CN114558150A - 一种用于pH可视化的磁共振成像纳米探针的制备方法 - Google Patents
一种用于pH可视化的磁共振成像纳米探针的制备方法 Download PDFInfo
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Abstract
本发明公开一种用于pH可视化的磁共振成像纳米探针的制备方法,其特征在于包括以下步骤:步骤1:超顺磁性纳米颗粒的制备;步骤2:将蛋白溶解在水中,然后按一定比例加入超顺磁性纳米颗粒水溶液,形成超顺磁性纳米颗粒@蛋白结构纳米颗粒;步骤3:将多酚‑铁溶液与步骤2制备的纳米颗粒混合在一起,充分溶解混合均匀,获得超顺磁性纳米颗粒@蛋白@多酚‑铁纳米颗粒;本发明以纳米颗粒为核心构建响应型分子影像探针在有望实现对病灶部位高灵敏精准成像。其中以超顺磁性磁性纳米颗粒(T2造影效果)为核心构建的MRI造影剂,在表面修饰铁离子络合物后,到达酸性微环境时通过pH响应释放铁离子(T1造影效果),从而可以实现pH响应激活的T1/T2磁共振比率成像。
Description
技术领域
本发明属于磁共振成像探针的领域,特别涉及一种用于pH可视化的磁共振成像纳米探针的制备方法。
背景技术
过去几年,越来越多的研究证明,与正常细胞相比,病灶部位由于肿瘤细胞或炎症状态的存在导致周围组织细胞的供氧量不足,只能利用无氧呼吸的糖酵解产生能量,因此产生的大量乳酸、氢离子以及二氧化碳堆积,导致病灶部位的微环境酸化,pH值在6.5-6.8之间,这不仅对炎症有促进作用,同样提高肿瘤转移的风险。而近几年人们认为这种酸性微环境可以作为病灶部位的显影和治疗的有效靶标,与传统的受体-配体靶标方式相比,这种方式的优势非常明显,由于大多数病灶部位的微环境都呈酸性,因此利用病灶部位的pH异常状态,可以作为疾病诊断的重要指标之一。
磁共振成像(MRI)相比于其他的成像技术,由于成像方式多样,成像原理更加复杂,使其得到的成像信息更加丰富,空间分辨率更高,另外由于磁共振成像是磁场成像,无放射性,对人体无害,同时磁共振成像摆脱了光学成像穿透性不强的缺点,不受组织穿透能力的限制,可通过不同序列(T1、T2)在病灶部位进行成像,更加适用于临床疾病的诊断,广泛用于肿瘤的检查。
人体内病灶部位由于细胞代谢改变引起pH低的特点,导致免疫抑制、炎症等,加速了疾病的进展,通过pH值分布的影像检测,可以对病灶部位的准确定位。
现有技术中,缺少一种磁共振成像纳米探针实现成像的同时还可以实现病灶微环境pH在体可视化。
发明内容
为解决上述技术问题,本发明提供一种用于pH可视化的磁共振成像纳米探针的制备方法,以纳米颗粒为核心构建响应型分子影像探针在有望实现对病灶部位高灵敏精准成像。其中以超顺磁性磁性纳米颗粒(T2造影效果)为核心构建的MRI造影剂,在表面修饰铁离子络合物后,到达酸性微环境时通过pH响应释放铁离子(T1造影效果),从而可以实现pH响应激活的T1/T2磁共振比率成像。
本发明提供的技术方案如下:
一种用于pH可视化的磁共振成像纳米探针的制备方法,包括以下步骤:
步骤1:超顺磁性纳米颗粒的制备
步骤2:将蛋白溶解在水中,然后按一定比例加入超顺磁性纳米颗粒水溶液,形成超顺磁性纳米颗粒@蛋白结构纳米颗粒;
步骤3:将多酚-铁溶液与步骤2制备的纳米颗粒混合在一起,充分溶解混合均匀,获得超顺磁性纳米颗粒@蛋白@多酚-铁纳米颗粒;
所述用于pH可视化的磁共振成像纳米探针的制备方法,包括以下步骤:
步骤1)超顺磁性纳米颗粒的制备
