技术领域technical field
本发明涉及一种纳米凝胶的制备方法及应用，具体涉及一种基于聚合物四唑衍生物和含甲基丙烯酸酯基团交联剂的纳米凝胶制备方法，以及该纳米凝胶在医药领域的应用。The invention relates to a preparation method and application of a nanogel, in particular to a preparation method of a nanogel based on a polymer tetrazole derivative and a methacrylate group-containing crosslinking agent, and the application of the nanogel in medicine field applications.
背景技术Background technique
在过去几十年，基于抗体、细胞因子、酶以及转录因子的蛋白质药物已被广泛地用于糖尿病、心血管疾病和恶性肿瘤等疾病的高效治疗（Vermonden T, Censi R, HenninkWE. Chem. Rev. 2012, 112, 2853-2888; Walsh G. Nat. Biotech. 2000, 18, 831-833）。与具有较高毒副作用的化疗药物相比，蛋白质药物通常具有较高的特异性，较好的治疗效果和较低的毒副作用，在临床上展现出了优越的疾病治疗功效。但这些临床使用的蛋白质药物都是在细胞外起作用。虽然在细胞内起作用的蛋白质药物很多，但还没有一个进入临床使用，这主要是因为这些蛋白药物的血浆半衰期短、体内降解快、细胞内吞效率低、胞内运输过程慢等原因所致（Lu Y, Sun W, Gu Z. J. Controlled Release 2014, 194,1-19）。纳米凝胶通常指由亲水性或两亲性高分子链通过物理或者化学交联形成的三维网状结构的水凝胶微粒，具有高含水量、良好的生物相容性和多孔结构，纳米凝胶可通过物理交联和化学交联制备。物理交联包括憎水作用、静电作用、氢键作用等，物理凝胶制备通常条件温和，不会明显损伤包载药物的活性；但存在稳定性较差、释放包裹的药物较快的缺点。化学交联包括自由基聚合，迈克尔加成反应，酰胺化反应，酶催化反应，铜催化点击化学反应等。但这些化学交联反应通常不具备专一性，这可能使药物参与交联反应，导致变性。而且，尽管已有很多报道使用纳米凝胶输送药物，但很少能够实现治疗药物在体内的靶向高效输送。所以需要开发专一性强、高效快速、无需催化的交联反应方法，用于制备具有靶向性的生物可降解纳米凝胶载体，使其更好地应用于药物控制释放载体等领域。In the past few decades, protein drugs based on antibodies, cytokines, enzymes, and transcription factors have been widely used for the effective treatment of diseases such as diabetes, cardiovascular disease, and malignant tumors (Vermonden T, Censi R, HenninkWE. Chem. Rev . 2012, 112, 2853-2888; Walsh G. Nat. Biotech. 2000, 18, 831-833). Compared with chemotherapy drugs with higher toxic and side effects, protein drugs usually have higher specificity, better therapeutic effect and lower toxic and side effects, and have demonstrated superior disease treatment efficacy in clinical practice. But these clinically used protein drugs all work outside the cell. Although there are many protein drugs that work in cells, none of them have entered clinical use, mainly because of the short plasma half-life of these protein drugs, rapid degradation in vivo, low endocytosis efficiency, and slow intracellular transport process. (Lu Y, Sun W, Gu Z. J. Controlled Release 2014, 194, 1-19). Nanogel usually refers to a three-dimensional network structure of hydrogel particles formed by hydrophilic or amphiphilic polymer chains through physical or chemical cross-linking, with high water content, good biocompatibility and porous structure, nano Gels can be prepared by physical crosslinking and chemical crosslinking. Physical crosslinking includes hydrophobic interaction, electrostatic interaction, hydrogen bond interaction, etc. Physical gel preparation is usually under mild conditions, which will not significantly damage the activity of the encapsulated drug; however, it has the disadvantages of poor stability and faster release of the encapsulated drug. Chemical crosslinking includes free radical polymerization, Michael addition reaction, amidation reaction, enzyme-catalyzed reaction, copper-catalyzed click chemistry reaction, etc. However, these chemical cross-linking reactions are usually not specific, which may cause the drug to participate in the cross-linking reaction, resulting in denaturation. Moreover, although there have been many reports on the use of nanogels for drug delivery, few have been able to achieve targeted and efficient delivery of therapeutic drugs in vivo. Therefore, it is necessary to develop a cross-linking reaction method with strong specificity, high efficiency and fast, and no catalysis, for the preparation of targeted biodegradable nanogel carriers, so that it can be better used in the fields of drug controlled release carriers.
发明内容Contents of the invention
本发明的目的是提供一种专一性强、高效快速、无需催化的光控“四唑-烯”点击化学方法制备纳米凝胶的方法，用该方法制备的纳米凝胶在药物控制释放载体和组织工程支架材料等领域中具有很好的应用前景。The purpose of the present invention is to provide a method for preparing nanogel by light-controlled "tetrazolene" click chemistry method with strong specificity, high efficiency and rapidity, and without catalysis. The nanogel prepared by this method is used in drug controlled release carrier It has a good application prospect in the fields of tissue engineering scaffold materials and the like.
为达到上述发明目的，本发明采用的技术方案是：一种自发荧光纳米凝胶的制备方法，包括以下步骤：将聚合物四唑衍生物和含甲基丙烯酸酯基团的交联剂加入水或者缓冲液中得到混合液；然后将混合液注射到有机溶剂中，得到悬浮液；然后进行光照反应得到自发荧光纳米凝胶；In order to achieve the above-mentioned purpose of the invention, the technical solution adopted in the present invention is: a preparation method of autofluorescent nanogel, comprising the following steps: adding polymer tetrazole derivatives and cross-linking agents containing methacrylate groups to water Or obtain a mixed solution in a buffer; then inject the mixed solution into an organic solvent to obtain a suspension; then perform a light reaction to obtain an autofluorescent nanogel;
所述聚合物四唑衍生物具有式I结构：The polymer tetrazole derivative has a formula I structure:
式Ⅰ；Formula I;
其中n ≥ 2；where n ≥ 2;
R为H、NH2、NMe2、OMe、NO2、Cl、Br、Me、CO2Me或者PhNHBoc；R is H, NH2 , NMe2 , OMe, NO2 , Cl, Br, Me, CO2 Me or PhNHBoc;
P为透明质酸、透明质酸赖氨酸化合物、透明质酸胱胺化合物、葡聚糖、壳聚糖、胶原蛋白、聚乙二醇或者聚乙二醇-聚酯；所述聚乙二醇为线性或者多臂聚乙二醇，表示为PEG-x-OH，x=2，4，6或8；所述聚酯为聚丙交酯、聚（丙交酯-co-乙交酯）、聚己内酯或者聚碳酸酯；所述聚酯的聚合度为1～20；P is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, dextran, chitosan, collagen, polyethylene glycol or polyethylene glycol-polyester; the polyethylene glycol The alcohol is linear or multi-arm polyethylene glycol, expressed as PEG-x-OH, x=2, 4, 6 or 8; the polyester is polylactide, poly(lactide-co-glycolide) , polycaprolactone or polycarbonate; the degree of polymerization of the polyester is 1-20;
所述含甲基丙烯酸酯基团的交联剂具有式II结构：The cross-linking agent containing methacrylate group has formula II structure:
式II；Formula II;
其中m ≥ 2；where m ≥ 2;
CL为透明质酸、透明质酸胱胺化合物、透明质酸赖氨酸化合物、壳聚糖、葡聚糖、胶原蛋白、聚乙二醇、聚乙二醇-聚酯、丁二胺、己二胺、胱胺、胱氨酸或者赖氨酸。CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, dextran, collagen, polyethylene glycol, polyethylene glycol-polyester, butylenediamine, hexamethylene glycol Diamine, cystamine, cystine or lysine.
上述技术方案中，所述聚乙二醇为线性或者多臂聚乙二醇，表示为PEG-x-OH，x=2，4，6或8；所述聚酯为聚丙交酯、聚（丙交酯-co-乙交酯）、聚己内酯或者聚碳酸酯；所述聚酯的聚合度为1～20；所述聚乙二醇的分子量为2～100 kg/mol。In the above technical solution, the polyethylene glycol is linear or multi-arm polyethylene glycol, expressed as PEG-x-OH, x=2, 4, 6 or 8; the polyester is polylactide, poly( lactide-co-glycolide), polycaprolactone or polycarbonate; the degree of polymerization of the polyester is 1-20; the molecular weight of the polyethylene glycol is 2-100 kg/mol.
