技术领域Technical field
本发明属于材料领域，涉及一种大分子造影剂及其制备方法和应用。The invention belongs to the field of materials and relates to a macromolecular contrast agent and its preparation method and application.
背景技术Background technique
2020年，全世界约有1930万新发癌症病例，预计到2040年将有2840万例新发癌症患者，比2020年增长47％。因此，癌症仍是全球的重大公共卫生问题，严重威胁人类健康，并且发病率和死亡率不断上升。早期诊断和治疗将是降低癌症病发率和死亡率的有效措施。MRI技术由于具有无电离辐射以及较高的空间分辨率和对比度等诸多突出优点，已成为当今临床诊断中最有力的检测手段之一。然而，尽管MRI的空间分辨率很高，但是单纯使用MRI成像的灵敏度却不高。某些不同组织或肿瘤部位的弛豫时间相互重叠使MRI发现早期微小肿瘤面临挑战。因而需要使用造影剂辅助成像，以增强信号对比度和提高组织图像的分辨率。目前应用于临床上的造影剂存在弛豫低、在血液中的循环时间和在组织中的驻留时间较短、没有靶向性等缺点，无法满足肿瘤早期诊断的需求。In 2020, there were approximately 19.3 million new cancer cases worldwide, and it is expected that there will be 28.4 million new cancer patients by 2040, an increase of 47% from 2020. Therefore, cancer remains a major global public health problem that seriously threatens human health, with increasing morbidity and mortality. Early diagnosis and treatment will be effective measures to reduce the incidence and mortality of cancer. MRI technology has become one of the most powerful detection methods in today's clinical diagnosis due to its many outstanding advantages such as no ionizing radiation and high spatial resolution and contrast. However, despite the high spatial resolution of MRI, the sensitivity of MRI imaging alone is not high. The overlapping relaxation times of some different tissues or tumor sites make it challenging for MRI to detect early-stage micro tumors. Therefore, it is necessary to use contrast agents to assist imaging to enhance signal contrast and improve the resolution of tissue images. The contrast agents currently used in clinical applications have shortcomings such as low relaxation, short circulation time in the blood and residence time in tissues, and lack of targeting properties, which cannot meet the needs of early diagnosis of tumors.
β-环糊精(β-CD)是一种α-D型吡喃葡萄糖单元通过α-1，4糖苷键首尾相连而成的环状糖，CD具有疏水的空腔和亲水的表面，可以作为主体分子与无机、有机和生物等客体分子相结合形成饱合物。环糊精在高分子聚合物、大分子自组装、生物医学等领域有着广泛的应用。大分子自组装是指聚合物在输水作用、范德华力、氢键等非共价作用力的推动下，自发地形成特殊结构或形貌的聚集体的过程。并且由于环糊精具有较低的毒性以及良好的生物相容性，在此方面得到了充分的利用。支化聚乙烯亚胺(PEI)是一种树状大分子，其具有良好的水溶性，分子量分布范围窄，单个分子即具有纳米尺寸，具有多价位效应，并且易于通过修饰表面基团改变其理化性质。可以应用于大分子造影剂的制备。大分子造影剂不仅可以将靶向基团和小分子造影剂同时连接起来，增强特定细胞的吞噬能力，从而提高造影的特异性，另一方面还能够连接多种小分子造影剂，能够进行多种成像，为临床诊断和治疗能够提供更加丰富而准确的信息。β-Cyclodextrin (β-CD) is a cyclic sugar in which α-D-glucopyranose units are connected end-to-end through α-1,4 glycosidic bonds. CD has a hydrophobic cavity and a hydrophilic surface. It can be used as a host molecule to combine with inorganic, organic and biological guest molecules to form a saturate. Cyclodextrins are widely used in the fields of polymers, macromolecule self-assembly, biomedicine and other fields. Macromolecule self-assembly refers to the process in which polymers spontaneously form aggregates with special structures or morphologies driven by non-covalent forces such as water transport, van der Waals forces, and hydrogen bonds. And because cyclodextrin has low toxicity and good biocompatibility, it has been fully utilized in this aspect. Branched polyethyleneimine (PEI) is a dendritic macromolecule with good water solubility and a narrow molecular weight distribution range. A single molecule has a nanometer size, has a multivalent effect, and can be easily changed by modifying surface groups. Physical and chemical properties. Can be used in the preparation of macromolecular contrast agents. Macromolecular contrast agents can not only connect targeting groups and small molecule contrast agents at the same time to enhance the phagocytosis ability of specific cells, thereby improving the specificity of contrast, but can also connect a variety of small molecule contrast agents, enabling multiple This kind of imaging can provide richer and more accurate information for clinical diagnosis and treatment.
发明内容Contents of the invention
根据以上现有技术的不足，本发明提供一种大分子造影剂及其制备方法和应用，将环糊精构建于高分子主体中，而功能基团构建于侧链上，通过主客体自组装可以简易制备造影剂，使其应用于肿瘤成像技术中。Based on the above deficiencies in the prior art, the present invention provides a macromolecular contrast agent and its preparation method and application. The cyclodextrin is constructed into the polymer body, and the functional groups are constructed on the side chains, through host-guest self-assembly. Contrast agents can be easily prepared and used in tumor imaging technology.