采用改进的水热法制备油酸铁:在搅拌下将4.8gNaOH、60mL水、80mL乙醇和120mL油酸混合以形成均相溶液,然后在磁搅拌条件下加入10.8gFeCl3·6H2O和140mL环己烷,将混合物在70℃下搅拌约4小时,并冷却至室温,将所得混合物用水洗涤去除其他残余物,通过旋蒸获得油酸铁;
疏水性Fe3O4纳米颗粒的合成:
将3.6g上述步骤制备的油酸铁和1.13g油酸溶解在25mL的1-十八烯中,将所得溶液以3.3℃/min的速率加热至310℃,然后在氮气保护下保持310℃30min,通过将反应混合物冷却至室温终止制备,用乙醇沉淀得到疏水性Fe3O4纳米颗粒,通过磁选收集,用乙醇洗涤,最后再分散在环己烷中,备用;
亲水性Fe3O4纳米颗粒水溶液的制备:
将上述制备的疏水性Fe3O4纳米颗粒环己烷分散液测量氧化铁浓度(邻菲罗啉法),取含有10mg的Fe3O4纳米颗粒的环己烷分散液,用无水乙醇沉淀、离心,除去上清液后,所得沉淀用1ml环己烷溶解,再加入10mL环己烷、7mL叔丁醇、2mL水和1mL质量百分比为5wt%K2CO3的混合物在室温下搅拌20min,然后逐滴添加4mL Lemieuxvon Rudloff试剂,将所得混合物在40℃下搅拌4小时,再通过离心分离亲水性Fe3O4纳米颗粒,并分别用去离子水、丙酮和乙醇洗涤,将产物分散在50mL pH值为4-5的盐酸中,并将混合物搅拌30min,再将亲水性Fe3O4纳米颗粒通过使用30kDa MWCO超滤管的水超滤2个循环进行纯化,并分散在水中备用,测量所得溶液的氧化铁浓度;
其中,所述Lemieuxvon Rudloff试剂由5.7mM高锰酸钾和0.105M NaIO4水溶液。
步骤2)Fe3O4@BSA纳米颗粒的制备
将BSA溶解于经Milli-Q处理的水中得到BSA水溶液,将制备好的亲水性Fe3O4纳米颗粒水溶液添加到BSA水溶液中,混合后最终浓度为:亲水性Fe3O4纳米颗粒0.2mg/ml,BSA10mg/ml,以形成Fe3O4@BSA纳米颗粒,其中,Fe3O4与BSA的物质的量的比例为1:10,然后使用100kDa MWCO超滤管通过超滤去除游离BSA,得到Fe3O4@BSA纳米颗粒水溶液;
分别将TA水溶液和FeCl3溶液加入到Fe3O4@BSA纳米颗粒水溶液中,使得混合后最终TA浓度为0.5mg/ml,Fe3+浓度为0.08mg/ml,Fe3O4@BSA、TA、Fe3+的物质的量的比例是1:40:200,然后使用100kDa MWCO超滤管通过超滤去除游离TA-FeIII络合物,超滤2个循环进行纯化,最终形成纳米颗粒溶液。
本发明浓度单位mM是mmol/L的简写,M是mol/L的简写。
本发明所述TA为单宁酸。
本发明以超顺磁性纳米颗粒为核心,由于纳米颗粒带负电荷,在缓冲液中不稳定,所以在纳米颗粒表面通过静电作用吸附一层蛋白作为“蛋白冠”,在蛋白外通过配位结合多酚-铁络合物。当纳米颗粒进入血液中后,由于铁离子的存在,纳米颗粒会自发吸附血浆中的转铁蛋白,由于病灶部位的细胞膜会过表达转铁蛋白受体,因而纳米探针会靶向病灶部位。
当探针进入病灶部位后,在核磁共振成像仪的磁场中,超顺磁性磁性纳米颗粒具有(T2造影效果),由于病灶部位微环境的pH低于正常组织,酸性条件会T1造影效果。由于病灶组织内部pH分布不均匀,所以铁离子释放率不同,T1信号值则不同,通过T1/T2的比率,可以实现对病灶组织的可视化成像。