上述技术方案中，甲基丙烯酸酯基团与四唑基团的摩尔比为1∶1；混合液中，聚合物四唑衍生物浓度为0.5～10mg/mL。In the above technical solution, the molar ratio of the methacrylate group to the tetrazole group is 1:1; in the mixed solution, the concentration of the polymer tetrazole derivative is 0.5-10 mg/mL.
上述技术方案中，所述光照反应为紫外光照反应；所述紫外光的波长为302-390nm，强度为0.8～100 mW/cm2，时间为90～180s。In the above technical solution, the light reaction is an ultraviolet light reaction; the ultraviolet light has a wavelength of 302-390 nm, an intensity of 0.8-100 mW/cm2 , and a time of 90-180 s.
上述技术方案中，所述缓冲液包括磷酸盐（PB）缓冲液，4-（2-羟乙基）-1-哌嗪乙烷磺酸半钠盐（HEPES）缓冲溶液，三羟甲基氨基甲烷（Tris）缓冲溶液，2-吗啉乙磺酸（MES）缓冲溶液等；优选pH 为7.4的PB缓冲液；所述有机溶剂包括丙酮、乙腈、乙醇等；优选丙酮。In the above technical scheme, the buffer includes phosphate (PB) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hemisodium salt (HEPES) buffer solution, trihydroxymethylamino Methane (Tris) buffer solution, 2-morpholineethanesulfonic acid (MES) buffer solution, etc.; preferably PB buffer solution with a pH of 7.4; the organic solvent includes acetone, acetonitrile, ethanol, etc.; preferably acetone.
本发明公开了根据上述方法制备得到的自发荧光纳米凝胶；可以称为光控“四唑-烯”点击化学制备的自发荧光纳米凝胶。The invention discloses an autofluorescent nanogel prepared by the above method; it can be called an autofluorescent nanogel prepared by light-controlled "tetrazole-ene" click chemistry.
本发明中，聚合物四唑衍生物以聚合物作为主链，四唑基团通过O或者NH随机接在聚合物主链末端或侧链上；式I结构中，P表示聚合物，n ≥ 2是指四唑基团接在聚合物主链末端或侧链上的数量为复数个，比如本发明实施例一中，聚合物为透明质酸赖氨酸化合物，其重复单元上接有多个四唑基团。In the present invention, the polymer tetrazole derivatives use the polymer as the main chain, and the tetrazole group is randomly connected to the end or side chain of the polymer main chain through O or NH; in the structure of formula I, P represents the polymer, and n ≥ 2 means that the number of tetrazole groups connected to the end of the polymer main chain or the side chain is plural. For example, in Example 1 of the present invention, the polymer is a hyaluronic acid lysine compound, and its repeating unit is connected to multiple a tetrazole group.
本发明中，含甲基丙烯酸酯基团的交联剂可以为大分子也可以为小分子，m ≥ 2是指交联剂中甲基丙烯酸酯基团的数量为复数个；式II结构中，CL为小分子时，甲基丙烯酸酯基团接在小分子两端，如本发明实施例三的结构；CL为聚合物时，甲基丙烯酸酯基团通过O或者NH接在聚合物主链末端或侧链上，如本发明实施例二的结构，聚合物为透明质酸胱胺化合物，其重复单元上接有多个甲基丙烯酸酯基团。In the present invention, the cross-linking agent containing methacrylate group can be a macromolecule or a small molecule, and m ≥ 2 means that the number of methacrylate groups in the cross-linking agent is plural; in the formula II structure , when CL is a small molecule, the methacrylate group is connected to both ends of the small molecule, such as the structure of Example 3 of the present invention; when CL is a polymer, the methacrylate group is connected to the polymer main body through O or NH On the chain end or side chain, as in the structure of Example 2 of the present invention, the polymer is hyaluronic acid cystamine compound, and multiple methacrylate groups are attached to its repeating unit.
本发明中，聚合物四唑衍生物的制备方法为，先向四唑溶液中加入缩合剂与催化剂，反应得到活化的四唑溶液；然后把聚合物水溶液滴加到活化的四唑溶液中，室温反应得到聚合物四唑衍生物；所述聚合物为透明质酸、透明质酸赖氨酸化合物、透明质酸胱胺化合物、葡聚糖、壳聚糖、胶原蛋白、聚乙二醇或者聚乙二醇-聚酯。Among the present invention, the preparation method of polymer tetrazole derivative is, add condensing agent and catalyst to tetrazole solution earlier, react to obtain activated tetrazole solution; Then polymer aqueous solution is added dropwise in the tetrazole solution of activation, React at room temperature to obtain polymer tetrazole derivatives; the polymer is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, dextran, chitosan, collagen, polyethylene glycol or Polyethylene glycol-polyester.
上述技术方案中，四唑溶液的溶剂优选为二甲基亚砜、二甲基亚砜与水的混合溶液、二氯甲烷或氯仿；聚合物为含有氨基或羟基的水溶性聚合物，优选为透明质酸、透明质酸赖氨酸化合物、透明质酸胱胺化合物、壳聚糖、葡聚糖、聚乙二醇或者聚乙二醇-寡聚酯；其中所述聚乙二醇为线性或者多臂聚乙二醇；所述聚酯为聚丙交酯、聚（丙交酯-co-乙交酯）、聚己内酯或者聚碳酸酯。In the above-mentioned technical scheme, the solvent of the tetrazole solution is preferably dimethyl sulfoxide, a mixed solution of dimethyl sulfoxide and water, dichloromethane or chloroform; the polymer is a water-soluble polymer containing an amino group or a hydroxyl group, preferably Hyaluronic acid, hyaluronic acid lysine compound, hyaluronan cystamine compound, chitosan, dextran, polyethylene glycol or polyethylene glycol-oligoester; wherein said polyethylene glycol is a linear or multi-arm polyethylene glycol; the polyester is polylactide, poly(lactide-co-glycolide), polycaprolactone or polycarbonate.
上述技术方案中，水溶性聚合物中的羟基或者胺基与四唑的摩尔比优选为1∶0.1～2；四唑与缩合剂、催化剂的摩尔比优选为1∶2∶0.1。In the above technical solution, the molar ratio of hydroxyl or amine groups in the water-soluble polymer to tetrazole is preferably 1:0.1-2; the molar ratio of tetrazole to condensing agent and catalyst is preferably 1:2:0.1.
比如：先向四唑小分子（Tet）的DMSO溶液中加入缩合剂二环己基碳二亚胺（DCC）、4-二甲氨基吡啶（DMAP），反应过夜得到羧基活化的四唑溶液；然后把含有氨基或羟基的聚合物水溶液逐滴滴加到上述活化的四唑溶液中，再在室温下搅拌反应18～28小时，得到所述的聚合物四唑衍生物（P-Tetn）；具体反应过程如下：For example: first add the condensing agent dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to the DMSO solution of the tetrazole small molecule (Tet), and react overnight to obtain a carboxyl-activated tetrazole solution; then Add the aqueous polymer solution containing amino groups or hydroxyl groups dropwise to the above-mentioned activated tetrazole solution, and then stir and react at room temperature for 18-28 hours to obtain the polymer tetrazole derivative (P-Tetn ); Concrete reaction process is as follows:
其中R=H、Cl、Br、Me、NH2、NMe2、NO2或者OMe。where R=H, Cl, Br, Me,NH2 ,NMe2 ,NO2 or OMe.
本发明进一步公开了上述自发荧光纳米凝胶在制备组织工程支架中的应用；上述自发荧光纳米凝胶在制备蛋白质药物中的应用；上述自发荧光纳米凝胶作为药物缓释载体的应用。The invention further discloses the application of the above-mentioned autofluorescent nano-gel in preparing tissue engineering scaffold; the application of the above-mentioned auto-fluorescent nano-gel in the preparation of protein medicine; and the application of the above-mentioned auto-fluorescent nano-gel as a drug slow-release carrier.
