【技术领域】【Technical field】
本发明属于纳米医药技术领域，特别涉及一种用于负载贵金属颗粒的纳米凝胶及其制备方法与应用。The invention belongs to the technical field of nanomedicine, and in particular relates to a nanogel for loading noble metal particles, a preparation method and application thereof.
【背景技术】【Background technique】
医用凝胶体系在临床应用方面有大量研究，包括各种可注射医用凝胶，温度敏感微凝胶等。活性成分如肿瘤化疗药物与纳米凝胶载体的结合有多种形式，包括均匀分散(即药物等活性成分均匀的分散于凝胶载体体系中，随着凝胶的降解或解离而进行药物的缓控制释放)、胶囊式载药(即药物分布在纳米载体体系的内核，外部进行凝胶聚合物的包裹，随着凝胶体系的理化性质变化而释放药物)和化学键交联载药(即将活性分子与载体通过化学键进行结合，将药物成分变成凝胶载药体系的一部分，随便化学键的断裂进行药物的有效释放)。活性成分的选取多种多样，可以是疏水性的分子或亲水性的小分子，也可以是基因药物、蛋白药物或无机金属材料颗粒等。将贵金属纳米材料如银用于凝胶体系，可以药用杀灭细菌；将金棒等纳米颗粒进行化学吸附，可利用金颗粒的光热转化实现温敏凝胶的智能控制；将无机介孔材料置于纳米凝胶体系，可以增加介孔材料的生物相容性，更好的实现药物的有效控制释放。Medical gel systems have been extensively studied in clinical applications, including various injectable medical gels, temperature-sensitive microgels, etc. The combination of active ingredients such as tumor chemotherapy drugs and nanogel carriers has various forms, including uniform dispersion (that is, active ingredients such as drugs are uniformly dispersed in the gel carrier system, and the drug is degraded or dissociated as the gel is degraded or dissociated. Slow and controlled release), capsule drug loading (that is, the drug is distributed in the inner core of the nanocarrier system, and the gel polymer is wrapped on the outside, and the drug is released as the physicochemical properties of the gel system change) and chemical bond cross-linking drug loading (that is, The active molecule is combined with the carrier through chemical bonds, and the drug component becomes a part of the gel drug-loading system, and the drug is released effectively by breaking the chemical bond). The active ingredients can be selected in a variety of ways, such as hydrophobic molecules or hydrophilic small molecules, gene drugs, protein drugs, or inorganic metal material particles. The use of precious metal nanomaterials such as silver in the gel system can be used to kill bacteria in medicine; the chemical adsorption of nanoparticles such as gold rods can use the photothermal conversion of gold particles to realize the intelligent control of temperature-sensitive gels; the inorganic mesoporous materials placed in The nanogel system can increase the biocompatibility of the mesoporous material and better realize the effective controlled release of drugs.
随着材料学以及医学的发展，将凝胶体系与金颗粒复合而开发新的功能化凝胶成为新的研究方向。凝胶的形成有很多种，如自由基引发、两亲性聚合物自组装以及聚合物内部正负电荷相互吸引等。凝胶作为一大类生物相容性好的材料，在药物装载以及组织工程方面具有多种用途。将聚合物凝胶与贵金属如金纳米棒结合，通过金纳米棒的光热转化，实现凝胶体系的温度敏感响应，完成药物释放或环境监测。在已有的技术和方法中，凝胶体系较大，一般都在500nm以上，不利于广泛的生物医学应用。With the development of materials science and medicine, it has become a new research direction to develop new functional gels by combining gel systems with gold particles. There are many ways to form gels, such as free radical initiation, self-assembly of amphiphilic polymers, and mutual attraction of positive and negative charges inside the polymer. As a large class of biocompatible materials, gels have multiple uses in drug loading and tissue engineering. Combining polymer gel with noble metals such as gold nanorods, through the photothermal conversion of gold nanorods, the temperature-sensitive response of the gel system can be realized to complete drug release or environmental monitoring. In the existing technologies and methods, the gel system is relatively large, generally above 500 nm, which is not conducive to a wide range of biomedical applications.
【发明内容】【Content of invention】
本发明的首要目的在于克服现有技术存在的不足，提供一种用于负载贵金属颗粒的纳米凝胶的制备方法。The primary purpose of the present invention is to overcome the deficiencies of the prior art and provide a preparation method for nanogel loaded with noble metal particles.
本发明的另一目的在于提供由上述制备方法得到的用于负载贵金属颗粒的纳米凝胶。Another object of the present invention is to provide the nanogel for supporting noble metal particles obtained by the above preparation method.
本发明的再一目的在于提供所述的用于负载贵金属颗粒的纳米凝胶的应用。Another object of the present invention is to provide the application of the nanogel for loading noble metal particles.