本发明所述的一种大分子造影剂的制备方法，按照以下步骤进行：The preparation method of a macromolecular contrast agent according to the present invention is carried out according to the following steps:
(1)β-环糊精经CDI活化后与支化PEI反应，得到PEI-CD聚合物；(1) β-cyclodextrin is activated by CDI and reacts with branched PEI to obtain PEI-CD polymer;
(2)用N3-PEGn-NH2末端胺基引发赖氨酸NCA的开环聚合，并用金刚烷酰氯封端；(2) Use N3 -PEGn-NH2 terminal amine group to initiate ring-opening polymerization of lysine NCA, and end-capped with adamantane acid chloride;
(3)脱去苄基保护，再将DTPA-Gd修饰到聚赖氨酸上，制备出具有MRI成像能力的侧链N3-PEGn-PLL(DTPA-Gd)-AD；(3) Remove the benzyl protection, and then modify DTPA-Gd onto polylysine to prepare side chain N3 -PEGn-PLL(DTPA-Gd)-AD with MRI imaging capabilities;
(4)通过环糊精与金刚烷间的主客体相互作用，将PEI-CD与N3-PEGn-PLL(DTPA-Gd)-AD进行主客体作用组装，构建大分子造影剂；(4) Through the host-guest interaction between cyclodextrin and adamantane, PEI-CD and N3 -PEGn -PLL(DTPA-Gd)-AD are assembled by host-guest interaction to construct a macromolecular contrast agent;
(5)将靶向基团通过DBCO与N3的点击化学反应修饰到所合成的聚合物上。(5) Modify the targeting group to the synthesized polymer through the click chemical reaction of DBCO andN3 .
其中，作为优选方案，步骤(1)中β-环糊精与PEI的摩尔比为1：1～6。Among them, as a preferred embodiment, the molar ratio of β-cyclodextrin and PEI in step (1) is 1:1-6.
作为优选方案，步骤(2)中N3-PEGn-NH2中PEG分子量取值范围为n＝400～20000。As a preferred version, the molecular weight range of PEG in N3 -PEGn -NH2 in step (2) is n=400~20000.
作为优选方案，步骤(2)中N3-PEGn-NH2末端胺基与赖氨酸NCA的摩尔比为1：20～70。As a preferred embodiment, the molar ratio of N3 -PEGn -NH2 terminal amino group to lysine NCA in step (2) is 1:20-70.
作为优选方案，步骤(4)中PEI-CD与N3-PEGn-PLL(DTPA-Gd)-AD的质量比为1：10～20。As a preferred solution, the mass ratio of PEI-CD and N3 -PEGn -PLL(DTPA-Gd)-AD in step (4) is 1:10-20.
作为优选方案，步骤(5)中靶向基团为叶酸。As a preferred embodiment, the targeting group in step (5) is folic acid.
本发明还提供了一种大分子造影剂，按照上述的方法制备而成。The invention also provides a macromolecular contrast agent prepared according to the above method.
本发明还提供了大分子造影剂在肿瘤成像中的应用。The present invention also provides the application of macromolecular contrast agents in tumor imaging.
本发明的优点在于：The advantages of the present invention are:
(1)结构的可调性。主体结构PEI-CD中可调节PEI与CD比例来调控CD的含量，从而调整可与AD反应的位点数量。侧链结构PEG与PLL的比例可控性，从而调整钆的密度及整体结构的尺寸。(1) Adjustability of structure. In the main structure PEI-CD, the ratio of PEI to CD can be adjusted to control the content of CD, thereby adjusting the number of sites that can react with AD. The ratio of the side chain structure PEG and PLL is controllable, thereby adjusting the density of gadolinium and the size of the overall structure.
(2)靶向基团修饰的便捷性。生物正交反应绿色环保，反应速度快。本发明设计利用生物正交反应将靶向基团修饰于聚合物上，反应简单。(2) Convenience of targeting group modification. Bioorthogonal reaction is green, environmentally friendly and has fast reaction speed. The present invention is designed to use bioorthogonal reactions to modify targeting groups on polymers, and the reaction is simple.
附图说明Description of drawings
图1为本发明的反应路线图。Figure 1 is a reaction route diagram of the present invention.
图2为实施例6检测的PEI800-CyD的核磁氢谱图。Figure 2 is the hydrogen nuclear magnetic spectrum of PEI800 -CyD detected in Example 6.
图3为实施例6检测的N3-PEG2000-Lys(Z)-AD的核磁氢谱图。Figure 3 is the hydrogen nuclear magnetic spectrum of N3 -PEG2000 -Lys(Z)-AD detected in Example 6.
图4为实施例6检测的N3-PEG2000-PLL-AD及N3-PEG2000-PLL(DTPA)-AD的核磁氢谱图；其中A为N3-PEG2000-PLL-AD，B为N3-PEG2000-PLL(DTPA)-AD。Figure 4 is the hydrogen nuclear magnetic spectrum of N3 -PEG2000 -PLL-AD and N3 -PEG2000 -PLL(DTPA)-AD detected in Example 6; where A is N3 -PEG2000 -PLL-AD, and B is N3 -PEG2000 -PLL(DTPA)-AD.