本发明还公开了一种抗肿瘤药物，包括上述自发荧光纳米凝胶以及蛋白质药物。本发明公开的纳米凝胶与包载的药物，尤其是蛋白质药物和细胞不反应，能很好的保持药物、蛋白质和细胞的功效，实现完全、可控的释放，从而可作为蛋白质等药物的优良缓释载体；到达病灶处，纳米凝胶缓释药物，达到切实有效的治疗效果，不会造成现有技术中药物浪费的问题。The invention also discloses an antitumor drug, including the above-mentioned autofluorescence nano gel and protein drug. The nanogel disclosed by the present invention does not react with the drug loaded, especially the protein drug and the cell, and can well maintain the efficacy of the drug, protein and cell, and realize complete and controllable release, so it can be used as a drug for protein and other drugs. Excellent slow-release carrier; when it reaches the lesion, the nano-gel releases the drug slowly to achieve a practical and effective therapeutic effect without causing the problem of drug waste in the prior art.
由于上述技术方案运用，本发明与现有技术相比具有下列优点：Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:
1. 本发明公开的纳米凝胶的制备方法具有“点击化学”的强专一性、快速高效和反应条件温和的特点，同时无需铜盐等毒性催化剂；特别的，本发明通过设计前驱体原料以及结合注射、紫外反应，得到的纳米凝胶稳定性强，用于包载蛋白质时，与药物结合稳定，保证药物在体内循环不受干扰。1. The preparation method of the nanogel disclosed in the present invention has the characteristics of strong specificity, rapidity and high efficiency, and mild reaction conditions of "click chemistry", and does not require toxic catalysts such as copper salts; in particular, the present invention designs precursor raw materials And combined with injection and ultraviolet reaction, the obtained nanogel has strong stability, and when used to entrap proteins, it can be stably combined with drugs to ensure that the drug circulation in the body is not disturbed.
2. 本发明公开的纳米凝胶的交联方法具有很强的选择性，与包载的药物，尤其是蛋白质药物和细胞不反应，能很好的保持药物、蛋白质和细胞的功效，实现完全、可控的释放，从而可作为蛋白质等药物的优良缓释载体；达到病灶处，纳米凝胶缓释药物，到达切实有效的治疗效果，不会造成现有技术中药物浪费的问题。2. The cross-linking method of the nanogel disclosed in the present invention has strong selectivity, does not react with entrapped drugs, especially protein drugs and cells, and can well maintain the efficacy of drugs, proteins and cells, and realize complete , controllable release, so that it can be used as an excellent slow-release carrier for drugs such as proteins; when it reaches the lesion, the nanogel releases the drug slowly to achieve a practical and effective therapeutic effect, and will not cause the problem of drug waste in the prior art.
3. 本发明制备的纳米凝胶具有自发荧光性能，可被用来观察纳米凝胶载体在体外内吞进入细胞和在体内穿透病灶组织的行为，为药物摄取监控、输送观察提供有利的条件，克服了现有技术还需要另加荧光剂监视药物输送的缺陷。3. The nanogel prepared by the present invention has autofluorescence properties, which can be used to observe the behavior of nanogel carriers endocytosis into cells in vitro and penetration into lesion tissues in vivo, providing favorable conditions for drug uptake monitoring and delivery observation , overcoming the defect that the prior art still needs to add fluorescent agent to monitor drug delivery.
4. 本发明公开的纳米凝胶前驱体材料具有良好的生物相容性和生物降解性，而且来源广泛、制备简单、成本较低；制备过程可控，无需催化剂，节约资源的同时使得产物更纯净，是一种适用于工业化的制备方法。4. The nanogel precursor material disclosed in the present invention has good biocompatibility and biodegradability, and has a wide range of sources, simple preparation, and low cost; the preparation process is controllable, no catalyst is needed, and the product is more efficient while saving resources. Pure, it is a preparation method suitable for industrialization.
附图说明Description of drawings
图1是实施例一中透明质酸赖氨酸四唑衍生物的氢核磁谱图；Fig. 1 is the proton magnetic spectrogram of hyaluronic acid lysine tetrazole derivative in embodiment one;
图2是实施例二中透明质酸胱胺甲基丙烯酸酯衍生物的氢核磁谱图；Fig. 2 is the proton magnetic spectrogram of hyaluronic acid cystamine methacrylate derivative in embodiment two;
图3是实施例三中胱氨酸甲基丙烯酸酯衍生物的氢核磁谱图；Fig. 3 is the proton magnetic spectrum of cystine methacrylate derivative in embodiment three;
图4是实施例四中纳米凝胶的基本性质表征；Fig. 4 is the basic property characterization of nanogel in embodiment four;
图5是实施例六中含自发荧光纳米凝胶的杂化水凝胶的制备；Fig. 5 is the preparation of the hybrid hydrogel containing autofluorescence nanogel in embodiment six;
图6是实施例七中纳米凝胶体外控制释放蛋白质药物的行为，以及释放出的蛋白质药物的生物活性表征图；Fig. 6 is the behavior of nanogel in vitro controlled release of protein drug in Example 7, and the biological activity characterization diagram of the released protein drug;
图7是实施例八中纳米凝胶和载蛋白质药物的纳米凝胶的细胞实验表征图；Figure 7 is a cell experiment characterization diagram of nanogel and protein-loaded drug nanogel in Example 8;
图8是实施例九中载蛋白纳米凝胶对小鼠皮下人乳腺癌移植瘤的治疗表征图；Figure 8 is a characterization diagram of the treatment of protein-loaded nanogels on mouse subcutaneous human breast cancer xenografts in Example 9;
图9是实施例十中载蛋白纳米凝胶对小鼠原位肺癌移植瘤的治疗表征图；Fig. 9 is a characterization diagram of the treatment of orthotopic lung cancer transplanted tumors in mice by protein-loaded nanogels in Example 10;
图10是实施例十中载蛋白纳米凝胶治疗小鼠原位肺癌移植瘤的组织学分析图。Fig. 10 is a diagram of histological analysis of orthotopic lung cancer xenografts in mice treated with protein-loaded nanogels in Example 10.
具体实施方式detailed description
下面结合附图以及实施例对本发明作进一步描述：Below in conjunction with accompanying drawing and embodiment the present invention will be further described:
实施例一透明质酸赖氨酸四唑衍生物（HA-Lys-Tet）的合成Example 1 Synthesis of Hyaluronic Acid Lysine Tetrazole Derivatives (HA-Lys-Tet)
在氮气保护条件下，50 mL两颈瓶中加入四唑（608 mg），二甲亚砜10mL， DCC （120 mg）搅拌24小时，HA-Lys-NH2（Mn=35 K, 0.4 g）溶于30 mL 甲酰胺中，溶解后加入四唑溶液中，搅拌10 min，加入DMAP （80 mg，），反应48小时，过滤，滤液用水和二甲亚砜混合溶剂透析后换成纯水透析，冷冻干燥后得产品HA-Lys-Tet（产率69 %）；HA-Lys-Tet核磁表征见附图1，1H NMR (D2O/DMSO-d6): HA: δ 1.82, 2.70–3.68, and 4.23–4.38; Lys: δ 0.92, 1.06,1.52, 2.97, 3.61 and 3.95; Tet: δ 7.91,7.92 and 6.79, 6.80。Under nitrogen protection, add tetrazole (608 mg), 10 mL of dimethyl sulfoxide, and DCC (120 mg) into a 50 mL two-neck flask and stir for 24 hours. HA-Lys-NH2 (Mn =35 K, 0.4 g ) was dissolved in 30 mL formamide, added into tetrazole solution after dissolving, stirred for 10 min, added DMAP (80 mg,), reacted for 48 hours, filtered, and the filtrate was dialyzed with water and dimethyl sulfoxide mixed solvent and replaced with pure water After dialysis and freeze-drying, the product HA-Lys-Tet was obtained (yield 69 %); HA-Lys-Tet NMR characterization is shown in Figure 1,1 H NMR (D2 O/DMSO-d6 ): HA: δ 1.82, 2.70–3.68, and 4.23–4.38; Lys: δ 0.92, 1.06,1.52, 2.97, 3.61 and 3.95; Tet: δ 7.91,7.92 and 6.79, 6.80.