相较于现有技术,本发明提供一种用于pH可视化的磁共振成像纳米探针的制备方法,通过步骤1:超顺磁性纳米颗粒的制备;步骤2:将蛋白溶解在水中,然后按一定比例加入超顺磁性纳米颗粒水溶液超顺磁性纳米颗粒@蛋白结构纳米颗粒;步骤3:将多酚-铁溶液与步骤2制备的纳米颗粒混合在一起,充分溶解混合均匀,获得超顺磁性纳米颗粒@蛋白@多酚-铁纳米颗粒;制备得到的纳米探针,用于磁共振成像,通过计量病灶部位微环境pH的T1/T2磁共振比率可以实现病灶微环境pH在体可视化。
本发明提供的纳米探针,以具有T2造影效果的高品质超顺磁性纳米颗粒为核心,在纳米颗粒表面负载具有酸性微环境中响应释放能力的多酚-顺磁性金属离子配合物,铁离子具有T1造影效果,在pH值下顺磁性金属离子的释放率不同,因而在不同pH值的微环境下T1造影效果不同,从而实现对pH值分布的影像检测,以及对病灶部位的准确定位。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图2为Fe3+在不同pH下的释放动力学曲线;
图3为不同pH值下的纳米探针弛豫率随Fe3+释放变化线性回归拟合;
图4为T1及T2 MRI加权像及T1/T2比率计量肿瘤区域pH成像图。
具体实施方式
为了使本领域的技术人员更好地理解本发明中的技术方案,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
须知,本说明书附图所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整,在不影响本发明所能产生的功效及所能达成的目的下,均应仍落在本发明所揭示的技术内容得能涵盖的范围内。
用料说明
六水三氯化铁(FeCl3·6H2O,99.0%)购自山东西亚化工有限公司(中国山东)。
牛白蛋白购自北京阳光科技有限公司(中国北京)。
单宁酸(TA,≥99.7%购自国药集团化学试剂有限公司(中国上海)。
氢氧化钠(NaOH,≥99.7%购自富晨化学试剂有限公司(中国天津)。
油酸(技术级,90%)购自西格玛-奥尔德里奇有限公司(中国上海)。
1-十八烯(技术级,90%)购自西格玛-奥尔德里奇有限公司(中国上海)。
环己烷(≥99.7%)购自达茂化学试剂有限公司(中国天津)。
实施例1
用于pH可视化的磁共振成像纳米探针的制备方法,包括以下步骤:
步骤1)超顺磁性纳米颗粒的制备
采用改进的水热法制备油酸铁:在搅拌下将4.8gNaOH、60mL水、80mL乙醇和120mL油酸混合以形成均相溶液,然后在磁搅拌条件下加入10.8gFeCl3·6H2O和140mL环己烷,将混合物在70℃下搅拌约4小时,并冷却至室温,将所得混合物用水洗涤去除其他残余物,通过旋蒸获得油酸铁;
疏水性Fe3O4纳米颗粒的合成:
将3.6g上述步骤制备的油酸铁和1.13g油酸溶解在25mL的1-十八烯中,将所得溶液以3.3℃/min的速率加热至310℃,然后在氮气保护下保持310℃30min,通过将反应混合物冷却至室温终止制备,用乙醇沉淀得到疏水性Fe3O4纳米颗粒,通过磁选收集,用乙醇洗涤,最后再分散在环己烷中,备用;
亲水性Fe3O4纳米颗粒水溶液的制备:
将上述制备的疏水性Fe3O4纳米颗粒环己烷分散液测量氧化铁浓度(邻菲罗啉法),取含有10mg的Fe3O4纳米颗粒的环己烷分散液,用无水乙醇沉淀、离心,除去上清液后,所得沉淀用1ml环己烷溶解,再加入10mL环己烷、7mL叔丁醇、2mL水和1mL质量百分比为5wt%K2CO3的混合物在室温下搅拌20min,然后逐滴添加4mL Lemieuxvon Rudloff试剂,将所得混合物在40℃下搅拌4小时,再通过离心分离亲水性Fe3O4纳米颗粒,并分别用去离子水、丙酮和乙醇洗涤,将产物分散在50mL pH值为4-5的盐酸中,并将混合物搅拌30min,再将亲水性Fe3O4纳米颗粒通过使用30kDa MWCO超滤管的水超滤2个循环进行纯化,并分散在水中备用,测量所得溶液的氧化铁浓度;