图5为实施例7检测的PC/AD-PEG2000-PLL(DTPA-Gd)-N3弛豫率。Figure 5 shows the relaxation rate of PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 detected in Example 7.
图6为实施例7检测的PC/AD-PEG2000-PLL(DTPA-Gd)-N3在不同钆浓度下的溶液成像图。Figure 6 is a solution imaging diagram of PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 detected in Example 7 under different gadolinium concentrations.
图7为实施例8检测的大分子造影剂的细胞毒性结果分析图。Figure 7 is an analysis chart of the cytotoxicity results of the macromolecular contrast agents tested in Example 8.
图8为实施例9检测的大分子造影剂的组织毒性发结果分析图。Figure 8 is an analysis chart of the tissue toxicity results of the macromolecular contrast agent tested in Example 9.
图9为实施例10检测的大分子造影剂在裸鼠体内的体内磁共振成像图Figure 9 is an in vivo magnetic resonance imaging image of the macromolecular contrast agent detected in Example 10 in nude mice.
图10为实施例10检测的大分子造影剂在裸鼠体内的MRI量化分析图。Figure 10 is an MRI quantitative analysis chart of the macromolecular contrast agent detected in Example 10 in nude mice.
图11为实施例11检测的大分子造影剂在裸鼠体内的组织残留图。Figure 11 is a diagram of tissue residues of the macromolecular contrast agent detected in Example 11 in nude mice.
具体实施方式Detailed ways
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
实施例1：Example 1:
PEI800-CyD的制备：Preparation of PEI800 -CyD:
(1)重结晶β-CD：将β-CD置于茄形瓶中，加入超纯水，油浴加热至110℃直到β-CD完全溶解。自然冷却使其完全析出，抽滤得到样品，重复操作三次，得到重结晶β-CD粗产品，60℃真空干燥，干燥后得到产物。(1) Recrystallize β-CD: Place β-CD in an eggplant-shaped bottle, add ultrapure water, and heat in an oil bath to 110°C until β-CD is completely dissolved. Allow natural cooling to completely precipitate, obtain a sample by suction filtration, repeat the operation three times to obtain a crude recrystallized β-CD product, and vacuum-dry at 60°C to obtain the product after drying.
(2)活化β-CD：将重结晶的β-CD(2.1g，1.85mmol)和CDI(2.4g，14.8mmol)溶于10mL超干DMF中，通入N2保护。室温下反应1h后，用冷乙醚沉淀，将沉淀后的固体再用乙醚洗涤沉淀3次，过滤沉淀溶于超干DMSO中。(2) Activation of β-CD: Dissolve recrystallized β-CD (2.1g, 1.85mmol) and CDI (2.4g, 14.8mmol) in 10 mL of ultra-dry DMF, and pass in N2 for protection. After reacting for 1 hour at room temperature, precipitate with cold ether. Wash the precipitated solid with ether three times. Filter the precipitate and dissolve it in ultra-dry DMSO.
(3)PEI800-CyD的合成：取PEI800(2.9g，3.63mmol)溶于超干DMSO中，加入三乙胺(4.14mL，29.6mmol)，再将(2)中活化后的环糊精DMSO溶液置于平衡漏斗中，缓慢滴加至上述溶液中，滴加时间至少不低于1.5h。反应12h后，用Mw＝14000的透析袋透析3天后过滤，冻干，产物在-20℃保存。(3) Synthesis of PEI800 -CyD: Dissolve PEI800 (2.9g, 3.63mmol) in ultra-dry DMSO, add triethylamine (4.14mL, 29.6mmol), and then add the activated cyclopaste in (2) Place the refined DMSO solution in a balance funnel and slowly add it dropwise to the above solution. The dropping time should be at least 1.5 hours. After reacting for 12 hours, dialyze with a dialysis bag with Mw=14000 for 3 days, filter, and freeze-dry. The product was stored at -20°C.
实施例2：Example 2:
赖氨酸NCA的合成：Synthesis of Lysine NCA:
在250mL三颈烧瓶中，将H-Lys(Z)-OH(10g，1.36mol)分散于80mL干燥的THF中，在加热至50℃。将三光气(4.8g，16.17mmol)溶于20mL干燥的THF后，置于恒压漏斗逐滴滴入上述反应溶液中。反应一段时间后，溶液逐渐凝固，磁子无法搅动。随着三光气的不断加入，固体渐渐溶解直至形成澄清溶液。然后，通入N2半小时，并用NaOH溶液进行尾气处理。最后，浓缩溶液并用石油醚沉淀，收集沉淀，用THF溶解，再沉淀，重复操作3次，过滤得到白色固体粉末。In a 250 mL three-neck flask, H-Lys(Z)-OH (10 g, 1.36 mol) was dispersed in 80 mL of dry THF and heated to 50°C. Dissolve triphosgene (4.8 g, 16.17 mmol) in 20 mL of dry THF, then place it in a constant pressure funnel and add it drop by drop into the above reaction solution. After reacting for a period of time, the solution gradually solidified and the magnet could not stir. With the continuous addition of triphosgene, the solid gradually dissolves until a clear solution is formed. Then,N2 was passed for half an hour, and the tail gas was treated with NaOH solution. Finally, the solution was concentrated and precipitated with petroleum ether. The precipitate was collected, dissolved with THF, and precipitated again. Repeat the operation three times, and filter to obtain a white solid powder.