四臂聚乙二醇四唑衍生物（PEG-Tet4）、壳聚糖四唑衍生物（Chit-Tet）可更换聚合物制备得到，结构式如下：Four-arm polyethylene glycol tetrazole derivatives (PEG-Tet4) and chitosan tetrazole derivatives (Chit-Tet) can be prepared by replacing polymers. The structural formula is as follows:
。 .
实施例二透明质酸胱胺甲基丙烯酸酯衍生物（HA-Cy-MA）的合成Example 2 Synthesis of Hyaluronic Acid Cystamine Methacrylate Derivatives (HA-Cy-MA)
HA-Cy-MA分两步合成，首先胱胺的甲基丙烯酸衍生物（MA-Cy-NH2）由Boc-Cy-NH2和甲基丙烯酰氯反应后脱Boc保护获得，然后，在氮气保护条件下，将MA-Cy-NH2 (14.5 mg, 66μmol)的溶液加入到HA (50 mg, 1.43 μmol) 被EDC (75.9 mg, 0.396 mmol) 和NHS(22.8 mg, 0.198 mmol) 活化的5 mL二次水中，置于40℃油浴中避光反应24小时，然后用水透析，冷冻干燥，产率92 %；HA-Cy-MA核磁表征见附图2，1H NMR (D2O): HA: δ 2.00,2.86–3.88, and 4.44–4.52; Cys: δ 2.70, 3.11–3.15 and 3.56; MA: δ 1.92, 5.45and 5.69。HA-Cy-MA was synthesized in two steps. First, the methacrylic acid derivative of cystamine (MA-Cy-NH2 ) was obtained by de-Boc protection after the reaction of Boc-Cy-NH2 and methacryloyl chloride, and then, under nitrogen A solution of MA-Cy-NH2 (14.5 mg, 66 μmol) was added to HA (50 mg, 1.43 μmol) activated by EDC (75.9 mg, 0.396 mmol) and NHS (22.8 mg, 0.198 mmol) under protective conditions. mL of secondary water, placed in an oil bath at 40°C in the dark for 24 hours, then dialyzed with water, and freeze-dried, with a yield of 92%; HA-Cy-MA NMR characterization is shown in Figure 2,1 H NMR (D2 O) : HA: δ 2.00,2.86–3.88, and 4.44–4.52; Cys: δ 2.70, 3.11–3.15 and 3.56; MA: δ 1.92, 5.45and 5.69.
实施例三胱胺酸甲基丙烯酰胺衍生物（MA-Cys-MA）的合成Example Synthesis of Tricystine Methacrylamide Derivatives (MA-Cys-MA)
在冰水浴条件下，将胱氨酸（1.2 g，5.0 mmol）的NaOH（1.5 M，10 mL）溶液滴加到甲基丙烯酰氯（2.0 mL，20.6 mmol)的DCM（10 mL）中，在冰水浴条件下反应4 h，反应期间用NaOH溶液调控pH为9.0。反应结束后，用分液漏斗分出水层，再向其逐滴加入约3 mL HCl（2 M），过滤，真空干燥，得到白色粉末1.72 g，产率91%。MA-Cys-MA核磁表征见附图3，1H NMR（400MHz，DMSO-d6）：MA (δ 5.72，5.39和1.85)，Cys (δ 12.92，8.24，4.53，3.18 和3.03)。In an ice-water bath, a solution of cystine (1.2 g, 5.0 mmol) in NaOH (1.5 M, 10 mL) was added dropwise to methacryloyl chloride (2.0 mL, 20.6 mmol) in DCM (10 mL). The reaction was carried out in an ice-water bath for 4 h, and the pH was adjusted to 9.0 with NaOH solution during the reaction. After the reaction, the aqueous layer was separated with a separatory funnel, and about 3 mL of HCl (2 M) was added dropwise to it, filtered, and vacuum-dried to obtain 1.72 g of white powder with a yield of 91%. The NMR characterization of MA-Cys-MA is shown in Figure 3,1 H NMR (400MHz, DMSO-d6 ): MA (δ 5.72, 5.39 and 1.85), Cys (δ 12.92, 8.24, 4.53, 3.18 and 3.03).
实施例四 大分子交联制备透明质酸纳米凝胶Example 4 Preparation of hyaluronic acid nanogel by macromolecule crosslinking
用大分子交联剂综合利用反相纳米沉淀法和光控“四唑-烯”交联法制备透明质酸纳米凝胶，制备如下：将实施例一和实施例二制备的HA-Lys-Tet 和HA-Cy-MA以摩尔比1:1溶于的PB（pH 7.4，10 mM）中，制备得到浓度为1.25 mg/mL的聚合物溶液，然后混合溶液注射到100mL丙酮中，用紫外光（波长320-390 nm，光强60 mW/cm2）辐射3 min，旋蒸除去丙酮后用PB透析，冷冻干燥得到纳米凝胶。参见附图4，纳米凝胶的粒径可用动态光散射（DLS）测量，通过此种方法获得的纳米凝胶粒径可控（150-343 nm），PDI很小（0.10-0.17）（图4a），同时纳米凝胶的形貌可以用透射电镜（TEM）观察得到，该法制备的纳米凝胶为球形（图4a）；另外，制备得到的纳米凝胶在405nm的UV激发光辐射下，在450 nm处有较强的绿色荧光（图4b）；利用纳米凝胶的自发荧光性能来观察纳米凝胶的细胞内吞和在体内分布情况，该纳米凝胶还表现出很好的稳定性，放置在PB（pH 7.4，10 mM）和10% FBS模拟体内血液环境均能保持粒径稳定（图4c）；但是把纳米凝胶放置在10 mM GSH 的 PB (pH 7.4, 10 mM)中4小时就可以观察到显著的粒径变化，随时间增加，粒径开始快速变大，表明此种纳米凝胶有快速的还原响应性质（图4d）。这可以被用来实现药物在细胞内的响应性快速释放，大大增加包载药物的治疗效果。Using a macromolecular cross-linking agent to comprehensively utilize the reversed-phase nanoprecipitation method and the light-controlled "tetrazolium-ene" cross-linking method to prepare hyaluronic acid nanogel, the preparation is as follows: the HA-Lys- Tet and HA-Cy-MA were dissolved in PB (pH 7.4, 10 mM) at a molar ratio of 1:1 to prepare a polymer solution with a concentration of 1.25 mg/mL, and then the mixed solution was injected into 100 mL of acetone, and the Light (wavelength 320-390 nm, light intensity 60 mW/cm2 ) was irradiated for 3 minutes, acetone was removed by rotary evaporation, dialyzed with PB, and freeze-dried to obtain nanogels. See Figure 4, the particle size of the nanogel can be measured by dynamic light scattering (DLS), the particle size of the nanogel obtained by this method is controllable (150-343 nm), and the PDI is very small (0.10-0.17) (Fig. 4a), at the same time, the morphology of the nanogel can be observed by transmission electron microscopy (TEM), and the nanogel prepared by this method is spherical (Figure 4a); , has strong green fluorescence at 450 nm (Figure 4b); using the autofluorescence properties of nanogels to observe the endocytosis and distribution in vivo of nanogels, the nanogels also showed good stability However, nanogels placed in 10 mM GSH in PB (pH 7.4, 10 mM) Significant particle size changes can be observed within 4 hours, and the particle size begins to increase rapidly as time increases, indicating that this nanogel has a fast reduction response property (Figure 4d). This can be used to achieve responsive and rapid release of drugs in cells, greatly increasing the therapeutic effect of entrapped drugs.