本发明的目的通过以下技术方案实现：一种用于负载贵金属颗粒的纳米凝胶的制备方法，包括如下步骤：The object of the present invention is achieved through the following technical solutions: a method for preparing a nanogel for loading noble metal particles, comprising the steps of:
(1)纳米凝胶的表面修饰：将硫辛酸按5～50mg/ml溶于二甲亚砜中，加入二环己基二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)，10～40℃活化过夜，得到活化液；将活化液加入肝素-聚乙烯亚胺纳米凝胶水溶液中，室温反应36h，过滤并透析，得到肝素-聚乙烯亚胺-硫辛酸纳米凝胶；所述硫辛酸、二环己基二亚胺和N-羟基琥珀酰亚胺的摩尔比为1:1.5:1.5～1:10:12，所述肝素-聚乙烯亚胺纳米凝胶水溶液中的水与所述二甲亚砜的体积比为2～5；(1) Surface modification of nanogel: Dissolve lipoic acid in dimethyl sulfoxide at 5-50 mg/ml, add dicyclohexyldiimide (DCC) and N-hydroxysuccinimide (NHS), 10 Activation at ~40°C overnight to obtain an activation solution; adding the activation solution to an aqueous solution of heparin-polyethyleneimine nanogel, reacting at room temperature for 36 hours, filtering and dialysis to obtain a heparin-polyethyleneimine-lipoic acid nanogel; The molar ratio of lipoic acid, dicyclohexyldiimide and N-hydroxysuccinimide is 1:1.5:1.5～1:10:12, and the water in the heparin-polyethyleneimine nanogel aqueous solution and the The volume ratio of said dimethyl sulfoxide is 2～5;
(2)还原剂处理纳米凝胶：往步骤(1)的肝素-聚乙烯亚胺-硫辛酸纳米凝胶加入0.5～5wt％还原剂，氮气保护下0～40℃搅拌10min～24h后透析，得到用于负载贵金属颗粒的纳米凝胶。(2) Treating nanogel with reducing agent: add 0.5-5wt% reducing agent to the heparin-polyethyleneimine-lipoic acid nanogel in step (1), and dialyze after stirring at 0-40°C for 10min-24h under nitrogen protection, A nanogel for loading noble metal particles is obtained.
步骤(1)中：In step (1):
所述肝素-聚乙烯亚胺纳米胶束采用以下方法进行制备：将500～5000mg支链聚乙烯亚胺溶于5～50mL去离子水中后以2～20滴/min加入20～100mL、浓度为5～100mg/mL肝素钠溶液中，室温搅拌12～36h，过滤并透析，得到肝素-聚乙烯亚胺纳米凝胶水溶液。The heparin-polyethyleneimine nanomicelle is prepared by the following method: dissolve 500-5000 mg of branched-chain polyethyleneimine in 5-50 mL of deionized water and add 20-100 mL at 2-20 drops/min at a concentration of 5-100 mg/mL heparin sodium solution, stirred at room temperature for 12-36 hours, filtered and dialyzed to obtain heparin-polyethyleneimine nanogel aqueous solution.
优选的，所述肝素-聚乙烯亚胺纳米胶束采用以下方法进行制备：将2g支链聚乙烯亚胺溶于15mL去离子水中，然后加入50mL、浓度为100mg/mL肝素钠溶液中，室温搅拌12h，0.22μm滤膜过滤并透析(截留分子量3000Da)，得到肝素-聚乙烯亚胺纳米凝胶水溶液；Preferably, the heparin-polyethyleneimine nanomicelle is prepared by the following method: dissolve 2g of branched-chain polyethyleneimine in 15mL of deionized water, then add 50mL of heparin sodium solution with a concentration of 100mg/mL at room temperature Stir for 12 hours, filter with a 0.22 μm filter membrane and dialyze (molecular weight cut-off 3000Da), to obtain a heparin-polyethyleneimine nanogel aqueous solution;
所述支链聚乙烯亚胺的分子量优选为600、1800或25000Da；The molecular weight of the branched polyethyleneimine is preferably 600, 1800 or 25000Da;
所述肝素钠溶液优选采用以下方法进行活化：将肝素钠按5～50mg/ml加入100mL的2-吗啉乙磺酸水溶液中，分别加入N-羟基琥珀酰亚胺和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，活化2～12h，得到活化后的肝素钠溶液；所述肝素钠、N-羟基琥珀酰亚胺和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔比为1:1.5:1.5～1:12:10；The sodium heparin solution is preferably activated by the following method: add sodium heparin at 5 to 50 mg/ml into 100 mL of 2-morpholineethanesulfonic acid aqueous solution, add N-hydroxysuccinimide and 1-ethyl-( 3-dimethylaminopropyl) carbodiimide hydrochloride, activated for 2 to 12 hours, to obtain activated heparin sodium solution; the heparin sodium, N-hydroxysuccinimide and 1-ethyl-( The molar ratio of 3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1.5:1.5～1:12:10;
所述2-吗啉乙磺酸水溶液(MES水溶液)优选为pH为5.5的、含有0.05M2-吗啉乙磺酸的水溶液；The 2-morpholineethanesulfonic acid aqueous solution (MES aqueous solution) is preferably an aqueous solution containing 0.05M 2-morpholineethanesulfonic acid at a pH of 5.5;
所述过滤优选采用0.45μm或0.22μm滤膜过滤；The filtration preferably adopts 0.45 μm or 0.22 μm membrane filtration;
所述透析优选采用截留分子量大于3000Da的透析袋进行透析；The dialysis preferably uses a dialysis bag with a molecular weight cut-off greater than 3000Da for dialysis;
步骤(2)中：In step (2):
所述还原剂优选为硼氢化钠或二硫苏糖醇(DTT)；The reducing agent is preferably sodium borohydride or dithiothreitol (DTT);
所述透析优选采用截留分子量为3500Da的透析袋进行透析；The dialysis preferably adopts a dialysis bag with a molecular weight cut-off of 3500Da for dialysis;
所述贵金属优选为金。The noble metal is preferably gold.