其中,所述Lemieuxvon Rudloff试剂由5.7mM高锰酸钾和0.105M NaIO4水溶液。
步骤2)Fe3O4@BSA纳米颗粒的制备
将BSA溶解于经Milli-Q处理的水中得到BSA水溶液,将制备好的亲水性Fe3O4纳米颗粒水溶液添加到BSA水溶液中,混合后最终浓度为:亲水性Fe3O4纳米颗粒0.2mg/ml,BSA10mg/ml,以形成Fe3O4@BSA纳米颗粒,其中,Fe3O4与BSA的物质的量的比例为1:10,然后使用100kDa MWCO超滤管通过超滤去除游离BSA,得到Fe3O4@BSA纳米颗粒水溶液;
分别将TA水溶液和FeCl3溶液加入到Fe3O4@BSA纳米颗粒水溶液中,使得混合后最终TA浓度为0.5mg/ml,Fe3+浓度为0.08mg/ml,Fe3O4@BSA、TA、Fe3+的物质的量的比例是1:40:200,然后使用100kDa MWCO超滤管通过超滤去除游离TA-FeIII络合物,超滤2个循环进行纯化,最终形成纳米颗粒溶液。
实验例
1、纳米探针的透射电镜(TEM)电镜图
利用透射电子显微镜(TEM,JEM-2100)监测了纳米粒子在合成过程中的形态变化,分别检索了磁性氧化铁(A)、Fe3O4@BSA(B)、(C)。如图1所示,BSA电晕形成后,纳米颗粒的形态没有显著变化,这意味着蛋白质电晕不会破坏Fe3O4纳米颗粒的结构。相反,在颗粒表面络合TA-FeIII后,可以在Fe3O4表面观察到明显的涂层,这清楚地证实了BSA蛋白冠及TA-FeIII层的形成。
2、Fe3+在不同pH值下释放动力学曲线
将纳米颗粒放在不同pH的Tris缓冲液中孵育两小时后超滤,通过普鲁士蓝法测定滤液中的铁浓度,既为释放的铁离子浓度。如图2所示,在中性条件下(pH=7.4)纳米探针非常稳定,而在病灶部位微环境pH值(即pH=6.5)下,Fe3+在最初15分钟内释放量占总量的8.1%,随后不再释放。相对于溶酶体pH值(pH 4.5),在初始阶段可以观察到Fe3+的快速释放过程,其中约占总铁含量的18.6%的铁离子在最初的30分钟内迅速释放,之后释放速率减慢,两小时后该值达到约19.6%。这些结果表明,在酸性条件下,酚羟基被质子化,导致TA与Fe3+的结合亲和力显著降低,纳米探针可以在酸性环境下的病变区域快速触发,但在中性pH的正常组织中保持稳定,这也突出了纳米探针的响应灵敏度和生物安全性。
3、在不同pH值下释放的Fe3+体外弛豫率测量
在7.0T磁共振扫描仪上评估纳米探针,通过对实验数据进行线性回归拟合得到图3,在中性条件下(pH值7.4)纵向弛豫率(r1)为0.79mM-1s-1,当pH值降低到4.5时,弛豫率达到1.14mM-1s-1,显著增加了44.3%。而在pH变化前后横向弛豫率(r2)几乎保持不变(~150mM-1s-1)。纳米探针的磁性可以随pH依赖性Fe 3+释放行为而变化,这使纳米探针具有可激活的磁共振成像(MRI)性能。因此,由于顺磁性Fe 3+释放导致pH值降低而激活的T1 MRI信号使探针仅在病灶部位特异性激活增强T1加权MRI成像,这有助于提高对病灶部位诊断的灵敏度。
4、磁共振MRI成像分析