实施例五 小分子交联制备透明质酸纳米凝胶Example 5 Preparation of Hyaluronic Acid Nanogels by Small Molecule Crosslinking
用小分子交联剂综合利用反相纳米沉淀法和光控“四唑-烯”交联法制备透明质酸纳米凝胶，将1 mL HA-OEG-Tet（1 mg/mL）和MA-Cys-MA的PB（pH 8.5，10 mM）溶液（Tet和MA基团的摩尔比控制在1: 1）注射到20 mL乙腈中，然后将该溶液置于紫外暗箱（320-390 nm，37.5mW/cm2）光照90 s。旋转蒸发除去丙酮，用PB（pH 7.4，10 mM）透析12 h（MWCO 7000 Da），得到纳米凝胶。动态光散射（DLS）结果表明该法制备的纳米凝胶的平均粒径165 nm，且呈现单峰分布。TEM图片说明该纳米凝胶具有球形结构，并且大部分的纳米粒子尺寸约为100 nm，TEM图片中的纳米凝胶的粒径比DLS测出的粒径小，可能因为TEM制样过程中，纳米凝胶中水分蒸发，纳米凝胶皱缩，而使拍出来的粒径要小。Hyaluronic acid nanogels were prepared by comprehensively utilizing reversed-phase nanoprecipitation method and light-controlled "tetrazolium-ene" cross-linking method with small molecule cross-linking agents. 1 mL HA-OEG-Tet (1 mg/mL) and MA- A solution of Cys-MA in PB (pH 8.5, 10 mM) (the molar ratio of Tet and MA groups was controlled at 1:1) was injected into 20 mL of acetonitrile, and then the solution was placed in a UV dark box (320-390 nm, 37.5 mW/cm2) light for 90 s. The acetone was removed by rotary evaporation, and the nanogel was obtained by dialyzing against PB (pH 7.4, 10 mM) for 12 h (MWCO 7000 Da). The results of dynamic light scattering (DLS) showed that the average particle size of the nanogel prepared by this method was 165 nm, and it showed a unimodal distribution. The TEM picture shows that the nanogel has a spherical structure, and most of the nanoparticles are about 100 nm in size. The particle size of the nanogel in the TEM picture is smaller than that measured by DLS, which may be due to the fact that during the TEM sample preparation process, The water in the nanogel evaporates, and the nanogel shrinks, so that the particle size of the shot is smaller.
实施例六光控“四唑-烯”点击化学纳米凝胶用于生长因子的包载和复合水凝胶的制备Example 6 Light-controlled "tetrazolium-ene" click chemistry nanogel for growth factor entrapment and preparation of composite hydrogel
以包载血管内皮生长因子（VEGF）为例，首先将VEGF、HA-OEG-Tet和HA-Cy-MA溶解到PB（pH 7.4，10 mM）缓冲溶液中，使聚合物总浓度为1.25 mg/mL。然后将该溶液注射到丙酮中，再用功率极低的手提式紫外分析仪（302 nm，0.88 mW/cm2）光照180秒。最后旋转蒸发除去丙酮，用水透析12 h，得到包载VEGF的纳米凝胶（VEGF-NGs）溶液。DLS结果显示包载VEGF的纳米凝胶（VEGF-NGs）呈现单峰分布，且其平均粒径为173 nm。TEM观察显示VEGF-NGs具有球形结构，并且纳米凝胶的直径约为100 nm。该包载VEGF的纳米凝胶具有较好的注射性，可直接注射使用或者与水凝胶结合制备水凝胶复合物。Taking vascular endothelial growth factor (VEGF) as an example, first dissolve VEGF, HA-OEG-Tet and HA-Cy-MA into PB (pH 7.4, 10 mM) buffer solution, so that the total polymer concentration is 1.25 mg /mL. The solution was then injected into acetone and irradiated for 180 seconds with a very low power hand-held UV analyzer (302 nm, 0.88 mW/cm2 ). Finally, the acetone was removed by rotary evaporation and dialyzed with water for 12 h to obtain the VEGF-loaded nanogel (VEGF-NGs) solution. DLS results showed that VEGF-encapsulated nanogels (VEGF-NGs) exhibited a unimodal distribution with an average particle size of 173 nm. TEM observation showed that VEGF-NGs had a spherical structure, and the diameter of the nanogel was about 100 nm. The VEGF-loaded nanogel has good injectability, and can be used for direct injection or combined with hydrogel to prepare a hydrogel complex.
水凝胶复合物的制备是先将巯基功能化的胶原蛋白（Col-SH）和低聚碳酸酯-聚乙二醇-低聚碳酸酯（OAC-PEG-OAC）分别溶于100 µL的PB（pH 7.4，100 mM）中，待完全溶解后，把两种溶液和VEGF-NGs溶液在室温下混合均匀，再置于37℃摇床反应形成复合水凝胶。该复合水凝胶的从溶液状态变成水凝胶的形成过程见图5a。同时，复合水凝胶的储能模量（G`）和损耗模量（G``）随时间的变化可用流变仪测定。结果表明该复合水凝胶的模量可达2000 Pa，凝胶时间约为3分钟（图5b）。该包载VEGF生长因子的复合水凝胶作为组织工程支架在缺血心肌修复中有广泛的应用前景。The hydrogel composite was prepared by first dissolving thiol-functionalized collagen (Col-SH) and oligocarbonate-polyethylene glycol-oligocarbonate (OAC-PEG-OAC) in 100 µL of PB (pH 7.4, 100 mM), after complete dissolution, the two solutions and VEGF-NGs solution were mixed evenly at room temperature, and then placed on a shaker at 37°C to react to form a composite hydrogel. The formation process of the composite hydrogel from solution state to hydrogel is shown in Fig. 5a. At the same time, the changes of storage modulus (G`) and loss modulus (G``) of the composite hydrogel with time can be measured with a rheometer. The results show that the modulus of the composite hydrogel can reach 2000 Pa, and the gelation time is about 3 min (Fig. 5b). The composite hydrogel encapsulating VEGF growth factor has broad application prospects in the repair of ischemic myocardium as a tissue engineering scaffold.
实施例七光控“四唑-烯”点击化学纳米凝胶用于蛋白质药物的包载和体外控制释放Example 7 Light-controlled "tetrazolium-ene" click chemistry nanogel for entrapment and controlled release of protein drugs in vitro
以包载细胞色素C（CC）为例，将一定量的CC（理论载药量为10%）加入到聚合物总浓度为1.25 mg/mL的HA-OEG-Tet和HA-Cy-MA的PB（pH 7.4，10 mM）溶液中，然后将该溶液注射到丙酮中，再用紫外（320-390 nm，50 mW/cm2）光照90秒。最后旋转蒸发除去丙酮，用水透析12h，得到包载CC的纳米凝胶（CC-NGs）溶液。用类似的方法可以实现对治疗蛋白果粒酶B（GrB）的高效包裹，得到包载GrB的纳米凝胶（GrB-NGs）。蛋白质CC的释放实验于37℃在两种不同的释放介质中进行，即PB（pH 7.4，10 mM）和10 mM GSH的 PB（pH 7.4，10 mM）溶液。取1 mL包载CC-NGs样品于蛋白释放袋（MWCO 350 K）中，并置于25 mL相应的PB释放介质中。在每个取样时间点，取出5 mL释放介质，并补充相应的新鲜介质。将各时间点取出的样品冻干，复溶，用紫外（CC紫外吸收波长是410 nm）测定。每组释放试验平行进行三次，最终显示结果为实验所得平均值±标准方差。图6是上述纳米凝胶体外控制释放蛋白质药物的行为，以及释放出的蛋白质药物的生物活性表征图；蛋白质的体外释放实验表明在生理条件下（pH 7.4，37℃）CC能很好地被包裹在HA-NGs中，经48 h后，释放量约30%（图6a）。相反，在含有10 mMGSH的还原条件下，10 h后从纳米凝胶中释放出了超过80%的CC。这说明CC-NGs在细胞质的还原环境下能快速释放出包裹的蛋白质药物。Taking Cytochrome C (CC) as an example, a certain amount of CC (10% theoretical drug loading) was added to HA-OEG-Tet and HA-Cy-MA with a total polymer concentration of 1.25 mg/mL. PB (pH 7.4, 10 mM) solution, and then inject the solution into acetone, and then irradiate with ultraviolet light (320-390 nm, 50 mW/cm2 ) for 90 seconds. Finally, the acetone was removed by rotary evaporation and dialyzed against water for 12 hours to obtain the CC-loaded nanogel (CC-NGs) solution. A similar method can be used to achieve high-efficiency encapsulation of the therapeutic protein granzyme B (GrB), and to obtain GrB-encapsulated nanogels (GrB-NGs). The release experiments of protein CC were carried out in two different release media, PB (pH 7.4, 10 mM) and 10 mM GSH in PB (pH 7.4, 10 mM) at 37 °C. Take 1 mL of loaded CC-NGs sample in a protein release bag (MWCO 350 K), and place it in 25 mL of corresponding PB release medium. At each sampling time point, remove 5 mL of release medium and supplement with corresponding fresh medium. The samples taken out at each time point were freeze-dried, reconstituted, and measured by ultraviolet (CC ultraviolet absorption wavelength is 410 nm). Each group of release tests was carried out three times in parallel, and the final results were shown as the mean ± standard deviation obtained from the experiments. Figure 6 is the behavior of the nanogel in vitro controlled release of protein drugs, as well as the biological activity characterization of the released protein drugs; the in vitro release experiments of proteins show that under physiological conditions (pH 7.4, 37 ℃) CC can be well absorbed Wrapped in HA-NGs, after 48 h, the release amount was about 30% (Fig. 6a). In contrast, more than 80% of CC was released from the nanogel after 10 h under reducing conditions containing 10 mMGSH. This indicates that CC-NGs can rapidly release the encapsulated protein drug under the reducing environment of cytoplasm.