一种用于负载贵金属颗粒的纳米凝胶，由上述制备方法得到。A nanogel for loading noble metal particles is obtained by the above preparation method.
所述用于负载贵金属颗粒的纳米凝胶可应用于环境监测、医学诊断学或肿瘤光热治疗等技术领域中。The nanogel for loading noble metal particles can be applied in technical fields such as environmental monitoring, medical diagnostics, or tumor photothermal therapy.
本发明的发明机理：本发明以阴离子聚合物与阳离子聚合物的相互作用为基础，以极负的肝素作为合成骨架，通过其中的羧基形成酰胺键，在其上引入带正电荷的聚乙烯亚胺，由于正负电的相互作用，形成具有纳米尺度、表面带正点的纳米凝胶体系，在纳米凝胶表面的氨基位置进行小分子硫辛酸修饰，在还原剂的作用下，使得纳米凝胶表面的基团由氨基转变为巯基，完成对金纳米颗粒的吸附。Invention mechanism of the present invention: the present invention is based on the interaction between anionic polymer and cationic polymer, uses extremely negative heparin as a synthetic skeleton, forms an amide bond through the carboxyl group in it, and introduces a positively charged polyethylene oxide on it. Amines, due to the interaction of positive and negative charges, form a nano-gel system with nanoscale and positive points on the surface. Small molecule lipoic acid is modified at the amino position on the surface of the nano-gel. Under the action of a reducing agent, the nano-gel The groups on the surface changed from amino groups to mercapto groups to complete the adsorption of gold nanoparticles.
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
(1)本发明以肝素钠为合成模板，通过与聚乙烯亚胺结合形成纳米凝胶，并在表面进行硫辛酸修饰，得到用于负载贵金属颗粒的纳米凝胶。该纳米凝胶粒径小，稳定性好，便于通过控制原料组成而控制纳米凝胶的粒径，可通过加入硼氢化钠或DTT进行二硫键的还原，用于贵金属纳米颗粒的稳定吸附。(1) The present invention uses heparin sodium as a synthetic template, forms nanogels by combining with polyethyleneimine, and modifies the surface with lipoic acid to obtain nanogels for loading noble metal particles. The nanogel has small particle size and good stability, and it is convenient to control the particle size of the nanogel by controlling the composition of raw materials, and can be used for stable adsorption of noble metal nanoparticles by adding sodium borohydride or DTT to reduce disulfide bonds.
(2)本发明的制备方法简便、反应条件温和，重现性好，成本低，形貌均一且毒性低，得到的纳米凝胶具有很好的生物相容性，可应用于环境监测、医学诊断学或肿瘤光热治疗等技术领域中，具有良好的市场应用前景。(2) The preparation method of the present invention is simple, the reaction conditions are mild, the reproducibility is good, the cost is low, the appearance is uniform and the toxicity is low, and the obtained nanogel has good biocompatibility, and can be applied to environmental monitoring, medicine In technical fields such as diagnostics or tumor photothermal therapy, it has a good market application prospect.
【附图说明】【Description of drawings】
图1是本发明中纳米凝胶合成与修饰过程的示意图。Fig. 1 is a schematic diagram of the nanogel synthesis and modification process in the present invention.
图2是实施例1的肝素-聚乙烯亚胺(高分子量，25kDa)纳米凝胶TEM检测结果图(磷钨酸负染)。Fig. 2 is a graph of TEM detection results of heparin-polyethyleneimine (high molecular weight, 25kDa) nanogel in Example 1 (negative staining with phosphotungstic acid).
图3是实施例1的肝素-聚乙烯亚胺(25kD)-硫辛酸纳米凝胶DTT处理后吸附金棒复合物TEM检测结果图。Fig. 3 is a graph showing the TEM detection results of the heparin-polyethyleneimine (25kD)-lipoic acid nanogel complex after DTT treatment in Example 1.