实施例3：Example 3:
N3-PEG2000-PLL-AD的合成：Synthesis of N3 -PEG2000 -PLL-AD:
(1)合成N3-PEG2000-Lys-PLL(Z)：N3-PEG2000-NH2,(500mg，0.25mmol)接入油泵抽真空，通过甲苯与水共沸除去原料中的水分。随后加入10mL超干DMF以完全溶解，将制备好的赖氨酸NCA(3.03g，9.8mmol)加入到溶液中，在油浴40℃和N2保护下反应48h。(1) Synthesis of N3 -PEG2000 -Lys-PLL (Z): N3 -PEG2000 -NH2 , (500 mg, 0.25 mmol) is connected to an oil pump for vacuuming, and the moisture in the raw material is removed by azeotroping toluene and water. Then 10 mL of ultra-dry DMF was added to completely dissolve, and the prepared lysine NCA (3.03 g, 9.8 mmol) was added to the solution and reacted for 48 h in an oil bath at 40°C andN2 protection.
(2)合成N3-PEG2000-Lys(Z)-AD：金刚烷酰氯(0.5g，2.5mmol)添加到上述反应中，继续在N2保护40℃加热下反应24h。反应完成后，将反应液用乙醚沉淀，离心得到的沉淀再用乙醚洗3次。通过真空干燥得到淡黄色产物。(2) Synthesis of N3 -PEG2000 -Lys(Z)-AD: Add adamantane acyl chloride (0.5 g, 2.5 mmol) to the above reaction, and continue the reaction under N2 protection and heating at 40°C for 24 hours. After the reaction is completed, the reaction solution is precipitated with diethyl ether, and the precipitate obtained by centrifugation is washed three times with diethyl ether. A pale yellow product was obtained by vacuum drying.
(3)脱保护合成N3-PEG2000-PLL-AD：N3-PEG2000-Lys(Z)-AD溶于20mL TFA中，加入5mL 33wt.％的溴化氢。充N2气球保护，在室温条件下反应6h使产物完全脱除保护基团。反应液经乙醚沉淀并洗涤3次，用水溶解，透析(MW＝3500)3天，过滤冻干后得到淡黄色产物。(3) Deprotection synthesis of N3 -PEG2000 -PLL-AD: N3 -PEG2000 -Lys(Z)-AD was dissolved in 20 mL of TFA, and 5 mL of 33 wt.% hydrogen bromide was added. Fill withN2 balloon for protection, and react at room temperature for 6 hours to completely remove the protective group of the product. The reaction solution was precipitated with diethyl ether and washed three times, dissolved in water, dialyzed (MW=3500) for 3 days, filtered and freeze-dried to obtain a light yellow product.
实施例4：Example 4:
N3-PEG2000-PLL(DTPA-Gd)-AD的合成：Synthesis of N3 -PEG2000 -PLL(DTPA-Gd)-AD:
(1)合成DTPA-NHS(活化DTPA)：将二乙烯三胺五乙酸(12g，30.5mmol)溶于220mL乙腈中，加入17mL三乙胺，在N2保护下，油浴加热至50℃，反应30min。将整个反应体系冰浴10min后，将N-羟基琥珀亚酰胺(2.108g，18.3mmol)溶于50mL乙腈中，N,N-二环己基碳酰亚胺(3.781g，18.3mmol)溶于80mL乙腈中，分别置于恒压漏斗中，逐滴加入上述反应体系中，滴加完成后在室温下反应8h。反应完成后，除去溶液中的DCU，得到淡黄色粘稠固体，用20mL干燥DMSO溶解，置于-20℃保存。(1) Synthesis of DTPA-NHS (activated DTPA): Dissolve diethylenetriaminepentacetic acid (12g, 30.5mmol) in 220mL acetonitrile, add 17mL triethylamine, and heat the oil bath to 50°C underN2 protection. React for 30 minutes. After the entire reaction system was kept on ice for 10 minutes, N-hydroxysuccinimide (2.108g, 18.3mmol) was dissolved in 50mL acetonitrile, and N,N-dicyclohexylcarbonimide (3.781g, 18.3mmol) was dissolved in 80mL acetonitrile, placed in constant pressure funnels, and added dropwise to the above reaction system. After the dropwise addition was completed, react at room temperature for 8 hours. After the reaction is completed, remove DCU from the solution to obtain a light yellow viscous solid, which is dissolved in 20 mL of dry DMSO and stored at -20°C.
(2)合成N3-PEG2000-PLL(DTPA)-AD：将N3-PEG2000-PLL-AD(0.3g，0.045mmol)溶解于20mL干燥DMSO中，加入0.68mL三乙胺，加入5.35mL DTPA-NHS的DMSO溶液，反应12h后。再加入0.68mL三乙胺和5.35mL DTPA-NHS，反应12h后，再次加入0.68mL三乙胺和5.35mL DTPA-NHS，继续反应12h。反应结束后，将溶液用Mw＝3500的透析袋透析三天，过滤冻干，得到淡黄色固体。(2) Synthesis of N3 -PEG2000 -PLL(DTPA)-AD: Dissolve N3 -PEG2000 -PLL-AD (0.3g, 0.045mmol) in 20mL of dry DMSO, add 0.68mL of triethylamine, and add 5.35 mL of DMSO solution of DTPA-NHS, after 12 hours of reaction. Then add 0.68mL triethylamine and 5.35mL DTPA-NHS. After reacting for 12h, add 0.68mL triethylamine and 5.35mL DTPA-NHS again and continue the reaction for 12h. After the reaction, the solution was dialyzed with a dialysis bag of Mw=3500 for three days, filtered and freeze-dried to obtain a light yellow solid.
(3)合成N3-PEG2000-PLL(DTPA-Gd)-AD：将N3-PEG2000-PLL(DTPA)-AD(1.4g，0.15mmol)溶解于20mL水中，GdCl3·6H2O溶解于10mL水并加入至上述溶液中，用1M NaOH调节溶液的pH值为4-5后，油浴加热到40℃反应过夜。反应结束后，将溶液用Mw＝3500的透析袋在含有EDTA的水溶液中透析三天，除去未反应的Gd3+，再在水溶液中透析三天后，冻干得到淡黄色产物。(3) Synthesis of N3 -PEG2000 -PLL(DTPA-Gd)-AD: Dissolve N3 -PEG2000 -PLL(DTPA)-AD (1.4g, 0.15mmol) in 20mL water, GdCl3 ·6H2 O Dissolve in 10 mL of water and add to the above solution. After adjusting the pH value of the solution to 4-5 with 1M NaOH, heat the oil bath to 40°C for overnight reaction. After the reaction, the solution was dialyzed in an aqueous solution containing EDTA for three days using a dialysis bag with Mw=3500 to remove unreacted Gd3+ . After another three days of dialysis in an aqueous solution, the solution was freeze-dried to obtain a light yellow product.
实施例5：Example 5:
PC/AD-PEG2000-PLL(DTPA-Gd)-N3的合成：Synthesis of PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 :
(1)DBCO-FA的合成：将叶酸(5.6mg，0.0127mmol)溶解于5mL干燥DMF中，加入0.007mL DIPEA和PyBOP(10.29mg，0.019mmol)，将DBCO-PEG2000-amine(25.4mg，0.0127mmol)溶解于2mLDMF中并加入到上述溶液中，反应过夜。反应结束后，用乙醚沉淀3次，完全除去乙醚后，用100μL水溶解。(1) Synthesis of DBCO-FA: Dissolve folic acid (5.6mg, 0.0127mmol) in 5mL dry DMF, add 0.007mL DIPEA and PyBOP (10.29mg, 0.019mmol), add DBCO-PEG2000- amine (25.4mg, 0.0127 mmol) was dissolved in 2 mL DMF and added to the above solution, and the reaction was carried out overnight. After the reaction, precipitate with diethyl ether three times. After completely removing the diethyl ether, dissolve it in 100 μL of water.
(2)PC/AD-PEG2000-PLL(DTPA-Gd)-N3：将PC(5mg)溶解于10mL水中，N3-PEG2000-PLL(DTPA-Gd)-AD(60mg)溶解于10mL水并逐滴加入上述溶液中，反应12h后，加入DBCO-FA(1mg)，继续搅拌过夜。最后，用Mw＝50000的超滤离心管离心洗涤溶液，除去未反应的原料，浓缩后的到微黄色溶液。(2) PC/AD-PEG2000- PLL(DTPA-Gd)-N3 : Dissolve PC (5 mg) in 10 mL of water, and N3 -PEG2000 -PLL(DTPA-Gd)-AD (60 mg) in 10 mL Water was added dropwise to the above solution. After reacting for 12 hours, DBCO-FA (1 mg) was added and stirring was continued overnight. Finally, use an ultrafiltration centrifuge tube with Mw = 50,000 to centrifuge the washed solution to remove unreacted raw materials and concentrate to obtain a slightly yellow solution.
实施例6：Example 6:
核磁共振波谱检测：Nuclear magnetic resonance spectroscopy detection:
利用核磁共振波谱仪(400M)测试PEI800-CyD、N3-PEG2000-Lys(Z)-AD、N3-PEG2000-PLL-AD和N3-PEG2000-PLL(DTPA)-AD的氢谱图，以相适应的氘代试剂作为溶剂，TMS为内标。Using nuclear magnetic resonance spectrometer (400M) to test PEI800 -CyD, N3 -PEG2000 -Lys(Z)-AD, N3 -PEG2000 -PLL-AD and N3 -PEG2000 -PLL(DTPA)-AD Hydrogen spectrum, using a suitable deuterated reagent as the solvent and TMS as the internal standard.
PEI800-CyD的核磁结果如图2所示。在图中出现δ4.92(d，1H)，4.03～2.84是CyD的特征峰，δ2.14～2.81是PEI的特征峰。通过比较CyD和PEI的峰面积比至，计算出β-CD和PEI的比值为1:2.3。The NMR results of PEI800-CyD are shown in Figure 2. In the figure, δ4.92 (d, 1H) appears, 4.03~2.84 is the characteristic peak of CyD, and δ2.14~2.81 is the characteristic peak of PEI. By comparing the peak area ratio of CyD and PEI, the ratio of β-CD to PEI was calculated to be 1:2.3.