蛋白质的电子转移活性的测试是通过检测其对ABTS转变为ABTS+的催化效率得到的。首先释放出的CC用PBS溶液稀释至浓度为0.004 mg/mL。同时配置相同浓度的、未经任何处理的CC，取相同量两种溶液放入石英样品池中，向两种溶液中加入相同量的含有10 μL的0.045 M的过氧化氢溶液和100 μL的1 mg/mL的ABTS的PBS溶液。倒置使之混合好并马上用UV分光光度计读在410 nm处的吸收值，并以此为零点，每个15秒测一次。每个时间点对应的紫外吸收值减去第一个点的吸收值从而得到吸收值的变化（∆A），以∆A对时间作图表示其活性变化随时间的变化。附图6b为上述释放出的蛋白活性检测图，结果显示从纳米凝胶里释放出来的CC仍然能较快地催化ABTS的氧化，催化速率和未经任何处理的CC的接近，证实了从纳米凝胶中释放出的蛋白质仍能较好地保持活性。The electron transfer activity of the protein was tested by detecting its catalytic efficiency for the conversion of ABTS to ABTS+. The first released CC was diluted with PBS solution to a concentration of 0.004 mg/mL. Prepare the same concentration of CC without any treatment at the same time, put the same amount of the two solutions into the quartz sample cell, add the same amount of 0.045 M hydrogen peroxide solution containing 10 μL and 100 μL of 1 mg/mL of ABTS in PBS. Invert it to mix well and immediately read the absorbance value at 410 nm with a UV spectrophotometer, and use this as the zero point, measuring once every 15 seconds. The UV absorption value corresponding to each time point is subtracted from the absorption value of the first point to obtain the change of absorption value (∆A). Plotting ∆A against time shows the change of its activity over time. Accompanying drawing 6b is the detection chart of the protein activity released above, and the results show that the CC released from the nanogel can still catalyze the oxidation of ABTS relatively quickly, and the catalytic rate is close to that of the CC without any treatment, confirming that the CC released from the nanogel The proteins released from the gel still retain their activity well.
实施例八空纳米凝胶和包载有蛋白质药物的纳米凝胶的细胞实验Example 8 Cell experiment of empty nanogel and nanogel loaded with protein drug
以实施例四中的制备的纳米凝胶为例，测试空纳米凝胶的细胞相容性。将成纤维细胞（L929）、乳腺癌细胞（MCF-7）、脑胶质瘤细胞（U87）和肺癌细胞（A549）分别铺在96孔细胞培养板上，每个孔大约5000个细胞，再加入含有10 %小牛血清、1%谷氨酸盐、抗生素青霉素（100 IU/mL）和链霉素（100 μg/mL）的DMEM培养基，再置于37℃，5%二氧化碳条件下培养12h。然后，加20 μL 纳米凝胶的PB（10 mM，pH 7.4）溶液（最终纳米凝胶的浓度为0.2，0.4，0.6，0.8和1.0 mg/mL）到每孔中，再在37℃，5%二氧化碳条件下培养48 h。随后，向每孔加入3-（4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐（MTT）的PBS溶液（15 μL，5 mg/mL），并放入培养箱中继续培养。4 h后，移除含有MTT的培养液，再加入150 μL DMSO用于溶解活细胞与MTT生成的紫色结晶甲瓒，并用酶标仪（Bio Tek）测定每个孔在492 nm处的紫外吸收。细胞相对存活率通过与只有空白细胞的对照孔在492 nm处的吸收相比得到，每组实验平均进行4次。图7是上述纳米凝胶和载蛋白质药物的纳米凝胶的细胞实验表征图。Taking the nanogel prepared in Example 4 as an example, the cytocompatibility of the empty nanogel was tested. Spread fibroblasts (L929), breast cancer cells (MCF-7), brain glioma cells (U87) and lung cancer cells (A549) on 96-well cell culture plates, with about 5000 cells per well, and then add DMEM medium containing 10% calf serum, 1% glutamate, antibiotics penicillin (100 IU/mL) and streptomycin (100 μg/mL), cultured at 37°C and 5% carbon dioxide for 12h . Then, add 20 μL of nanogel in PB (10 mM, pH 7.4) solution (final nanogel concentration is 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) to each well, and then incubate at 37°C for 5 % CO2 for 48 h. Subsequently, a PBS solution (15 μL, 5 mg/mL) of 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, and Put into the incubator to continue culturing. After 4 h, the medium containing MTT was removed, and 150 μL of DMSO was added to dissolve the purple crystalline formazan produced by living cells and MTT, and the ultraviolet absorption of each well at 492 nm was measured with a microplate reader (Bio Tek) . The relative viability of the cells was obtained by comparing the absorbance at 492 nm of the control wells with only blank cells, and each group of experiments was performed an average of 4 times. Fig. 7 is a cell experiment characterization diagram of the above-mentioned nanogel and the protein-loaded nanogel.
附图7a为细胞存活率图，可以看出，48小时后加入纳米凝胶细胞的存活率均达到90 %以上，说明透明质酸纳米凝胶没有毒性。Accompanying drawing 7a is the graph of cell viability, it can be seen that after 48 hours, the survival rate of cells added with nanogel reached more than 90%, indicating that the hyaluronic acid nanogel has no toxicity.