图4是实施例2的肝素-聚乙烯亚胺(1800Da)-硫辛酸纳米凝胶DTT处理后吸附金纳米棒的TEM图。4 is a TEM image of the heparin-polyethyleneimine (1800Da)-lipoic acid nanogel of Example 2 after DTT treatment and adsorption of gold nanorods.
图5是实施例3的肝素-聚乙烯亚胺(1800Da)-硫辛酸纳米凝胶硼氢化钠处理后吸附金棒的TEM图。5 is a TEM image of the heparin-polyethyleneimine (1800Da)-lipoic acid nanogel of Example 3 after being treated with sodium borohydride and adsorbed on gold rods.
【具体实施方式】【Detailed ways】
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的保护范围并不限于此。The present invention will be described in further detail below in conjunction with the embodiments and drawings, but the protection scope of the present invention is not limited thereto.
实施例1Example 1
(1)金纳米棒的合成(1) Synthesis of gold nanorods
种子金纳米颗粒的合成：将5mL 0.2mol/L的十六烷基三甲基溴化铵(CTAB)与5mL 0.5mmol/L的氯金酸混合，400转/分钟边搅拌边加入0.6mL0℃保存的0.01mol/L的硼氢化钠，1000转/分钟搅拌5min，室温静置2h，得到种子金纳米颗粒，命名为GNP-R0。Synthesis of seed gold nanoparticles: Mix 5mL 0.2mol/L cetyltrimethylammonium bromide (CTAB) with 5mL 0.5mmol/L chloroauric acid, add 0.6mL 0°C while stirring at 400 rpm The stored 0.01 mol/L sodium borohydride was stirred at 1000 rpm for 5 min, and allowed to stand at room temperature for 2 h to obtain seed gold nanoparticles, which were named GNP-R0.
CTAB模板法合成短的金纳米棒：取7个25mL的样品瓶(分别标记为GNP-R1、GNP-R2、GNP-R3、GNP-R4、GNP-R5、GNP-R6、GNP-R7)，分别加入5mL 0.2mol/L的CTAB水溶液，对应编号加入不同量4mmol/L的AgNO3水溶液(0.050、0.10、0.15、0.20、0.25、0.30和0.40mL)，再分别加入5mL 1mmol/L的氯金酸水溶液，此时溶液颜色变成金黄色，接着分别加入70μL 0.0788mol/L的抗坏血酸，溶液变成无色。最后分别加入12μL的种子金纳米颗粒分散液，在27℃静置反应2h，得到金纳米棒，室温、避光保存，备用。Synthesis of short gold nanorods by CTAB template method: take seven 25mL sample vials (marked as GNP-R1, GNP-R2, GNP-R3, GNP-R4, GNP-R5, GNP-R6, GNP-R7), Add 5mL of 0.2mol/L CTAB aqueous solution respectively, add different amounts of 4mmol/L AgNO3 aqueous solution (0.050, 0.10, 0.15, 0.20, 0.25, 0.30 and 0.40mL) corresponding to the number, and then add 5mL of 1mmol/L gold chloride respectively At this time, the color of the solution turns golden yellow, and then 70 μL of 0.0788 mol/L ascorbic acid is added respectively, and the solution turns colorless. Finally, 12 μL of seed gold nanoparticle dispersion was added, and left to react at 27° C. for 2 h to obtain gold nanorods, which were stored at room temperature and protected from light for later use.
(2)肝素-聚乙烯亚胺纳米凝胶的制备(2) Preparation of heparin-polyethyleneimine nanogel
1)配制MES水溶液100mL(pH 5.5、0.05M)；1) Prepare 100mL of MES aqueous solution (pH 5.5, 0.05M);
2)肝素钠溶液的活化：取0.5g肝素钠溶解于50mL MES(100mL圆底烧瓶)中，加入300mg N-羟基琥珀酰亚胺和200mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，活化2h，得到活化后的肝素钠溶液；2) Activation of heparin sodium solution: Dissolve 0.5g heparin sodium in 50mL MES (100mL round bottom flask), add 300mg N-hydroxysuccinimide and 200mg 1-ethyl-(3-dimethylaminopropyl ) carbodiimide hydrochloride, activated for 2h, to obtain activated heparin sodium solution;
3)肝素-聚乙烯亚胺纳米凝胶的制备：将1g支链聚乙烯亚胺(分子量25kDa)溶于50mL去离子水中，并置于恒压漏斗中，加入步骤(2)的活化后的肝素钠溶液中，室温搅拌12h；过滤并离心，得到肝素-聚乙烯亚胺纳米凝胶；结果如图2所示；从图2可以看出，能形成纳米级凝胶，且分布均匀。3) Preparation of heparin-polyethyleneimine nanogel: Dissolve 1 g of branched-chain polyethyleneimine (molecular weight 25 kDa) in 50 mL of deionized water, place it in a constant pressure funnel, add the activated In heparin sodium solution, stir at room temperature for 12 hours; filter and centrifuge to obtain heparin-polyethyleneimine nanogel; the results are shown in Figure 2; it can be seen from Figure 2 that nanoscale gel can be formed and distributed evenly.
(3)纳米凝胶的表面硫辛酸修饰：(3) surface lipoic acid modification of nanogel:
将100mg硫辛酸溶解于20mL二甲亚砜中，加入200mg二环己基碳二亚胺和220mg N-羟基琥珀酰亚胺，过夜活化并滤纸过滤后加入上述得到的肝素-聚乙烯亚胺纳米胶束中，室温反应36小时，过滤并透析，得到肝素-聚乙烯亚胺-硫辛酸纳米凝胶。冻干样品，通过样品质量称重测定纳米凝胶产物的浓度。结果：能形成稳定的纳米凝胶，沉淀物较少。Dissolve 100mg of lipoic acid in 20mL of dimethylsulfoxide, add 200mg of dicyclohexylcarbodiimide and 220mg of N-hydroxysuccinimide, activate overnight and filter through filter paper, then add the heparin-polyethyleneimine nanogel obtained above In the bundle, react at room temperature for 36 hours, filter and dialyze to obtain heparin-polyethyleneimine-lipoic acid nanogel. The samples were lyophilized and the concentration of the nanogel product was determined by weighing the sample mass. Result: A stable nanogel can be formed with less precipitation.
(4)纳米凝胶表面巯基的释放与金纳米颗粒的吸附：(4) The release of sulfhydryl groups on the surface of the nanogel and the adsorption of gold nanoparticles:
往2mL步骤(3)中的肝素-聚乙烯亚胺-硫辛酸纳米凝胶加入0.1mM DTT300μL，对二硫键进行还原，然后加入2mL步骤(1)的金纳米棒，透析1天后，检测贵金属纳米颗粒的吸附效果；结果如图3所示，肝素-聚乙烯亚胺-硫辛酸纳米凝胶对金纳米棒具有良好的吸附效果。Add 0.1mM DTT300μL to 2mL of heparin-polyethyleneimine-lipoic acid nanogel in step (3) to reduce disulfide bonds, then add 2mL of gold nanorods in step (1), dialyze for 1 day, and detect precious metals The adsorption effect of nanoparticles; the results are shown in Figure 3, the heparin-polyethyleneimine-lipoic acid nanogel has a good adsorption effect on gold nanorods.
实施例2Example 2
(1)金纳米棒的合成(1) Synthesis of gold nanorods
种子金纳米颗粒的合成：将5mL 0.2mol/L的十六烷基三甲基溴化铵(CTAB)与5mL 0.5mmol/L的氯金酸混合，400转/分钟边搅拌边加入0.6mL0℃保存的0.01mol/L的硼氢化钠，600转/分钟搅拌5min，室温静置2h，得到种子金纳米颗粒，命名为GNP-R0。Synthesis of seed gold nanoparticles: Mix 5mL 0.2mol/L cetyltrimethylammonium bromide (CTAB) with 5mL 0.5mmol/L chloroauric acid, add 0.6mL 0°C while stirring at 400 rpm The preserved 0.01 mol/L sodium borohydride was stirred at 600 rpm for 5 min, and allowed to stand at room temperature for 2 h to obtain seed gold nanoparticles, which were named GNP-R0.
CTAB模板法合成短的金纳米棒：取7个25mL的样品瓶(分别标记为GNP-R1、GNP-R2、GNP-R3、GNP-R4、GNP-R5、GNP-R6、GNP-R7)，分别加入5mL 0.2mol/L的CTAB水溶液，对应编号加入不同量4mmol/L的AgNO3水溶液(0.050、0.10、0.15、0.20、0.25、0.30和0.40mL)，再分别加入5mL 1mmol/L的氯金酸水溶液，此时溶液颜色变成金黄色，接着分别加入70μL 0.0788mol/L的抗坏血酸，溶液变成无色。最后分别加入12μL的种子金纳米颗粒分散液，在27℃静置反应2h，得到金纳米棒，室温、避光保存，备用。Synthesis of short gold nanorods by CTAB template method: take seven 25mL sample vials (marked as GNP-R1, GNP-R2, GNP-R3, GNP-R4, GNP-R5, GNP-R6, GNP-R7), Add 5mL of 0.2mol/L CTAB aqueous solution respectively, add different amounts of 4mmol/L AgNO3 aqueous solution (0.050, 0.10, 0.15, 0.20, 0.25, 0.30 and 0.40mL) corresponding to the number, and then add 5mL of 1mmol/L gold chloride respectively At this time, the color of the solution turns golden yellow, and then 70 μL of 0.0788 mol/L ascorbic acid is added respectively, and the solution turns colorless. Finally, 12 μL of seed gold nanoparticle dispersion was added, and left to react at 27° C. for 2 h to obtain gold nanorods, which were stored at room temperature and protected from light for later use.
