CN116870202A - 壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂及制备方法 - Google Patents
壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂及制备方法 Download PDFInfo
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Abstract
本发明公开了一种壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂及制备方法，属于磁共振成像材料领域，通过将叶酸、壳聚糖、氧化钆三者相结合，制备出一种低毒性、弛豫率高且具有肿瘤靶向性的磁共振对比剂，制作方法简单合理、制作成本低，具有较高的临床应用前景。
Description
技术领域
本发明涉及磁共振成像材料领域，具体涉及一种壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂及其制备方法。
背景技术
高癌症死亡率往往与低筛查率、延误诊断和低质量的医疗保健有关。开发敏感、快速、安全、低成本的诊断技术将大大降低癌症死亡率。磁共振成像(MRI)是临床上最强大的成像技术之一，具有无创、无辐射、多核多参数成像和任意层扫描的优点。特别是对于其他方法无法识别的小肿瘤病变和中枢神经系统的神经血管造影，MRI是首选，因为它可以提供软组织的解剖图像。
但是核磁共振成像常规扫描对于某些疾病及早期肿瘤检出的敏感性较低，为了提高邻近组织与肿瘤的成像对比度使成像清晰，常使用磁共振对比剂来弥补其敏感性低的缺陷，并可借助磁共振对比剂对深部组织进行较准确的定位、定量分析等特性。磁共振对比剂又分为T1和T2对比剂，分别可通过缩短质子的纵向及横向弛豫时间来增强其所在病灶部位的信号对比度。由于T2成像易与出血、钙化、金属沉积等低信号区域混淆，从而导致临床检测过程中误诊、漏诊病灶，所以临床上多采用T1成像进行疾病检测。临床上采用的T1对比剂多为非特异性钆基大分子螯合物对比剂，如Magnevist(Gd-DTPA)、Dotarem(Gd-DOTA)等，其不具有肿瘤靶向特性，对于早期肿瘤的检出性较低，且研究显示，钆基大分子螯合物对比剂钆离子泄露会导致肾源性系统性纤维化以及脑部钆离子沉积。
钆基纳米颗粒与钆基螯合物相比具有钆离子泄露更低、比表面积更大等优点，其中氧化钆纳米颗粒因可以提供高弛豫率而获得广泛关注，高弛豫率可以增强对比度和提高敏感度，有助于提升临床疾病的检测出率。壳聚糖为天然多糖的一种，属于纯天然可再生资源，其本身具有许多优良的特性：i)零毒性带来的生物相容性以及与重金属粒子的亲和性；ii)容易通过共价键结合，结合后利用壳聚糖的长链缠绕特性将螯合的顺磁性金属离子或金属氧化物包在核心，同时增加弛豫效率；iii)进入体内后壳聚糖会降解，但需要一定时间，保证对比剂的金属离子在体内存留时处于包裹状态不外泄；iv)壳聚糖与人体血清蛋白等运送蛋白的结合能力强，可在成像测定结束后将未完全降解的对比剂代谢排出体外；v)壳聚糖自身还能提高生命体的免疫力，并具有一定的抗癌作用，有望增强协同作用；vi)壳聚糖易于加工成微胶囊，因此在剂型制备方面也有着优势。对于肿瘤特异性成像而言，虽然EPR效应可以实现部分的肿瘤靶向功能，但留存效率较低，通过主动靶向可以增强肿瘤对纳米颗粒的摄取效率。叶酸可参与人体多种生理活动、对人体无毒，且肿瘤细胞表面有大量的叶酸受体，因此叶酸可作为肿瘤靶向的介导物质。因此亟需制作一种生物相容性高、弛豫率高且可进行肿瘤靶向的壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂，以满足多种成像造影需求。
发明内容
有鉴于此，本发明的目的之一在于提供一种壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂的制备方法；本发明的目的之二在于提供所述方法制备得到的壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂。
为达到上述目的，本发明提供如下技术方案：
1、一种壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂的制备方法，包含如下制备步骤：
S1:称取氯化钆六水合物和氢氧化钠分别分散于三乙二醇中，将两种溶液加热至80℃直至前驱体溶解，然后混合两种溶液，加热至140℃持续2h，最后加热至180℃持续4h，待反应溶液冷却至室温后，用去离子水透析，得到氧化钆纳米颗粒；
S2:取步骤S1制得的氧化钆纳米颗粒分散于壳聚糖溶液中，在磁力搅拌下缓慢加入TPP溶液至反应完全，得到壳聚糖包被的氧化钆纳米颗粒；