CC-NGs和GrB-NGs的细胞毒性也是通过MTT法测定。将包载蛋白药物的纳米凝胶加入乳腺癌细胞（MCF-7，CD44受体高表达）, 肺癌细胞（A549，CD44受体高表达）和脑胶质瘤细胞（U87，CD44受体低表达）中，并培育4 h。然后将载蛋白的纳米凝胶的培养基吸走，再加入新鲜培养基于37℃，5%二氧化碳条件下继续培养92 h。培养结束后向每孔中加入10 μL MTT的PBS溶液（5 mg/mL）并放入培养箱中，继续培养4 h使MTT与活细胞充分作用。随后移除含有MTT的培养液，并加入150 μL DMSO用于溶解活细胞与MTT生成的紫色结晶甲瓒，并用酶标仪（Bio Tek）测定每个孔在492 nm处的紫外吸收。细胞相对存活率计算方法如上。封闭实验是先用HA（5 mg/mL）与MCF-7细胞孵育4 h，然后再加入包载包载蛋白质的纳米凝胶。结果表明CC-NGs对MCF-7细胞有较高的抗肿瘤活性，其细胞的半抑制浓度（IC50）为0.52 µM（图7b）。相反，自由CC即便在浓度高达6.2 µM时仍无明显的细胞毒性，这主要是因为CC内吞进入细胞的能力很差。另外，CC-NGs对CD44受体低表达的U87细胞表现出明显减小的凋亡活性，而且CC-NGs对预先封闭CD44受体的MCF-7细胞的抗肿瘤活性明显降低，这些结果说明CC-NGs是通过CD44受体介导内吞机理进入细胞。同时，GrB-NGs对CD44受体高表达的MCF-7和A549细胞都表现出了较高的体外抗肿瘤活性，其分别为3.0 nM和8.1 nM（图7c）。The cytotoxicity of CC-NGs and GrB-NGs was also determined by MTT method. Adding nanogels loaded with protein drugs to breast cancer cells (MCF-7, high expression of CD44 receptor), lung cancer cells (A549, high expression of CD44 receptor) and brain glioma cells (U87, low expression of CD44 receptor ) and incubated for 4 h. Then the medium of the protein-loaded nanogel was sucked away, and then fresh culture was added to continue culturing for 92 h at 37°C and 5% carbon dioxide. After the culture was over, 10 μL of MTT in PBS solution (5 mg/mL) was added to each well and placed in an incubator, and cultured for 4 h to allow MTT to fully interact with living cells. Subsequently, the culture medium containing MTT was removed, and 150 μL of DMSO was added to dissolve the purple crystalline formazan produced by living cells and MTT, and the ultraviolet absorption of each well at 492 nm was measured with a microplate reader (Bio Tek). The calculation method of relative cell viability was as above. In the blocking experiment, MCF-7 cells were first incubated with HA (5 mg/mL) for 4 h, and then the nanogel loaded with protein was added. The results showed that CC-NGs had high antitumor activity against MCF-7 cells, and the half-inhibitory concentration (IC50) of the cells was 0.52 µM (Fig. 7b). In contrast, free CC showed no significant cytotoxicity even at concentrations as high as 6.2 µM, mainly due to the poor ability of CC to endocytose into cells. In addition, CC-NGs showed significantly reduced apoptotic activity on U87 cells with low CD44 receptor expression, and the antitumor activity of CC-NGs on MCF-7 cells pre-blocked with CD44 receptors was significantly reduced. These results indicate that CC -NGs enter cells through CD44 receptor-mediated endocytosis mechanism. Meanwhile, GrB-NGs showed high in vitro antitumor activity against MCF-7 and A549 cells with high expression of CD44 receptor, which were 3.0 nM and 8.1 nM, respectively (Fig. 7c).
实施例九载蛋白纳米凝胶对小鼠皮下人乳腺癌移植瘤的治疗The treatment of embodiment nine apoprotein nanogels to subcutaneous human breast cancer xenografts in mice
首先使用皮下注射MCF-7细胞（1 × 107）的PBS溶液（50 μL）到裸鼠的右后侧建立了人乳腺癌皮下肿瘤模型。当肿瘤的体积达到30 mm3后，裸鼠被随机分成4组，每组5只。然后，通过静脉注射方法分别向每组荷瘤老鼠注射GrB-NGs （25 µg GrB equiv./kg）、GrB-NGs（100 µg GrB equiv./kg）、空纳米凝胶和PBS缓冲溶液，每三天给药一次，总共给药四次。肿瘤体积以及裸鼠体重每隔一天测量一次。肿瘤体积的计算公式：体积 = ½*a*b*c，a是肿瘤最长边，b是肿瘤最宽边，c是肿瘤的高度。附图8为皮下乳腺癌的治疗，可以发现PBS组和空纳米凝胶组的肿瘤体积生长快速。而GrB-NGs在剂量为25 µg GrB equiv./kg和100 µg GrBequiv./kg时都能有效地抑制肿瘤的生长，且剂量越高对肿瘤生长的抑制效果越明显。同时，GrB-NGs组与PBS组相似，都没有导致动物体重降低，说明GrB-NGs没有明显的毒副作用（参见图8)。Firstly, a human breast cancer subcutaneous tumor model was established by subcutaneously injecting MCF-7 cells (1 × 107 ) in PBS solution (50 μL) into the right posterior flank of nude mice. When the tumor volume reached 30 mm3 , the nude mice were randomly divided into 4 groups, 5 mice in each group. Then, each group of tumor-bearing mice was injected with GrB-NGs (25 µg GrB equiv./kg), GrB-NGs (100 µg GrB equiv./kg), empty nanogel and PBS buffer solution by intravenous injection, each Dosing once every three days, for a total of four administrations. Tumor volume and body weight of nude mice were measured every other day. The formula for calculating tumor volume: Volume = ½*a*b*c, where a is the longest side of the tumor, b is the widest side of the tumor, and c is the height of the tumor. Figure 8 shows the treatment of subcutaneous breast cancer. It can be found that the tumor volume of the PBS group and the empty nanogel group grows rapidly. However, GrB-NGs can effectively inhibit tumor growth at the dose of 25 μg GrB equiv./kg and 100 μg GrBequiv./kg, and the higher the dose, the more obvious the inhibitory effect on tumor growth. At the same time, similar to the PBS group, the GrB-NGs group did not cause the animals to lose weight, indicating that GrB-NGs had no obvious toxic side effects (see Figure 8).
实施例十载蛋白纳米凝胶对小鼠原位人肺癌移植瘤的治疗Example Decaprin Nanogel Treatment of Orthotopic Human Lung Cancer Transplanted Tumors in Mice
首先通过注射带luciferase的A549细胞（1 × 107）的PBS溶液（50 μL）到裸鼠的肺部建立了人肺癌原位肿瘤模型。当肿瘤细胞的荧光值达到20000 p/s/cm2/sr时，裸鼠被随机分成3组，每组6只。然后，每三天通过尾静脉注射分别向每组荷瘤老鼠注射GrB-NGs（150 µgGrB equiv./kg GrB）、空白纳米凝胶和PBS，总共给药四次。肿瘤出的荧光和裸鼠体重每三天记录一次，图9是载蛋白纳米凝胶对小鼠原位肺癌移植瘤的治疗表征图。附图9a-c为肺癌原位治疗图，PBS组和空纳米凝胶组的肿瘤处荧光强度随时间明显增强，表明肿瘤在快速生长。而GrB-NGs治疗中的肿瘤荧光强度较弱，这说明GrB-NGs能有效地抑制裸鼠肺癌的生长。同时，GrB-NGs组的小鼠体重变化并不显著（图9d），这一方面表明GrB-NGs无明细的毒副作用，另一方面也说明GrB-NGs能有效地抑制小鼠原位肺癌的生长，使小鼠生长状况良好。相反，PBS和空白纳米凝胶对照组的小鼠体重均显著降低，这是因为小鼠随着原位肺癌的快速生长，身体状况急剧下降。小鼠的生存实验也表明GrB-NGs治疗组的小鼠在整个观察期间（40天），没有死亡发生；而PBS和空白纳米凝胶对照组的小鼠在治疗观察结束后，全部死亡（图9e）。H&E染色可以发现GrB-NGs对体内主要脏器（心脏、肝脏、肾等）没有副作用（图10），这进一步表明了GrB-NGs不会引起明显的毒副作用。Firstly, an orthotopic tumor model of human lung cancer was established by injecting luciferase-containing A549 cells (1 × 107 ) in PBS solution (50 μL) into the lungs of nude mice. When the fluorescence value of the tumor cells reached 20000 p/s/cm2 /sr, the nude mice were randomly divided into 3 groups, 6 in each group. Then, tumor-bearing mice in each group were injected with GrB-NGs (150 µgGrB equiv./kg GrB), blank nanogel, and PBS via tail vein injection every three days for a total of four administrations. The fluorescence from the tumor and the body weight of the nude mice were recorded every three days. Figure 9 is a characterization diagram of the treatment of orthotopic lung cancer xenografts by the apoprotein nanogel. Figures 9a-c are pictures of in situ treatment of lung cancer. The fluorescence intensity of the tumors in the PBS group and the empty nanogel group increased significantly over time, indicating that the tumors were growing rapidly. However, the fluorescence intensity of tumors treated with GrB-NGs was weak, which indicated that GrB-NGs could effectively inhibit the growth of lung cancer in nude mice. At the same time, the body weight of the mice in the GrB-NGs group did not change significantly (Figure 9d), which on the one hand indicates that GrB-NGs has no detailed side effects, and on the other hand also indicates that GrB-NGs can effectively inhibit the growth of orthotopic lung cancer in mice. Grow so that the mice grow in good condition. In contrast, mice in the PBS and blank nanogel control groups had significantly reduced body weight, which was due to the sharp decline in body condition of the mice along with the rapid growth of orthotopic lung cancer. The survival experiment of mice also showed that the mice in the GrB-NGs treatment group did not die during the entire observation period (40 days); while the mice in the PBS and blank nanogel control groups all died after the treatment observation (Fig. 9e). H&E staining showed that GrB-NGs had no side effects on major organs (heart, liver, kidney, etc.) in the body (Figure 10), which further indicated that GrB-NGs would not cause obvious toxic side effects.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610536475.1ACN105968372B (en) | 2016-07-08 | 2016-07-08 | A kind of self-fluorescent nanogel and its preparation method and application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610536475.1ACN105968372B (en) | 2016-07-08 | 2016-07-08 | A kind of self-fluorescent nanogel and its preparation method and application |