(2)肝素-聚乙烯亚胺纳米凝胶的制备(2) Preparation of heparin-polyethyleneimine nanogel
1)配制MES水溶液100mL(pH 5.5、0.05M)；1) Prepare 100mL of MES aqueous solution (pH 5.5, 0.05M);
2)肝素钠溶液的活化：取0.5g肝素钠溶解于100mL MES(100mL圆底烧瓶)中，加入4.5mg N-羟基琥珀酰亚胺和7.2mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，活化12h，得到活化后的肝素钠溶液；2) Activation of heparin sodium solution: Dissolve 0.5g heparin sodium in 100mL MES (100mL round bottom flask), add 4.5mg N-hydroxysuccinimide and 7.2mg 1-ethyl-(3-dimethylamino Propyl) carbodiimide hydrochloride, activated for 12h, to obtain activated heparin sodium solution;
3)肝素-聚乙烯亚胺纳米凝胶的制备：将0.5g支链聚乙烯亚胺(分子量1800Da)溶于5mL去离子水中后加入步骤(2)的活化后的肝素钠溶液中，并置于恒压漏斗中，室温搅拌24h；过滤并透析(透析袋截留分子量3500Da)，得到肝素-聚乙烯亚胺纳米胶束。3) Preparation of heparin-polyethyleneimine nanogel: 0.5g branched polyethyleneimine (molecular weight 1800Da) was dissolved in 5mL deionized water, and then added to the activated heparin sodium solution in step (2), juxtaposed In a constant pressure funnel, stir at room temperature for 24 hours; filter and dialyze (dialysis bag molecular weight cut-off 3500Da) to obtain heparin-polyethyleneimine nanomicelles.
(3)纳米凝胶的表面硫辛酸修饰：(3) surface lipoic acid modification of nanogel:
将100mg硫辛酸溶解于2mL二甲亚砜中，加入150mg二环己基碳二亚胺和85mg N-羟基琥珀酰亚胺，过夜活化后加入上述得到的肝素-聚乙烯亚胺纳米胶束中，室温反应36小时，过滤并透析，得到肝素-聚乙烯亚胺-硫辛酸纳米凝胶。冻干样品，通过样品称重测定纳米凝胶产物的浓度。结果：能形成稳定的纳米凝胶，沉淀物较少。Dissolve 100 mg of lipoic acid in 2 mL of dimethyl sulfoxide, add 150 mg of dicyclohexylcarbodiimide and 85 mg of N-hydroxysuccinimide, and add it to the heparin-polyethyleneimine nanomicelle obtained above after overnight activation, React at room temperature for 36 hours, filter and dialyze to obtain heparin-polyethyleneimine-lipoic acid nanogel. The samples were lyophilized and the concentration of the nanogel product was determined by weighing the samples. Result: A stable nanogel can be formed with less precipitation.
(4)纳米凝胶表面巯基的释放与金纳米颗粒的吸附(4) Release of sulfhydryl groups on the surface of nanogels and adsorption of gold nanoparticles
往步骤(3)中的肝素-聚乙烯亚胺-硫辛酸纳米凝胶加入0.5mM DTT，对二硫键进行还原，透析1天后加入2mL步骤(1)的金纳米棒，使用TEM检测贵金属纳米颗粒的吸附效果,结果如图4所示；从图4可以看出，制备得到的肝素-聚乙烯亚胺-硫辛酸纳米凝胶对金纳米棒具有良好的吸附效果。Add 0.5mM DTT to the heparin-polyethyleneimine-lipoic acid nanogel in step (3) to reduce the disulfide bond, add 2mL of the gold nanorods in step (1) after 1 day of dialysis, and use TEM to detect the presence of precious metal nanoparticles. The adsorption effect of the particles is shown in Figure 4; it can be seen from Figure 4 that the prepared heparin-polyethyleneimine-lipoic acid nanogel has a good adsorption effect on the gold nanorods.
实施例3Example 3
(1)同实施例1的步骤(1)；(1) with the step (1) of embodiment 1;
(2)制备硫辛酸修饰的纳米凝胶：(2) Preparation of lipoic acid-modified nanogels:
1)配制MES水溶液100mL(pH 5.5、0.05M)；1) Prepare 100mL of MES aqueous solution (pH 5.5, 0.05M);
2)肝素钠溶液的活化：取5g肝素钠溶解于50mL MES(100mL圆底烧瓶)中，加入100mg N-羟基琥珀酰亚胺和150mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，过夜活化8h，得到活化后的肝素钠溶液；2) Activation of heparin sodium solution: Dissolve 5g of heparin sodium in 50mL MES (100mL round bottom flask), add 100mg of N-hydroxysuccinimide and 150mg of 1-ethyl-(3-dimethylaminopropyl) Carbodiimide hydrochloride was activated overnight for 8 hours to obtain activated heparin sodium solution;
3)肝素-聚乙烯亚胺纳米凝胶的制备：将5g支链聚乙烯亚胺(分子量1800Da)溶于50mL去离子水中，并置于恒压漏斗中，加入步骤(2)的活化后的肝素钠溶液中，室温搅拌12h；过滤并透析(透析袋截留分子量3500Da)，得到肝素-聚乙烯亚胺纳米凝胶。3) Preparation of heparin-polyethyleneimine nanogel: Dissolve 5g of branched-chain polyethyleneimine (molecular weight 1800Da) in 50mL of deionized water, place it in a constant pressure funnel, add the activated Stir in heparin sodium solution for 12 hours at room temperature; filter and dialyze (molecular weight cut-off of the dialysis bag is 3500Da) to obtain heparin-polyethyleneimine nanogel.
4)纳米凝胶的表面硫辛酸修饰：4) surface lipoic acid modification of nanogel:
将100mg硫辛酸溶解于10mL二甲亚砜中，加入1g二环己基碳二亚胺和700mg N-羟基琥珀酰亚胺，过夜活化并滤纸过滤后加入上述得到的肝素-聚乙烯亚胺纳米胶束中，室温反应36小时，过滤并透析，得到肝素-聚乙烯亚胺-硫辛酸纳米凝胶。冻干样品，通过样品质量称重测定纳米凝胶产物的浓度。结果：能形成稳定的纳米凝胶，沉淀物较少。Dissolve 100mg of lipoic acid in 10mL of dimethyl sulfoxide, add 1g of dicyclohexylcarbodiimide and 700mg of N-hydroxysuccinimide, activate overnight and filter with filter paper, then add the heparin-polyethyleneimine nanogel obtained above In the bundle, react at room temperature for 36 hours, filter and dialyze to obtain heparin-polyethyleneimine-lipoic acid nanogel. The samples were lyophilized and the concentration of the nanogel product was determined by weighing the sample mass. Result: A stable nanogel can be formed with less precipitation.
(3)纳米凝胶表面巯基的释放与金纳米颗粒的吸附(3) The release of sulfhydryl groups on the surface of the nanogel and the adsorption of gold nanoparticles
往步骤(2)中的肝素-聚乙烯亚胺-硫辛酸纳米凝胶加入0.5mM硼氢化钠，对二硫键进行还原，随后对纳米凝胶溶液透析出去硼氢化钠并加入2mL步骤(1)的金纳米棒，25℃、氮气保护下搅拌12小时，取澄清溶液进行TEM扫描，结果如图5所示；从图5可以看出，制备得到的肝素-聚乙烯亚胺-硫辛酸纳米凝胶对金纳米棒具有良好的吸附效果。Add 0.5mM sodium borohydride to the heparin-polyethyleneimine-lipoic acid nanogel in step (2) to reduce the disulfide bond, then dialyze the nanogel solution to remove sodium borohydride and add 2mL of step (1 ) gold nanorods, stirred for 12 hours at 25°C under the protection of nitrogen, took the clear solution for TEM scanning, the results are shown in Figure 5; as can be seen from Figure 5, the prepared heparin-polyethyleneimine-lipoic acid nanorods The gel has a good adsorption effect on gold nanorods.
对比实施例comparative example
(1)金纳米棒的合成(1) Synthesis of gold nanorods
种子金纳米颗粒的合成：将5mL 0.2mol/L的十六烷基三甲基溴化铵(CTAB)与5mL 0.5mmol/L的氯金酸混合，400转/分钟边搅拌边加入0.6mL0℃保存的0.01mol/L的硼氢化钠，600转/分钟搅拌5min，室温静置2h，得到种子金纳米颗粒，命名为GNP-R0。Synthesis of seed gold nanoparticles: Mix 5mL 0.2mol/L cetyltrimethylammonium bromide (CTAB) with 5mL 0.5mmol/L chloroauric acid, add 0.6mL 0°C while stirring at 400 rpm The preserved 0.01 mol/L sodium borohydride was stirred at 600 rpm for 5 min, and allowed to stand at room temperature for 2 h to obtain seed gold nanoparticles, which were named GNP-R0.