N3-PEG2000-Lys(Z)-AD的核磁结果如图3所示，通过比较ArCH2O中CH2(δ4.96)与PEG中OCH2CH2(δ3.5)的积分面积比，得到平均聚合度为29。The NMR results of N3 -PEG2000 -Lys(Z)-AD are shown in Figure 3. By comparing the integrated area ratio of CH2 (δ4.96) in ArCH2 O and OCH2 CH2 (δ3.5) in PEG , the average degree of polymerization is 29.
N3-PEG2000-PLL-AD的核磁结果如图4所示，脱保护后，δ7.43～6.89苯环特征峰消失，说明苄基完全被脱除。N3-PEG2000-PLL(DTPA)-AD的核磁结果如图3所示。在δ3.86ppm和δ3.58～3.00ppm出现新的峰属于DTPA亚甲基上的氢，表明DTPA已成功连接，并计算出接枝率为96％。The NMR results of N3- PEG2000 -PLL-AD are shown in Figure 4. After deprotection, the characteristic peak of the benzene ring at δ7.43~6.89 disappeared, indicating that the benzyl group was completely removed. The NMR results of N3 -PEG2000 -PLL(DTPA)-AD are shown in Figure 3. New peaks appearing at δ3.86ppm and δ3.58~3.00ppm belong to the hydrogen on the methylene group of DTPA, indicating that DTPA has been successfully connected, and the grafting rate is calculated to be 96%.
实施例7：Example 7:
弛豫率测定及T1加权溶液成像：Relaxation rate measurement and T1- weighted solution imaging:
将PC/AD-PEG2000-PLL(DTPA-Gd)-N3溶液配置成不同钆离子浓度梯度的(0.25，0.5，1，1.5，2mM)，各取100μL用脉冲核磁共振成像仪(0.5T)测试不同钆离子浓度的弛豫率。样品的弛豫率r1是通过以1/T1为纵坐标，以钆离子浓度为横坐标进行线性拟合得出。另外配制不同浓度的样品溶液进行T1加权成像，T1加权成像是利用自旋回波序列(CPMG)测量材料的横向弛豫时间，得到原始数据的恢复时间(t)及相应的幅度值M(t)。T1加权成像采用自旋回波序列(TE＝8.6ms，TR＝100ms)，测试温度保持在35℃。The PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 solution was configured into different gadolinium ion concentration gradients (0.25, 0.5, 1, 1.5, 2mM), and 100 μL of each was used with a pulsed nuclear magnetic resonance imaging instrument (0.5T ) Test the relaxation rate of different gadolinium ion concentrations. The relaxation rate r1 of the sample is obtained by linear fitting with 1/T1 as the ordinate and the gadolinium ion concentration as the abscissa. In addition, sample solutions of different concentrations are prepared for T1- weighted imaging. T1- weighted imaging uses a spin echo sequence (CPMG) to measure the transverse relaxation time of the material to obtain the recovery time (t) of the original data and the corresponding amplitude value M( t) . T1-weighted imaging uses spin echo sequence (TE=8.6ms, TR=100ms), and the test temperature is maintained at 35°C.
利用0.5T脉冲核磁共振成像仪，通过反转回波法测量PC/AD-PEG2000-PLL(DTPA-Gd)-N3的弛豫时间T1，以1/T1作为纵坐标，钆离子浓度作为横坐标进行线性拟合所得到的的斜率几位弛豫率r1。临床用的Gd-DTPA的弛豫率为4.25mM-1·S-1，所制备的造影剂的弛豫率如图5所示为10.71mM-1·S-1，相比较优于临床应用的造影剂，是其2.58倍。T1加权溶液成像更能够直观地显示出所制备的大分子造影剂的成像效果。如图6所示，所制备的造影剂的图像亮度随着钆离子浓度的升高而增加。Using a 0.5T pulsed nuclear magnetic resonance imaging instrument, the relaxation time T1 of PC/AD-PEG2000- PLL(DTPA-Gd)-N3 was measured by the inversion echo method, with 1/T1 as the ordinate, and the gadolinium ion concentration as The abscissa is the slope of the relaxation rate r1 obtained by linear fitting. The relaxation rate of Gd-DTPA for clinical use is 4.25mM-1 ·S-1 . The relaxation rate of the prepared contrast agent is 10.71mM-1 ·S-1 as shown in Figure 5, which is better than clinical application. contrast agent, which is 2.58 times. T1-weighted solution imaging can more intuitively display the imaging effect of the prepared macromolecular contrast agent. As shown in Figure 6, the image brightness of the prepared contrast agent increases with the increase in gadolinium ion concentration.
