrrhetinic acid propynamide derivative obtained above and dissolve it in 500 μL DMSO, and slowly drop it into 4 mL of the azide-modified polylysine self-assembled nano In the microsphere solution, the catalyst for the click chemical reaction (2 mmol/L Tris-triazoleamine, 15 mmol/L CuSO4 and 30 mmol/L sodium ascorbate) was finally added, and stirred at room temperature for 2 hours. After the reaction was completed, it was added to a Millipore ultrafiltration tube with a molecular weight cut-off of 3 kD, centrifuged at 3000 rpm for 30 minutes, the filtrate was discarded, washed three times with 5 mL of 0.2 mmol/L phosphate buffer solution, and the retentate was collected, which was the connection Fluorescent nanospheres with glycyrrhetinic acid drugs can be used for the capture and separation of target proteins.
实施例7、聚赖氨酸荧光纳米微球应用于细胞与活体成像Example 7, Polylysine Fluorescent Nanospheres Applied to Cell and Live Imaging
分别取上述实施例1和2所制备的聚赖氨酸荧光纳米自组装微球 30 µL，加入到人肺上皮BEAS-2B的培养细胞中，采用共聚焦显微镜TCS SP5观察，使用543 nm激发光，570 nm发射光检测细胞内的载药纳米微球的分布状况。结果如图5A所示，没有连接药物的纳米微球孵育3h后，将细胞用DAPI染色后，反复洗去游离的荧光染料，经观察只能看到细胞核DAPI的蓝色荧光，几乎看不到罗丹明的红色荧光。而偶联有牛蒡子苷元的纳米微球，同样经3小时孵育后，进行DAPI染色，并反复洗去游离的荧光染料，镜下观察发现细胞核呈蓝色，而胞浆内有着很强的红色荧光，但细胞膜和细胞核内均没有罗丹明的明显红色荧光（图5B）。结果表明，本专利所发明的载药纳米微球具有良好的生物相容性，可以有效地进入靶细胞内，为进一步靶点蛋白的捕获与富集提供了可行性。Take 30 µL of the polylysine fluorescent nano-self-assembled microspheres prepared in the above-mentioned Examples 1 and 2, respectively, and add them to the cultured cells of human lung epithelial BEAS-2B, observe with a confocal microscope TCS SP5, and use 543 nm excitation light , 570 nm emitted light to detect the distribution of drug-loaded nano-microspheres in cells. The results are shown in Figure 5A. After incubating the nanospheres without drugs for 3 hours, the cells were stained with DAPI, and the free fluorescent dye was repeatedly washed away. After observation, only the blue fluorescence of the nucleus DAPI could be seen, and almost nothing Red fluorescence of rhodamine. The nano-microspheres coupled with arctigenin were also stained with DAPI after 3 hours of incubation, and the free fluorescent dye was repeatedly washed away. Under the microscope, it was found that the nucleus was blue, and there was a strong cytoplasm. Red fluorescence, but there is no obvious red fluorescence of rhodamine in the cell membrane and nucleus (Fig. 5B). The results show that the drug-loaded nano-microspheres invented by this patent have good biocompatibility and can effectively enter target cells, which provides feasibility for further capture and enrichment of target proteins.
为了进一步观测所制备的载药纳米微球在小鼠体内的示踪变化，取昆明小鼠并尾静脉注射100 µL实施例2所制备的荧光纳米微球，并采用小动物活体成像系统观测纳米微球在小鼠体内的分布情况。结果如图5C所示，与给药前0 分钟的对照组相比，给药60 分钟后小鼠的腹腔下部有着明显的罗丹明荧光聚集，表明载药聚赖氨酸荧光纳米微球经过小鼠体液循环之后，被代谢并转移到了小鼠的膀胱中。In order to further observe the tracer changes of the prepared drug-loaded nanospheres in mice, Kunming mice were taken and injected with 100 µL of the fluorescent nanospheres prepared in Example 2 into the tail vein, and the small animal in vivo imaging system was used to observe the nanospheres. Distribution of microspheres in mice. The results are shown in Figure 5C. Compared with the control group at 0 minutes before administration, there was obvious rhodamine fluorescence aggregation in the lower abdominal cavity of mice after administration for 60 minutes, indicating that the drug-loaded polylysine fluorescent nanospheres passed through the small After the mouse body fluid circulates, it is metabolized and transferred to the bladder of the mouse.
实施例8、甘草次酸靶点蛋白的捕获与SDS-PAGE电泳分析Example 8, Glycyrrhetinic acid target protein capture and SDS-PAGE electrophoresis analysis
将培养的人肺上皮BEAS-2B细胞铺于六孔板中，待融合度达到80%以上，密度达到105以后，分别加入实施例5所制备的连接甘草次酸的聚赖氨酸荧光纳米微球100 µL。采用DMEM完全培养基继续培养48 小时；经胰酶消化30秒，收集细胞并将细胞悬浮于PBS中，冰浴中超声处理，破碎细胞。4℃，10,000 转离心10 分钟，弃去细胞碎片以及未破碎的细胞，取上清得到包含有靶点蛋白的聚赖氨酸纳米微球的混合溶液（图6，步骤1）。Spread the cultured human lung epithelial BEAS-2B cells in a six-well plate, and after the degree of fusion reaches more than 80% and the density reaches 105, add the polylysine fluorescent nanometers linked to glycyrrhetinic acid prepared in Example5 . Microspheres 100 µL. Continue culturing with DMEM complete medium for 48 hours; trypsinize for 30 seconds, collect the cells and suspend the cells in PBS, sonicate in an ice bath to break the cells. Centrifuge at 10,000 rpm for 10 minutes at 4°C, discard cell debris and unbroken cells, and take the supernatant to obtain a mixed solution of polylysine nanospheres containing the target protein (Figure 6, step 1).
将上述混合溶液加入到截留分子量为3 kD的Millipore超滤管中，3000 转离心，经生理盐水混悬并再次离心弃去小分子，分别收集滤液1和截留液（图6，步骤2）；将截留液加入到截留分子量为100 kD的Millipore超滤管中，3,000 转离心，经生理盐水混悬并再次离心弃去纳米微球未结合的大分子蛋白，分别收集滤液2和截留液（图6，步骤3）；向截留液中加入100 mM的DTT溶液100 µL，静置15分钟后再次加入到截留分子量为100 kD的Millipore超滤管中，3,000 转离心，收集滤液3和截留液4（图6，步骤4）；具体操作步骤如图6所示。Add the above mixed solution into a Millipore ultrafiltration tube with a molecular weight cut-off of 3 kD, centrifuge at 3000 rpm, suspend in saline and centrifuge again to discard small molecules, and collect filtrate 1 and retentate respectively (Figure 6, step 2); Add the retentate to a Millipore ultrafiltration tube with a molecular weight cut-off of 100 kD, centrifuge at 3,000 rpm, suspend in normal saline and centrifuge again to discard the unbound macromolecular protein of the nanospheres, collect the filtrate 2 and the retentate respectively (Fig. 6, step 3); add 100 µL of 100 mM DTT solution to the retentate, let it stand for 15 minutes, add it again to a Millipore ultrafiltration tube with a molecular weight cut-off of 100 kD, and centrifuge at 3,000 rpm to collect filtrate 3 and retentate 4 (Figure 6, step 4); the specific operation steps are shown in Figure 6.
分别收集上述的滤液和截留液，并采用10%SDS-PAGE电泳对捕获效果进行分析。结果如图7所示：第1泳道是蛋白Marker，第2泳道为细胞裂解液样品；经过3 kD的超滤管超滤，滤液中几乎没有明显的蛋白条带，如第3泳道所示；而将截留液再上100 kD的超滤管，滤液中可以检测到大量的蛋白条带，表明是未与纳米微球结合的细胞可溶性蛋白（第4泳道）；将截留液加入DTT还原之后，经过100 kD的超滤管，得到的滤液即为与甘草次酸结合的靶点蛋白以及相关蛋白（第6泳道）；而剩余的截留液，包括没有通过滤膜的纳米微球中几乎检测不到可溶性蛋白（第5泳道）。The above-mentioned filtrate and retentate were collected separately, and the capture effect was analyzed by 10% SDS-PAGE electrophoresis. The results are shown in Figure 7: the first lane is the protein marker, and the second lane is the cell lysate sample; after ultrafiltration with a 3 kD ultrafiltration tube, there are almost no obvious protein bands in the filtrate, as shown in the third lane; When the retentate was put on a 100 kD ultrafiltration tube, a large number of protein bands could be detected in the filtrate, indicating that it was a cell-soluble protein that was not bound to the nanospheres (lane 4); after adding the retentate to DTT for reduction, After passing through a 100 kD ultrafiltration tube, the obtained filtrate is the target protein combined with glycyrrhetinic acid and related proteins (lane 6); while the remaining retentate, including nanospheres that have not passed through the filter membrane, is almost undetectable to soluble protein (lane 5).
本实施例表明，本发明公开的聚赖氨酸荧光纳米自组装载药球，不但可以进行靶点蛋白的示踪定位，还可以进行靶点蛋白的捕获富集，在药物作用机制的研究方面有很好的应用前景。This example shows that the polylysine fluorescent nanometer self-assembled drug balls disclosed in the present invention can not only track and locate target proteins, but also capture and enrich target proteins. It has a good application prospect.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410391369.XACN104146964B (en) | 2014-08-11 | 2014-08-11 | Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410391369.XACN104146964B (en) | 2014-08-11 | 2014-08-11 | Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof |
| Publication Number | Publication Date |
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| CN104146964A CN104146964A (en) | 2014-11-19 |
| CN104146964Btrue CN104146964B (en) | 2017-01-18 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410391369.XAActiveCN104146964B (en) | 2014-08-11 | 2014-08-11 | Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109490523A (en)* | 2018-10-22 | 2019-03-19 | 北京纳晶生物科技有限公司 | Method for the nano material of label, nucleic acid probe and nucleic acid and nano material coupling |
| CN109741896B (en)* | 2019-01-07 | 2021-06-04 | 北京大学 | Functionalized magnetic nanoparticles for broad-spectrum bacterial capture and release of live bacteria |
| CN110237035B (en)* | 2019-06-28 | 2021-03-26 | 华中科技大学 | An active targeting amphiphilic polypeptide nano drug carrier and its preparation and application |
| CN112980786B (en)* | 2019-12-12 | 2023-04-28 | 深圳先进技术研究院 | T cell and nanoparticle connection method based on click chemistry |
| CN111138682A (en)* | 2019-12-12 | 2020-05-12 | 苏州永沁泉智能设备有限公司 | Fluorescence-labeled biological material and preparation method thereof |
| CN113061524B (en)* | 2020-06-17 | 2024-01-02 | 山东大学 | Double-layer microfluidic chip and breast cancer miRNA detection kit |
| CN111704716B (en)* | 2020-07-03 | 2021-07-13 | 浙江大学 | A kind of fluorescent polylysine dendrimer, its preparation method and application |
| CN115054701B (en)* | 2022-05-30 | 2024-06-28 | 浙江大学 | A protein-loaded hyperbranched polylysine micro/nano carrier and preparation method thereof |
| CN117658118B (en)* | 2023-12-05 | 2025-08-01 | 西安电子科技大学 | Preparation method of functional graphene oxide based on alcohol-alkyne click reaction |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100143258A1 (en)* | 2008-12-05 | 2010-06-10 | General Electric Company | Tumor margin imaging agents |
| CN103792346A (en)* | 2014-02-14 | 2014-05-14 | 赫利森（厦门）生物科技有限公司 | Polymeric chemiluminescence labeling reagent, preparation method and application thereof |
| Publication number | Publication date |
|---|---|
| CN104146964A (en) | 2014-11-19 |
| Publication | Publication Date | Title |
|---|---|---|
| CN104146964B (en) | Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof | |
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