CTAB模板法合成短的金纳米棒：取7个25mL的样品瓶(分别标记为GNP-R1、GNP-R2、GNP-R3、GNP-R4、GNP-R5、GNP-R6、GNP-R7)，分别加入5mL 0.2mol/L的CTAB水溶液，对应编号加入不同量4mmol/L的AgNO3水溶液(0.050、0.10、0.15、0.20、0.25、0.30和0.40mL)，再分别加入5mL 1mmol/L的氯金酸水溶液，此时溶液颜色变成金黄色，接着分别加入70μL 0.0788mol/L的抗坏血酸，溶液变成无色。最后分别加入12μL的种子金纳米颗粒分散液，在27℃静置反应2h，得到金纳米棒，室温、避光保存，备用。Synthesis of short gold nanorods by CTAB template method: take seven 25mL sample vials (marked as GNP-R1, GNP-R2, GNP-R3, GNP-R4, GNP-R5, GNP-R6, GNP-R7), Add 5mL of 0.2mol/L CTAB aqueous solution respectively, add different amounts of 4mmol/L AgNO3 aqueous solution (0.050, 0.10, 0.15, 0.20, 0.25, 0.30 and 0.40mL) corresponding to the number, and then add 5mL of 1mmol/L gold chloride respectively At this time, the color of the solution turns golden yellow, and then 70 μL of 0.0788 mol/L ascorbic acid is added respectively, and the solution turns colorless. Finally, 12 μL of seed gold nanoparticle dispersion was added, and left to react at 27° C. for 2 h to obtain gold nanorods, which were stored at room temperature and protected from light for later use.
(2)肝素-聚乙烯亚胺纳米凝胶的制备(2) Preparation of heparin-polyethyleneimine nanogel
1)配制MES水溶液100mL(pH 5.5、o.5mM)；1) Prepare 100mL of MES aqueous solution (pH 5.5, o.5mM);
2)肝素钠溶液的活化：取0.5g肝素钠溶解于50mL MES(100mL圆底烧瓶)中，加入300mg N-羟基琥珀酰亚胺和200mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐，活化2h，得到活化后的肝素钠溶液；2) Activation of heparin sodium solution: Dissolve 0.5g heparin sodium in 50mL MES (100mL round bottom flask), add 300mg N-hydroxysuccinimide and 200mg 1-ethyl-(3-dimethylaminopropyl ) carbodiimide hydrochloride, activated for 2h, to obtain activated heparin sodium solution;
3)肝素-聚乙烯亚胺纳米凝胶的制备：将2g支链聚乙烯亚胺(分子量1800Da)溶于100mL去离子水中后加入步骤(2)的活化后的肝素钠溶液中，并置于恒压漏斗中，室温搅拌24h；过滤并透析(透析袋截留分子量3500Da)，得到肝素-聚乙烯亚胺纳米胶束；3) Preparation of heparin-polyethyleneimine nanogel: dissolve 2g of branched-chain polyethyleneimine (molecular weight 1800Da) in 100mL of deionized water, add to the activated heparin sodium solution in step (2), and place In a constant pressure funnel, stir at room temperature for 24 hours; filter and dialyze (dialysis bag molecular weight cut-off 3500Da) to obtain heparin-polyethyleneimine nanomicelles;
(3)纳米凝胶的表面硫辛酸修饰：(3) surface lipoic acid modification of nanogel:
将100mg硫辛酸溶解于二甲亚砜中，加入200mg二环己基碳二亚胺和220mg N-羟基琥珀酰亚胺，过夜活化后加入上述得到的肝素-聚乙烯亚胺纳米胶束中，室温反应36小时，过滤并透析，得到肝素-聚乙烯亚胺-硫辛酸纳米凝胶。冻干样品，通过样品称重测定纳米凝胶产物的浓度。结果：能形成稳定的纳米凝胶，沉淀物较少。Dissolve 100mg of lipoic acid in dimethyl sulfoxide, add 200mg of dicyclohexylcarbodiimide and 220mg of N-hydroxysuccinimide, activate overnight and add to the heparin-polyethyleneimine nanomicelles obtained above, at room temperature React for 36 hours, filter and dialyze to obtain heparin-polyethyleneimine-lipoic acid nanogel. The samples were lyophilized and the concentration of the nanogel product was determined by weighing the samples. Result: A stable nanogel can be formed with less precipitation.
(4)纳米凝胶表面巯基的释放与金纳米颗粒的吸附(4) Release of sulfhydryl groups on the surface of nanogels and adsorption of gold nanoparticles
往步骤(3)中的肝素-聚乙烯亚胺-硫辛酸纳米凝胶加入2mM硼氢化钠，对二硫键进行还原，透析1天后加入2mL步骤(1)的金纳米棒，使用TEM检测贵金属纳米颗粒的吸附效果,无明显金棒附着。Add 2mM sodium borohydride to the heparin-polyethyleneimine-lipoic acid nanogel in step (3) to reduce the disulfide bonds, add 2mL of gold nanorods from step (1) after 1 day of dialysis, and use TEM to detect precious metals The adsorption effect of nanoparticles, no obvious gold rod attachment.
以上所述本发明的具体实施方式，并不构成对本发明保护范围的限定。任何根据本发明的技术构思所作出的各种其他相应的改变与变形，均应包含在本发明权利要求的保护范围内。The specific embodiments of the present invention described above do not constitute a limitation to the protection scope of the present invention. Any other corresponding changes and modifications made according to the technical concept of the present invention shall be included in the protection scope of the claims of the present invention.
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