S3:称取叶酸溶于无水二甲基亚砜中并活化，然后加入步骤S2制备的壳聚糖包被的氧化钆颗粒溶液中，在磁力搅拌下将叶酸嫁接到壳聚糖表层，随后用去离子水透析，得到壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂。
本发明优选的，步骤S1中，所述氯化钆六水合物和氢氧化钠的摩尔比为1﹕3，溶解氯化钆六水合物和氢氧化钠三乙二醇的体积比为3﹕1。
本发明优选的，步骤S1中，所述加热的升温速率为10℃/min。
本发明优选的，步骤S1中，所述透析使用的透析袋的分子量为Mw＝3500Da。
本发明优选的，步骤S2中，所述氧化钆纳米颗粒和壳聚糖的质量比为1﹕8。
本发明优选的，步骤S2中，所述壳聚糖与TPP质量比为3﹕1～6﹕1，更优选的壳聚糖与TPP质量比为3﹕1，所述磁力搅拌的转速为600r/min。
本发明优选的，步骤S3中，叶酸和壳聚糖的质量比为1﹕4.5。
本发明优选的，步骤S3中，所述活化为使用N-羟基琥珀酰亚胺(NHS)和碳酰二亚胺(EDC)对叶酸进行活化，且叶酸、NHS和EDC的摩尔比为1﹕2﹕2。
2、所述方法制备得到的壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂。
本发明的有益效果在于：
1)通过多元醇法合成氧化钆纳米颗粒，制备方法简单，且制备纳米颗粒粒径小、形状易于控制。通过离子交联制备壳聚糖包被的氧化钆纳米颗粒，制作方法简单合理、制作成本低，得到的纳米颗粒粒径均一、分散性好；
2)制备的Gd2O3/CS-TPP@FA纳米颗粒弛豫率可达9.707mM-1·s-1，大约为临床上使用的Gd-DTPA弛豫率的两倍；
3)制备的Gd2O3/CS-TPP@FA纳米颗粒对肿瘤具有靶向性，可以有效在肿瘤部位积累，对早期肿瘤的临床成像检出具有潜在应用价值；
4)壳聚糖是天然可再生资源，本身具有零毒性和生物降解性的特点，且可以包被氧化钆防止钆离子泄露。叶酸是人体生理活动的必需物质，其本身对人体并无危害，所以制备的Gd2O3/CS-TPP@FA纳米颗粒具有高度生物相容性。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为本发明中叶酸偶联壳聚糖包被氧化钆傅里叶红外(FTIR)谱图；
图2为本发明中Gd2O3(a)、Gd2O3/CS-TPP(b)、Gd2O3/CS-TPP@FA纳米颗粒(c)的SEM图，a图中右下插图为100nm视野下Gd2O3纳米颗粒；
图3为本发明Gd2O3(a)、Gd2O3/CS-TPP(b)、Gd2O3/CS-TPP@FA纳米颗粒(c)动态光散射(DLS)测得的水合动力直径；
图4为本发明中Gd2O3、Gd2O3/CS-TPP、Gd2O3/CS-TPP@FA纳米颗粒电位分析图；
图5为本发明中Gd2O3/CS-TPP@FA纳米颗粒的体外细胞毒性图；
图6为本发明中Gd2O3/CS-TPP@FA纳米颗粒Gd3+泄露测量曲线图；
图7为本发明中Gd2O3/CS-TPP@FA纳米颗粒弛豫率r1(b)及体外磁共振成像图T1加权像(a)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1、壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂的制备
氧化钆纳米颗粒的制备：
将15ml TEG(三乙二醇)注入三颈圆底烧瓶中，然后称取2mmol的氯化钆六水合物分散于注入TEG的圆底烧瓶中，将圆底烧瓶置于磁力搅拌器加热至80℃，直至前驱体溶解。称取6mmol的氢氧化钠，分散在5ml TEG中，加热直至氢氧化钠完全溶解，然后加入氯化钆前驱体溶液中，以10℃/min的升温速度加热至140℃持续2h，最后加热至180℃持续4h。加热完成后，待溶液冷却至室温，用去离子水透析4d后，取出备用。
壳聚糖包被的氧化钆纳米颗粒的制备：
称取壳聚糖溶于乙酸，称取三聚磷酸钠(TPP)溶于去离子水，取2.7mg/ml氧化钆纳米颗粒溶液0.5ml分散在1mg/ml的10ml壳聚糖(Chitosan，CS)溶液中，超声分散10min使颗粒分散均匀。取1mg/ml TPP(三聚磷酸钠)溶液，在磁力搅拌器600r/min的转速下以1ml/min的速度加入壳聚糖溶液中。壳聚糖与TPP质量比为3﹕1-6﹕1，磁力搅拌器转速为600r/min。更优选的壳聚糖与TPP质量比为3﹕1。为保证壳聚糖与TPP反应完全，TPP滴加结束后，待反应至30min后，关闭磁力搅拌器停止反应。停止反应后超声10min，使颗粒分散，放置于玻璃瓶中保存以备后续使用。
壳聚糖包被的氧化钆交联叶酸纳米颗粒的制备：
称取0.05mmol叶酸(folicacid，FA)溶解在10ml无水二甲基亚砜中，然后加入0.1mmol的N-羟基琥珀酰亚胺(NHS)和碳酰二亚胺(EDC)反应12h对叶酸进行活化，叶酸、NHS、EDC的摩尔比为1﹕2﹕2。取1ml活化完成后的叶酸缓慢加入壳聚糖包被的氧化钆纳米颗粒溶液中反应16h，反应完成后在去离子水中透析3d后保存以备后续使用。