实施例8：Example 8:
细胞毒性考察：Cytotoxicity investigation:
用WST法来测定所制备的造影剂对4T1细胞的毒性。首先，将4T1细胞以每个孔4000个细胞密度接种到96孔板中，每个孔板中加入100μL新鲜的DMEM培养基，在培养箱中放置培育24h。将造影剂PC/AD-PEG2000-PLL(DTPA-Gd)-N3水溶液稀释成不同钆离子浓度(6、3、1.5、0.75mM)。将不同Gd离子浓度的材料加入到相应的孔中，每个孔加入100μL，对照组加入100μL的培养基，继续培养24h。最后用移液枪将培养基全部吸出，在每个孔中加入100μL新鲜培养基和10μL CCK8，再置于培养箱中培养2h，用酶标仪测定450nm处的吸光度。每个钆浓度和对照组均做4个平行样。The WST method was used to determine the toxicity of the prepared contrast agent to 4T1 cells. First, 4T1 cells were seeded into a 96-well plate at a density of 4,000 cells per well, 100 μL of fresh DMEM medium was added to each well plate, and placed in an incubator for 24 h. The contrast agent PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 aqueous solution was diluted into different gadolinium ion concentrations (6, 3, 1.5, 0.75mM). Add materials with different Gd ion concentrations to the corresponding wells, add 100 μL to each well, and add 100 μL of culture medium to the control group, and continue culturing for 24 h. Finally, use a pipette to suck out all the culture medium, add 100 μL of fresh culture medium and 10 μL of CCK8 to each well, and then place it in an incubator for 2 h. Use a microplate reader to measure the absorbance at 450 nm. Four parallel samples were made for each gadolinium concentration and control group.
以利用公式计算得到的细胞相对存活率为纵坐标，以钆离子浓度为横坐标作图。结果如图7所示，PC/AD-PEG2000-PLL(DTPA-Gd)-N3对4T1细胞几乎没有毒性，当钆离子浓度达到6mM时，U87细胞的存活率保持在85％以上，表明该大分子钆基造影剂具有良好的生物相容性。The relative survival rate of cells calculated using the formula is plotted as the ordinate, and the gadolinium ion concentration is plotted as the abscissa. The results are shown in Figure 7. PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 has almost no toxicity to 4T1 cells. When the gadolinium ion concentration reaches 6mM, the survival rate of U87 cells remains above 85%, indicating that The macromolecular gadolinium-based contrast agent has good biocompatibility.
实施例9：Example 9:
组织毒性考察：Tissue toxicity investigation:
用苏木精-伊红染色法(H&E)来评估所制备的材料的安全性，考察造影剂PC/AD-PEG2000-PLL(DTPA-Gd)-N3的组织毒性。取9只裸鼠，在恒温和恒湿的室内的环境中，用标准颗粒饮食和纯净水适应性饲养1周，定期更换饲料并消毒。将裸鼠分为三组(n＝3)，其中两组分别进行腹腔注射100μL含PC/AD-PEG2000-PLL(DTPA-Gd)-N3的生理盐水，钆离子量分别为0.1mmol/kg和0.3mmol/kg。另外一组裸鼠作为对照组，腹腔注射100μL生理盐水。继续饲养2天后，用颈脱位法处死裸鼠，解剖并收集器官，包括心、肝、脾、肺和肾浸泡在福尔马林中，制成染色切片后置于显微镜下进行组织学分析并拍照。组织H&E染色后的显微镜照片如图8所示。实验结果表明，注射0.1mM/kg和0.3mM/kg的PC/AD-PEG2000-PLL(DTPA-Gd)-N3的小鼠组织器官均无明显损伤，在心细胞中没有坏死的情况，在肝细胞中没有炎症反应的现象，在肺细胞中没有纤维化的迹象，在肾细胞中没有形态变化的出现。所观察的心、肝、脾、肺、肾这些器官几乎无组织学变化，维持着正常组织学的形态。故可以判断PC/AD-PEG2000-PLL(DTPA-Gd)-N3具有良好的生物相容性。Hematoxylin-eosin staining (H&E) was used to evaluate the safety of the prepared materials, and the tissue toxicity of the contrast agent PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 was investigated. Nine nude mice were adaptively raised with standard pellet diet and purified water for 1 week in an indoor environment with constant temperature and humidity. The feed was changed and disinfected regularly. Nude mice were divided into three groups (n=3), and two groups were intraperitoneally injected with 100 μL of physiological saline containing PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 , and the amount of gadolinium ions was 0.1 mmol/ kg and 0.3mmol/kg. Another group of nude mice served as the control group and were injected intraperitoneally with 100 μL of normal saline. After continuing to be raised for 2 days, the nude mice were killed by cervical dislocation, and the organs were dissected and collected, including the heart, liver, spleen, lungs and kidneys, soaked in formalin, stained sections were made, and then placed under a microscope for histological analysis and photography. The micrograph of the tissue after H&E staining is shown in Figure 8. Experimental results showed that mice injected with 0.1mM/kg and 0.3mM/kg PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 had no obvious damage to their tissues and organs, and there was no necrosis in cardiac cells. There was no inflammatory response in liver cells, no signs of fibrosis in lung cells, and no morphological changes in kidney cells. The observed organs such as heart, liver, spleen, lung, and kidney had almost no histological changes and maintained normal histological morphology. Therefore, it can be judged that PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 has good biocompatibility.
实施例10：Example 10:
体内磁共振成像研究：In vivo magnetic resonance imaging studies:
将9只荷瘤裸鼠分成三组，腹腔均注射135μL的25％的乌拉坦溶液使荷瘤裸鼠麻醉，并将裸鼠固定到固定器上，置于4.7T的磁共振成像仪进行空白扫描，所得的图片作为空白参照。温度保持在35℃。然后，分别通过尾静脉注射PC/AD-PEG2000-PLL(DTPA-Gd)-RGD，PC/AD-PEG2000-PLL(DTPA-Gd)-N3，Gd-DTPA的生理盐水溶液，每组样品的钆离子浓度均为0.1mmol/kg。注射后，将裸鼠转移至4.7T磁共振成像仪观察肿瘤处的MRI成像情况。以每组的空白亮度为1，比较各组各个相同时间点的相对亮度。Nine tumor-bearing nude mice were divided into three groups. Each tumor-bearing nude mouse was anesthetized by intraperitoneal injection of 135 μL of 25% urethane solution. The nude mice were fixed to a holder and placed in a 4.7T magnetic resonance imaging machine for blanking. Scan, and the resulting image serves as a blank reference. The temperature is maintained at 35°C. Then, physiological saline solutions of PC/AD-PEG2000 -PLL(DTPA-Gd)-RGD, PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 and Gd-DTPA were injected through the tail vein respectively, in each group. The gadolinium ion concentration of the samples was all 0.1mmol/kg. After injection, the nude mice were transferred to a 4.7T magnetic resonance imaging machine to observe the MRI imaging of the tumor. Taking the blank brightness of each group as 1, compare the relative brightness of each group at the same time point.
结果如图9所示，可以看出，靶向组肿瘤部位的亮度随着时间的增加有明显的均亮于非靶向组，为了更直观的比较肿瘤部位的信号强度提升效果，用Image J处理图像，以每只裸鼠给药前肿瘤部位的亮度值转化为1，将1小时和2小时后肿瘤部位的亮度转化为具体数值，计算信号强度增长倍数，结果如图10所示。The results are shown in Figure 9. It can be seen that the brightness of the tumor site in the targeted group is significantly brighter than that in the non-targeted group as time increases. In order to more intuitively compare the signal intensity improvement effect of the tumor site, Image J was used The image was processed, and the brightness value of the tumor site before administration of each nude mouse was converted into 1, and the brightness value of the tumor site after 1 hour and 2 hours was converted into a specific value, and the signal intensity increase multiple was calculated. The results are shown in Figure 10.
实施例11：Example 11:
组织残留：Tissue residue:
取3只雄性裸鼠(5周龄，约20g)，尾静脉注射100μL 0.1mmol/kg钆离子浓度的所制备的造影剂PC/AD-PEG2000-PLL(DTPA-Gd)-N3的生理盐水溶液。在SPF环境下继续饲养10天后，用颈脱位法处死裸鼠，解剖并收集包括心，肺，肝，脾，肾，依次称重并记录。然后，将各器官和肿瘤取100mg左右放在4mL离心管中，加入1mL硝酸溶液，水浴加热至60℃维持1个小时。将溶液转移至15mL离心管中，再加入10mL双氧水，水浴90℃加热1小时。通过加热使其完全消化后赶酸，最后转移加水定容至60mL。每个样品取1mL，通过ICP-MS测定溶液中钆含量。最后，计算每个器官中平均的钆含量，并将其转换为每个器官、组织或肿瘤的注射剂量(ID)百分比。结果如图11所示。钆在心、肝、脾、肺和肾的残留量分别为0.01％、0.28％、0.03％、0.02％和0.02％每组织或器官。这些数据说明所制备的大分子造影剂在主要器官和组织中具有很少的长期残留，能够初步满足造影剂的体内长期安全性要求。Three male nude mice (5 weeks old, about 20g) were injected into the tail vein with 100 μL of the prepared contrast agent PC/AD-PEG2000 -PLL(DTPA-Gd)-N3 with a gadolinium ion concentration of 0.1mmol/kg. Saline solution. After being kept in the SPF environment for 10 days, the nude mice were sacrificed by cervical dislocation, and the heart, lungs, liver, spleen, and kidneys were dissected and collected, and their weights were recorded in turn. Then, place about 100 mg of each organ and tumor in a 4 mL centrifuge tube, add 1 mL of nitric acid solution, and heat the water bath to 60°C for 1 hour. Transfer the solution to a 15mL centrifuge tube, add 10mL hydrogen peroxide, and heat in a water bath at 90°C for 1 hour. Completely digest it by heating to drive out the acid, and finally transfer and add water to make the volume up to 60 mL. Take 1 mL of each sample and determine the gadolinium content in the solution by ICP-MS. Finally, the average gadolinium content in each organ is calculated and converted to a percentage of the injected dose (ID) for each organ, tissue, or tumor. The results are shown in Figure 11. The residual amounts of gadolinium in the heart, liver, spleen, lungs and kidneys are 0.01%, 0.28%, 0.03%, 0.02% and 0.02% per tissue or organ respectively. These data indicate that the prepared macromolecular contrast agent has very few long-term residues in major organs and tissues, and can initially meet the long-term safety requirements of contrast agents in vivo.
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