CN106474050A - 一种载有核酸适配体‑阿霉素结合物的温敏原位凝胶瘤内注射制剂及其制备方法 - Google Patents
一种载有核酸适配体‑阿霉素结合物的温敏原位凝胶瘤内注射制剂及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种载有MUC1黏蛋白适配体‑阿霉素结合物的温敏原位凝胶瘤内注射制剂及其制备方法。本发明将5’端有巯基修饰的MUC1黏蛋白适配体与阿霉素结合，通过物理包埋法将其载入壳聚糖/β‑甘油磷酸钠温敏凝胶中，制备成一种可注射的原位凝胶系统。该系统在室温下呈液态可直接瘤内注射，进入体内后可迅速(～3min)实现胶凝化而在肿瘤局部形成半固体药物储库并缓慢释放核酸适配体‑阿霉素结合物，其间凝胶三维网状结构可保护核酸适配体不易被生物体内的核酸酶降解，并进一步通过肿瘤细胞表面高表达的MUC1黏蛋白的特异性识别，实现阿霉素在MCF‑7细胞内的靶向摄取，提高阿霉素在相应肿瘤细胞内的浓度，以期为抗肿瘤药物的局部给药提供一种新的可供选择的剂型。
Description
技术领域
本发明涉一种载有核酸适配体-阿霉素结合物的的温敏原位凝胶瘤内注射制剂及其制备方法，属于药物制剂领域。
背景技术
肿瘤生物学标志物是指在肿瘤细胞中过量表达，而在正常细胞中不表达或表达量小于肿瘤细胞的生化分子。利用肿瘤生物学标志物的这一特点，将其作为靶标分子，筛选出能与其特异性结合的靶向分子，并将其与抗肿瘤药物结合或修饰在载体的表面，从而实现肿瘤细胞的靶向摄取，达到诊断与治疗的目的。其中，核酸适配体(aptamer)能够以较高亲和力与其相应靶标进行特异性结合的单链DNA或RNA。其大多是通过指数富集的配体系统进化技术从寡核苷酸序列库中随机筛选得到的能够和靶物质高特异性结合的寡核苷酸序列，与传统大分子靶向分子如抗体的作用类似，但同时又具有许多传统的抗体无法比拟的优点：特异性及亲和性高；稳定性好，不易降解，可长期保存，常温下运输；分子质量小，无免疫原性；适配体分子较小，易于透过细胞膜；易于合成，经修饰后可与药物分子结合，从而提要药物分子的靶向性。适配体作为一种极有前景的、可望取代抗体的药物，在肿瘤治疗领域引起广泛研究。作为一种极有前景的、可望取代抗体大分子的靶向分子，适配体在肿瘤治疗领域引起广泛研究。然而，核酸适配体注射入生物体内后，因其不可避免地易被生物体内大量的核酸酶降解，导致核酸适配体的体内应用具有一定的局限性。
黏蛋白1(Mucin 1，MUC1)是一种I型跨膜蛋白，在乳腺癌细胞、卵巢癌细胞等多种癌细胞中过量表达，而在相应的癌旁组织中不表达，是一种新型的肿瘤标志物。通过指数富集的配体系统进化技术筛选出的与黏蛋白1具有高亲和力高特异性的一段核酸适配体序列为5’-GCAGTTGATCCTTTGGATACCCTGG-3’。本发明拟将末端修饰后MUC1黏蛋白适配体与抗肿瘤药物DOX相结合，形成MUC1黏蛋白核酸适配体-DOX结合物，从而提高DOX的靶向性和特异性，减少DOX对正常细胞的毒副作用。同时考虑到MUC1适配体在体内的稳定性差，无法直接通过静脉注射给药而实现良好的靶向治疗效果，故本发明拟将得到的MUC1黏蛋白核酸适配体-DOX结合物通过物理包埋的方式载入凝胶的三维网状结构中，并将其注射埋植在肿瘤部位，以防止体循环中的核酸酶的降解，同时可实现MUC1黏蛋白核酸适配体-DOX结合物的缓慢释放。
原位凝胶又称在体凝胶，是指将其以溶液状态给药后，当接触到用药部位机体的生理条件或受到其他环境因素的影响后即发生相转变形成半固体凝胶状态的制剂。原位凝胶具有缓慢释放药物，避免药物的首过效应，提高药物生物利用度，降低药物毒副作用等优点。目前研究较多的是壳聚糖/β-甘油磷酸钠温敏原位凝胶系统，故本研究通过物理包埋法将MUC1黏蛋白核酸适配体-阿霉素结合物装载入由壳聚糖及β-甘油磷酸钠所构成凝胶三维网状结构中，其特征在于在室温下为液态可用于瘤内注射，且进入体内后在体温下可迅速实现胶凝化而形成药物储库，缓慢释放出核酸适配体-阿霉素结合物，并进一步通过肿瘤细胞表面高表达的核酸适配体受体的特异性识别，而实现阿霉素在肿瘤细胞内的靶向摄取而发挥抗肿瘤疗效。
发明内容
本发明的目的是为临床提供一种实用方便、稳定可靠的治疗肿瘤的载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位凝胶及其制备方法，旨在克服传统DOX制剂在肿瘤治疗方面所存在的不足。
为实现以上目的，本发明提供以下技术方案：
本发明将5’端有巯基修饰的MUC1黏蛋白适配体与DOX修饰物DOXO-EMCH在冰浴条件下反应24h，得到MUC1黏蛋白核酸适配体-DOX结合物。
本发明提供的载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位水凝胶瘤内注射制剂，其中壳聚糖溶液为的壳聚糖冰醋酸水溶液，用量为10.0～15.0mg/mL，壳聚糖分子量为10万，脱乙酰度≥90.0％，粘度为45mPa·s。
本发明提供的载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位水凝胶瘤内注射制剂，其中β-甘油磷酸钠的用量为100～200mg/mL。
本发明提供的载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位水凝胶瘤内注射制剂，其制备方法主要包括以下几个步骤：
(1)称取一定量的DOXO-EMCH，溶解于二甲基亚砜(Dimethyl sulfoxide，DMSO)中，配置成一定浓度的DOXO-EMCH溶液。
(2)将5’端有巯基修饰的MUC1黏蛋白适配体，溶解于pH 7.4PBS水溶液中，配置成一定浓度的MUC1黏蛋白适配体水溶液。
(3)将一定量的DOXO-EMCH溶液与MUC1黏蛋白适配体水溶液混合，冰浴条件下反应24h，反应液过交联葡聚糖凝胶柱(Sephadex G-25)除去未反应的DOXO-EMCH，而后冷冻干燥制得核酸适配体-阿霉素干燥粉末。
(4)称取一定量的壳聚糖，溶解于冰醋酸溶液中，配制成一定浓度的壳聚糖的冰醋酸水溶液，4℃储存备用。
(5)称取一定量的β-甘油磷酸钠溶于去纯化水中，配制成一定浓度的β-甘油磷酸钠水溶液。
(6)在冰浴条件下，向壳聚糖溶液中逐滴加入β-甘油磷酸钠溶液，继续搅拌使其混合均匀，制得空白温敏原位凝胶制剂。
(7)称取一定量的核酸适配体-阿霉素粉末，在冰浴搅拌下加入到空白温敏原位凝胶制剂中，继续搅拌使其混合均匀，即得本发明制剂。
本发明涉及的载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位凝胶瘤内注射制剂在室温下为液体，注射到肿瘤部位后，在生理温度下可迅速胶凝形成半固体凝胶，作为药物贮库缓慢释放药物，一方面壳聚糖温敏原位凝胶三维网状结构可以保护核酸适配体不被生物体内的核酸酶降解，并达到缓慢释药的目的，另一方面核酸适配体可帮助DOX进行精准的肿瘤细胞靶向及摄取从而达到了增加DOX对肿瘤细胞的杀伤力，同时降低对正常细胞的毒副作用，减小药物用量的目的。本发明有望作为一种新型的瘤内注射给药系统，通过缓慢释药达到治疗目的，操作方便，降低药物毒性，解决传统静脉及埋植给药方法上的不足。
附图说明
附图1：具体实施例1所得的MUC1黏蛋白核酸适配体-DOX结合物的质谱图。
附图2：具体实施例1所得的载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位凝胶瘤内注射制剂的体外胶凝图。
附图3：具体实施例2所得载有MUC1黏蛋白核酸适配体-DOX结合物的温敏原位凝胶瘤内注射制剂的体外药物释放曲线，其中APT-DOX-CS/GP代表载有MUC1黏蛋白核酸适配体-DOX结合物的壳聚糖/β-甘油磷酸钠温敏原位凝胶制剂。
附图4：具体实施例1所制备MUC1黏蛋白核酸适配体-DOX结合物和市售DOX溶液对人乳腺癌细胞MCF-7的细胞毒性评价实验结果。
具体实施方式
下面结合具体实施例对本发明作进一步解释和说明，应当理解，所给出的实施例只是举例说明性的，其不以任何方式对本发明的范围构成任何限制。
具体实施例1
称取一定量的DOXO-EMCH，溶解于DMSO中，配置成一定浓度的DOXO-EMCH溶液。将5’端有巯基修饰的MUC1黏蛋白适配体，溶解于pH 7.4PBS水溶液中，配置成一定浓度的MUC1黏蛋白适配体水溶液。将一定量的DOXO-EMCH溶液与MUC1适配体水溶液混合，冰浴条件下反应24h，反应液过交联葡聚糖凝胶柱(Sephadex G-25)除去未反应的DOXO-EMCH，而后冷冻干燥制得核酸适配体-阿霉素结合物干燥粉末。质谱图见附图1.
壳聚糖(0.04g)溶解于2mL的冰醋酸溶液(0.1M)中，在冰浴搅拌条件下，逐滴加入β-甘油磷酸钠溶液，继续搅拌使其混合均匀，继续在冰浴条件下，加入核酸适配体-阿霉素结合物粉末，继续搅拌使其溶解，即得载有阿霉素结合物的温敏原位凝胶制剂。该制剂在室温时为液体状态，通针性良好，37℃水浴条件下3min可胶凝。体外胶凝试验见附图2。
具体实施例2
对本发明具体实施例1所得的温敏原位凝胶进行体外释药行为试验，释放曲线如附图3所示。
精密量取1mL载药温敏原位凝胶，加入到10mL具塞试管中，37±1℃水浴条件下放置1h，待充分胶凝后，加入10mL pH 7.4的磷酸盐缓冲液，置于37±1℃，100rpm摇床中振荡。于固定的取样时间点取出全部释放介质，并补充相同体积的释放介质。经0.45μm滤膜过滤，取续滤液进样，在485nm波长下测定DOX含量，计算累计释药率。
具体实施例3
以市售DOX溶液为对照，进行体外细胞毒性试验，考察实施例1所得MUC1黏蛋白核酸适配体-DOX结合物的细胞毒性。
将人乳腺癌MCF-7细胞以密度为4×103细胞/孔接种于96孔板中，培养24h。此后，细胞用含有样品(阿霉素溶液和实施例1所得MUC1黏蛋白核酸适配体-阿霉素结合物溶液，相同的最终阿霉素浓度为0.5μM)的新鲜培养基(pH 7.4)处理2h(37℃)。细胞孵育4h后用新鲜PBS冲洗3次，将药物溶液替换成新鲜空白培养基继续培养46h。每孔加入20μL 5mg/mLMTT溶液，培养4h后弃去培养基，用新鲜PBS溶液小心清洗两遍后，每孔加入150μL DMSO溶液。用酶标仪在570nm条件下测定吸光度，以加入空白培养基的细胞作为对照组，计算细胞存活率。
实验结果如附图4所示：核酸适配体-阿霉素结合物组的细胞存活率比市售阿霉素组的细胞存活率低，图中*表明p＜0.05，两组制剂的细胞存活率有显著性差异。说明本发明所得的核酸适配体-阿霉素结合物，可以提高对MCF-7细胞的毒性，促进药物的跨细胞膜转运，增加药物在肿瘤内的累计而提高药效。
Claims (5)
1.一种载有核酸适配体-阿霉素结合物的温敏原位凝胶瘤内注射制剂及其制备方法，该系统系通过物理包埋法将核酸适配体-阿霉素结合物装载入由壳聚糖及β-甘油磷酸钠所构成的凝胶三维网状结构中，其特征在于在室温下为液态可用于瘤内注射，且进入体内后在体温下可迅速实现胶凝化而形成药物储库，缓慢释放出核酸适配体-阿霉素结合物，并进一步通过肿瘤细胞表面高表达的核酸适配体受体的特异性识别，而实现阿霉素在肿瘤细胞内的靶向摄取而发挥抗肿瘤疗效。
2.根据权利要求1中所述的载有核酸适配体-阿霉素结合物的温敏原位凝胶，其特征在于核酸适配体-阿霉素结合物是将5’端有巯基修饰的黏蛋白1(Mucin1，MUC1)适配体与阿霉素修饰物(Aldoxorubicin，DOXO-EMCH)在冰浴条件下反应24h制得。
3.根据权利要求1中所述的载有核酸适配体-阿霉素结合物的温敏原位凝胶，其特征在于体系中所用的壳聚糖溶液为的壳聚糖冰醋酸水溶液，用量为10.0～15.0mg/mL，壳聚糖分子量为10万～30万，脱乙酰度≥90.0％，粘度为45～140mPa·s。
4.根据权利要求1中所述的载有核酸适配体-阿霉素结合物的温敏原位凝胶，其特征在于体系中所用的β-甘油磷酸钠的用量为100～200mg/mL。
5.根据权利要求1～4所述的载有核酸适配体-阿霉素结合物的温敏原位凝胶，其制备方法具体包括以下几个步骤：
(1)称取一定量的DOXO-EMCH，溶解于二甲基亚砜(Dimethyl sulfoxide，DMSO)中，配置成一定浓度的DOXO-EMCH溶液。
(2)将5’端有巯基修饰的MUC1黏蛋白适配体，溶解于pH7.4PBS水溶液中，配置成一定浓度的MUC1黏蛋白适配体水溶液。
(3)将一定量的DOXO-EMCH溶液与MUC1黏蛋白适配体水溶液混合，冰浴条件下反应24h，反应液过交联葡聚糖凝胶柱(Sephadex G-25)除去未反应的DOXO-EMCH，而后冷冻干燥制得核酸适配体-阿霉素干燥粉末。
(4)称取一定量的壳聚糖，溶解于冰醋酸溶液中，配制成一定浓度的壳聚糖的冰醋酸水溶液，4℃储存备用。
(5)称取一定量的β-甘油磷酸钠溶于去纯化水中，配制成一定浓度的β-甘油磷酸钠水溶液。
(6)在冰浴条件下，向壳聚糖溶液中逐滴加入β-甘油磷酸钠溶液，继续搅拌使其混合均匀，制得空白温敏原位凝胶制剂。
(7)称取一定量的核酸适配体-阿霉素粉末，在冰浴搅拌下加入到空白温敏原位凝胶制剂中，继续搅拌使其混合均匀，即得本发明制剂。
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