实施例2、壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂的表征(红外、粒径电位、电镜图)
利用傅里叶红外光谱仪对壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂进行傅里叶红外图谱检测，结果如图1所示，在3200-3500cm-1处的峰值是O-H集团的拉伸震动所致，在1640和1540cm-1处为壳聚糖的酰胺带震动峰值，说明壳聚糖成功包被氧化钆，而在1609cm-1处显示也算特有的苯环震动峰值，说明叶酸的接连成功。
利用扫描电子显微镜进行形貌和粒径分析，结果如图2，a～c所示，本发明制作的Gd2O3/CS-TPP@FA纳米颗粒粒径在30nm左右，显示其粒径较小，有助于在体内留存。利用马尔文粒度仪进行水动力直径测量、电位测量，结果如图3、图4所示，水测量的动力直径为280nm，其电位为-38.3mv，显示其非常稳定，可长期保存。
实施例3、壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂细胞毒性验证及钆离子泄露测量
采用经典CCK-8法评价磁共振对比剂的体外细胞毒性，首先将细胞接种于96孔板中(1×104细胞/孔)。在细胞培养箱中孵育24h后，吸出96孔板中的旧培养基，加入含有不同浓度样品(0、0.125、0.25、0.5和1mM)的培养基溶液，并继续培养24h。吸出旧培养基，在每个孔中加入100μL含10μL CCK-8的DMEM溶液，并继续培养1-4h后，在酶标仪检测各孔的OD值(检测波长为460nm)，计算细胞存活率。
从图5可以看出，在1mM浓度下培养24h后，细胞存活率为91.71％，表明壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂细胞毒性低。
如图6所示，通过UV建立了Gd3+泄露的标准曲线图，通过标准曲线图，建立在573nm/433nm的校准曲线图。通过ICP-OES测得样品Gd浓度为8.9mM，测量样品的紫外曲线图获得了Gd2O3/CS-TPP@FA纳米颗粒573nm/433nm值，带入标准曲线图可得Gd3+泄露仅为10.51μΜ，泄露率为0.12％，证明制作的Gd2O3/CS-TPP@FA纳米颗粒有很好的生物相容性。
实施例4、壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂的体外成像
图7，(a)所示为壳聚糖交联叶酸包被氧化钆纳米磁共振对比剂的体外成像结果，图7，(b)显示弛豫率为9.707mM-1·s-1，可作为良好的T1成像对比剂。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (9)
1.一种壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂的制备方法，其特征在于，包含如下制备步骤：
S1:称取氯化钆六水合物和氢氧化钠分别分散于三乙二醇中，将两种溶液加热至80℃直至前驱体溶解，然后混合两种溶液，加热至140℃持续2h，最后加热至180℃持续4h，待反应溶液冷却至室温后，用去离子水透析，得到氧化钆纳米颗粒；
S2:取步骤S1制得的氧化钆纳米颗粒分散于壳聚糖溶液中，在磁力搅拌下缓慢加入TPP溶液至反应完全，得到壳聚糖包被的氧化钆纳米颗粒；
S3:称取叶酸溶于无水二甲基亚砜中并活化，然后加入步骤S2制备的壳聚糖包被的氧化钆颗粒溶液中，在磁力搅拌下将叶酸嫁接到壳聚糖表层，随后用去离子水透析，得到壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂。
2.根据权利要求1所述的方法，其特征在于，步骤S1中，所述氯化钆六水合物和氢氧化钠的摩尔比为1﹕3，溶解氯化钆六水合物和氢氧化钠三乙二醇的体积比为3﹕1。
3.根据权利要求1所述的方法，其特征在于，步骤S1中，所述加热的升温速率为10℃/min。
4.根据权利要求1所述的方法，其特征在于，步骤S1中，所述透析使用的透析袋的分子量为Mw＝3500Da。
5.根据权利要求1所述的方法，其特征在于，步骤S2中，所述氧化钆纳米颗粒和壳聚糖的质量比为1﹕8。
6.根据权利要求1所述的方法，其特征在于，步骤S2中，所述壳聚糖与TPP质量比为3：1～6：1，所述磁力搅拌的转速为600r/min。
7.根据权利要求1所述的方法，其特征在于，所述叶酸和壳聚糖的质量比为1﹕4.5。
8.根据权利要求1所述的方法，其特征在于，步骤S3中，所述活化为使用N-羟基琥珀酰亚胺(NHS)和碳酰二亚胺(EDC)对叶酸进行活化，且叶酸、NHS和EDC的摩尔比为1﹕2﹕2。
9.权利要求1～8任一项权利要求所述方法制备得到的壳聚糖交联叶酸包被氧化钆的肿瘤靶向磁共振对比剂。
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