根据pH响应性Fe3+释放和可激活性MRI的体外表现,用纳米探针检测植入4T1皮下肿瘤的BALB/c小鼠的肿瘤。根据图4所示的T1/T2加权成像结果,静脉注射7h后,肿瘤区域信号出现明显变化。这种变化表明,通过尾静脉递送的纳米探针在体内具有显著的肿瘤靶向能力。由于MRET效应,T1对比度增强不受纳米探针累积的影响,而仅与Fe3+的局部释放有关,在pH值较低的肿瘤区域,Fe3+释放较多,T1信号值较强,而T2信号与探针浓度呈正相关,通过对T1、T2信号的比率计量,可以计算出肿瘤区域的pH值。同时由于高T1信号区代表Fe3+离子大量释放的位置,可以看出Fe3+的释放在空间上是相当不均匀的,也就是说pH在肿瘤内分布不均,这也是众所周知的肿瘤特征,因此本发明的用于磁共振成像,可以实现pH在体可视化。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (2)
1.一种用于pH可视化的磁共振成像纳米探针的制备方法,其特征在于包括以下步骤:
步骤1:超顺磁性纳米颗粒的制备
步骤2:将蛋白溶解在水中,然后按一定比例加入超顺磁性纳米颗粒水溶液,形成超顺磁性纳米颗粒@蛋白结构纳米颗粒;
步骤3:将多酚-铁溶液与步骤2制备的纳米颗粒混合在一起,充分溶解混合均匀,获得超顺磁性纳米颗粒@蛋白@多酚-铁纳米颗粒。
2.如权利要求1所述的用于pH可视化的磁共振成像纳米探针的制备方法,其特征在于包括以下步骤:
步骤1)超顺磁性纳米颗粒的制备
采用改进的水热法制备油酸铁:在搅拌下将4.8gNaOH、60mL水、80mL乙醇和120mL油酸混合以形成均相溶液,然后在磁搅拌条件下加入10.8gFeCl3·6H2O和140mL环己烷,将混合物在70℃下搅拌约4小时,并冷却至室温,将所得混合物用水洗涤去除其他残余物,通过旋蒸获得油酸铁;
疏水性Fe3O4纳米颗粒的合成:
将3.6g上述步骤制备的油酸铁和1.13g油酸溶解在25mL的1-十八烯中,将所得溶液以3.3℃/min的速率加热至310℃,然后在氮气保护下保持310℃30min,通过将反应混合物冷却至室温终止制备,用乙醇沉淀得到疏水性Fe3O4纳米颗粒,通过磁选收集,用乙醇洗涤,最后再分散在环己烷中,备用;
亲水性Fe3O4纳米颗粒水溶液的制备:
将上述制备的疏水性Fe3O4纳米颗粒环己烷分散液测量氧化铁浓度,取含有10mg的Fe3O4纳米颗粒的环己烷分散液,用无水乙醇沉淀、离心,除去上清液后,所得沉淀用1ml环己烷溶解,再加入10mL环己烷、7mL叔丁醇、2mL水和1mL质量百分比为5wt%K2CO3的混合物在室温下搅拌20min,然后逐滴添加4mL Lemieuxvon Rudloff试剂,将所得混合物在40℃下搅拌4小时,再通过离心分离亲水性Fe3O4纳米颗粒,并分别用去离子水、丙酮和乙醇洗涤,将产物分散在50mL pH值为4-5的盐酸中,并将混合物搅拌30min,再将亲水性Fe3O4纳米颗粒通过使用30kDa MWCO超滤管的水超滤2个循环进行纯化,并分散在水中备用,测量所得溶液的氧化铁浓度;
其中,所述Lemieuxvon Rudloff试剂由5.7mM高锰酸钾和0.105M NaIO4水溶液。
步骤2)Fe3O4@BSA纳米颗粒的制备
将BSA溶解于经Milli-Q处理的水中得到BSA水溶液,将制备好的亲水性Fe3O4纳米颗粒水溶液添加到BSA水溶液中,混合后最终浓度为:亲水性Fe3O4纳米颗粒0.2mg/ml,BSA 10mg/ml,以形成Fe3O4@BSA纳米颗粒,其中,Fe3O4与BSA的物质的量的比例为1:10,然后使用100kDa MWCO超滤管通过超滤去除游离BSA,得到Fe3O4@BSA纳米颗粒水溶液;
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