| Publication Number | Publication Date |
|---|---|
| CN105968372Atrue CN105968372A (en) | 2016-09-28 |
| CN105968372B CN105968372B (en) | 2019-02-26 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610536475.1AActiveCN105968372B (en) | 2016-07-08 | 2016-07-08 | A kind of self-fluorescent nanogel and its preparation method and application |
| Country | Link |
|---|---|
| CN (1) | CN105968372B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105963703A (en)* | 2016-07-08 | 2016-09-28 | 苏州大学张家港工业技术研究院 | Preparation method of anti-tumor drug |
| WO2018010053A1 (en)* | 2016-07-10 | 2018-01-18 | 苏州大学张家港工业研究院 | Method for preparing antitumor drug |
| CN110251456A (en)* | 2019-06-06 | 2019-09-20 | 苏州大学 | A kind of preparation method and application of nano gel |
| CN111995745A (en)* | 2020-08-25 | 2020-11-27 | 华南理工大学 | A kind of double lock type polymer and its preparation method and application |
| CN112661929A (en)* | 2021-01-18 | 2021-04-16 | 丽水学院 | Polyurethane functionalized by click chemistry and preparation method thereof |
| US11262303B2 (en) | 2019-02-19 | 2022-03-01 | National Tsing Hua University | Oxygen content sensor, oxygen content sensor manufacturing method and using method |
| CN116712557A (en)* | 2023-05-11 | 2023-09-08 | 湖南省肿瘤医院 | EpCAM aptamer targeting Dox hyaluronic acid nanogel and its preparation method and application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101836970A (en)* | 2010-04-13 | 2010-09-22 | 南京大学 | Polyacrylic acid nano-gel microspheres, preparation method and application thereof |
| CN103396554A (en)* | 2013-07-02 | 2013-11-20 | 苏州大学 | Hydrogel, preparation method thereof and applications |
| CN103694479A (en)* | 2013-11-21 | 2014-04-02 | 中国科学院长春应用化学研究所 | Polymer, glucose-sensitive nanogel, glucose-sensitive drug-loading nanogel and their preparation methods |
| CN104940138A (en)* | 2015-06-16 | 2015-09-30 | 江南大学 | Preparation method of stimulation-sensitive hyaluronic acid in-situ gel |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101836970A (en)* | 2010-04-13 | 2010-09-22 | 南京大学 | Polyacrylic acid nano-gel microspheres, preparation method and application thereof |
| CN103396554A (en)* | 2013-07-02 | 2013-11-20 | 苏州大学 | Hydrogel, preparation method thereof and applications |
| CN103694479A (en)* | 2013-11-21 | 2014-04-02 | 中国科学院长春应用化学研究所 | Polymer, glucose-sensitive nanogel, glucose-sensitive drug-loading nanogel and their preparation methods |
| CN104940138A (en)* | 2015-06-16 | 2015-09-30 | 江南大学 | Preparation method of stimulation-sensitive hyaluronic acid in-situ gel |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105963703A (en)* | 2016-07-08 | 2016-09-28 | 苏州大学张家港工业技术研究院 | Preparation method of anti-tumor drug |
| CN105963703B (en)* | 2016-07-08 | 2019-05-10 | 苏州大学张家港工业技术研究院 | A kind of preparation method of antitumor drug |
| WO2018010053A1 (en)* | 2016-07-10 | 2018-01-18 | 苏州大学张家港工业研究院 | Method for preparing antitumor drug |
| US11262303B2 (en) | 2019-02-19 | 2022-03-01 | National Tsing Hua University | Oxygen content sensor, oxygen content sensor manufacturing method and using method |
| CN110251456A (en)* | 2019-06-06 | 2019-09-20 | 苏州大学 | A kind of preparation method and application of nano gel |
| CN111995745A (en)* | 2020-08-25 | 2020-11-27 | 华南理工大学 | A kind of double lock type polymer and its preparation method and application |
| CN111995745B (en)* | 2020-08-25 | 2021-07-20 | 华南理工大学 | A kind of double lock type polymer and its preparation method and application |
| CN112661929A (en)* | 2021-01-18 | 2021-04-16 | 丽水学院 | Polyurethane functionalized by click chemistry and preparation method thereof |
| CN112661929B (en)* | 2021-01-18 | 2022-07-12 | 丽水学院 | Polyurethane functionalized by click chemistry and preparation method thereof |
| CN116712557A (en)* | 2023-05-11 | 2023-09-08 | 湖南省肿瘤医院 | EpCAM aptamer targeting Dox hyaluronic acid nanogel and its preparation method and application |
| Publication number | Publication date |
|---|---|
| CN105968372B (en) | 2019-02-26 |
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Oxidation triggered formation of polydopamine-modified carboxymethyl cellulose hydrogel for anti-recurrence of tumor | |
| CN105968372A (en) | Self-fluorescence nanogel and preparation method and application thereof | |
| CN104497171B (en) | Hyaluronic-acid-based amphiphilic polymer as well as preparation method and application thereof | |
| CN110237035B (en) | An active targeting amphiphilic polypeptide nano drug carrier and its preparation and application | |
| CA3016655C (en) | Ovarian cancer specifically targeted biodegradable amphiphilic polymer, polymer vesicle prepared thereby and use thereof | |
| CN101897976A (en) | A drug solubilizing carrier and its preparation method and application | |
| CN113694023B (en) | Oxidation response type nano micelle and preparation method and application thereof | |
| CN110063933B (en) | A kind of dextran-based nanogel and its preparation method and application | |
| Yang et al. | CD44-mediated tumor homing of hyaluronic acid nanogels for hypoxia-activated photodynamic therapy against tumor | |
| CN110368501B (en) | RGD peptide modified boron drug-loading system and preparation and application thereof | |
| KR20180097707A (en) | Biodegradable amphipathic polymers, polymeric vehicles prepared thereby, and use in the manufacture of a therapeutic agent for lung cancer target | |
| CN103381273B (en) | Amycin prodrug and preparation method thereof and injectable compositions | |
| CN101665569B (en) | Hydrophilic polymer modified with lipoic acid and its preparation and application | |
| CN113827567A (en) | Application of small-molecule-drug-carrying polymer vesicle in preparation of drugs for treating acute lymphatic leukemia | |
| CN104474556A (en) | Application of carbon dot as anti-tumor medicine carrier | |
| CN102391517A (en) | Nanometer micelle capable of intelligently releasing medicine as well as preparation method and application thereof | |
| CN111870579A (en) | Tumor-targeting nanomicelle, preparation method and application as drug carrier | |
| CN112915205B (en) | A photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy and its preparation method | |
| CN101524326A (en) | Chlorin e6 chitosan-stearic acid graft micelle | |
| CN105963703A (en) | Preparation method of anti-tumor drug | |
| CN109876150B (en) | Polymer drug carrier, its drug-carrying composition, preparation method and application | |
| CN114533671A (en) | Preparation method and application of biodegradable hyperbranched polycarbonate shell-core polymer micelle | |
| CN112656951B (en) | Cross-linked acid-responsive natural polysaccharide polymer prodrug, preparation method and use | |
| CN110339368B (en) | Preparation method of reduction-responsive targeting polyethylene glycol-polycarbonate maytansine prodrug micelle | |
| CN110317281B (en) | Hyaluronic acid-g-folic acid amphiphilic polymer and application thereof |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |