CN106495225B - 一种用于磁共振显影的多糖杂化二氧化锰纳米粒子及其制法和用途 - Google Patents
一种用于磁共振显影的多糖杂化二氧化锰纳米粒子及其制法和用途 Download PDFInfo
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Abstract
本发明公开了一种用于磁共振显影的多糖杂化二氧化锰纳米粒子及其制法和用途。所述制备方法为:将具有优良的水溶性、生物相容性和可生物降解性的多糖同时用作化学还原剂和分散稳定剂,在水溶液中与高锰酸盐进行化学反应,一步法制得多糖杂化二氧化锰纳米粒子。所用天然多糖不仅具有优良的水溶性、生物相容性和可生物降解性,还具有一定的肿瘤靶向性。本发明制备的多糖杂化二氧化锰纳米粒子易于在水体系中均匀分散,安全低毒,且在肿瘤组织微环境条件下具有明显的磁弛豫增强效果,特别适用于胶质瘤、肝癌、乳腺癌等肿瘤组织部位的磁共振显影成像,还可化学偶连抗肿瘤药物,进而实现诊疗一体化。
Description
技术领域
本发明属于生物医用材料领域,具体涉及一种用于磁共振显影的多糖杂化二氧化锰纳米粒子及其制法和用途。
背景技术
锰是一种顺磁性过渡金属,可用作磁共振显影的新型对比剂。与临床上常用的钆类对比剂相比,锰类对比剂的生物安全性更好。锰可以锰盐、配位化合物、氧化物纳米粒等多种形式存在,其中二氧化锰纳米粒子最具应用前景。究其原因,主要是因为二氧化锰纳米粒子作为磁共振显影的对比剂在肿瘤组织微环境中能够转化成具有弛豫增强效果的二价锰离子,进而实现针对肿瘤部位的特异性成像(Nature Nanotechnology,2016,doi:10.1038/nnano.2016.72)。但二氧化锰粒子本身易团聚、难以在水体系中均匀分散,因而很难直接用作磁共振成像的对比剂。为此,目前国内外多采用聚烯丙基胺、聚乙二醇衍生物等水溶性合成类聚合物修饰改性二氧化锰纳米粒子(Prasad P,et al.ACS Nano,2014,8:3202;Kim T,et al.Journal of the American Chemical Society,2011,133:2955)。但尚存在以下主要问题:一是所用水溶性聚合物大都难以生物降解,一些阳离子聚合物还具有一定的生物毒性;二是有关修饰改性涉及多步反应,过程繁杂,难以分离纯化和批量生产;三是修饰改性后的二氧化锰纳米粒子均缺乏肿瘤靶向性,实际应用时还需借助另外的化学反应引入肿瘤靶向因子。
发明内容
为克服上述不足,本发明的目的之一是提供一种磁共振显影用多糖杂化二氧化锰纳米粒子的制备方法,即利用具有优良水溶性、生物相容性和可生物降解性的透明质酸或透明质酸钠在水溶液中原位还原高锰酸盐,一步法制备多糖杂化二氧化锰纳米粒子。其中,所用透明质酸或透明质酸钠既作为有关杂化型纳米粒子制备的还原剂,也作为有关杂化型纳米粒子在水体系中分散的稳定剂,同时还作为肿瘤靶向因子,具有多功能特征。所述的透明质酸或透明质酸钠靶向性是由于其特异性受体CD44在胶质瘤、肝癌、乳腺癌等肿瘤组织部位中呈现高表达。
本发明的另一目的在于提供一种由上述制备方法制得的用于磁共振显影的多糖杂化二氧化锰纳米粒子。
本发明的另一目的是提供上述用于磁共振显影的多糖杂化二氧化锰纳米粒子的应用。
本发明目的通过以下技术方案实现:
一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法,包括以下步骤:将多糖水溶液与高锰酸盐溶液在一定温度下,按照一定比例充分混合均匀;然后进行水热反应;反应完成后,将反应生成的分散液透析洗涤,即得到多糖杂化二氧化锰纳米粒子。
所述多糖水溶液中的多糖与高锰酸盐溶液中的高锰酸盐的质量比为1:0.01~1。多糖水溶液与高锰酸盐溶液优选在室温20-40℃混合。
所述水热反应的温度优选为20~90℃,时间优选为0.2~3h。
所述多糖水溶液质量分数为0.05%~10%。所述多糖水溶液是通过将多糖在20~70℃、pH=3~9下用水充分溶解制得。
所述多糖水溶液中的多糖优选为具有优良水溶性、生物相容性和可生物降解性的透明质酸或透明质酸钠。
所述多糖的分子量优选为5~500KDa。
所述高锰酸盐溶液质量分数优选为0.01%~5%。
所述高锰酸盐溶液中的高锰酸盐优选为高锰酸钠或高锰酸钾。
本发明还提供了一种由上述制备方法制得的用于磁共振显影的多糖杂化二氧化锰纳米粒子。所得多糖杂化二氧化锰纳米粒子可在水中均匀分散,安全低毒。
上述用于磁共振显影的多糖杂化二氧化锰纳米粒子适用于胶质瘤、肝癌、乳腺癌等肿瘤组织部位的磁共振成像,还可通过所用多糖组分化学偶联抗肿瘤药物,达到诊疗一体化的目的。
与现有技术相比,本发明具有以下优点及有益效果:
本发明所涉及的制备方法和流程简单、易于操作、反应条件温和、成本低廉,可应用于工业化大批量生产。
本发明所制备的二氧化锰杂化型纳米粒子可以较好地分散在水溶液中,并且保持较长时间的稳定,同时具有较好的生物相容性且自带肿瘤靶向性。此外,本发明制备的多糖杂化二氧化锰纳米粒子在肿瘤组织微环境(偏酸、高含量谷胱甘肽)条件下能够转化成为具有明显磁弛豫增强效果的二价锰离子(实施例4),特别适用于胶质瘤、肝癌、乳腺癌等肿瘤组织部位的磁共振成像,还可通过所用多糖组分化学偶联抗肿瘤药物,达到诊疗一体化的目的。
附图说明
图1为透明质酸钠和高锰酸钾混合溶液吸光度随反应时间变化曲线。
图2为透明质酸钠杂化二氧化锰纳米粒子的XPS谱图。
图3为透明质酸杂化二氧化锰纳米粒子在水溶液中的粒径分布(动态光散射法测定)和透射电镜照片。
图4为透明质酸杂化二氧化锰纳米粒子的细胞毒性。
图5为透明质酸钠杂化二氧化锰纳米粒子在体外磁共振成像的T1和T2加权图像。
图6为透明质酸钠杂化二氧化锰纳米粒子在体外磁共振成像的T1弛豫率。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。除非特别说明,实施例中所涉及的试剂、方法均为本领域常用的试剂和方法。
实施例1
将0.1g的透明质酸钠(HA,分子量20~50KDa)25℃下温和溶解于20mL水中,将1mg高锰酸钾溶于水5mL水中,将多糖水溶液与高锰酸钾溶液在室温下混合均匀,所得的混合物在40摄氏度下进行水热反应15分钟,利用紫光可见光谱仪实时测定溶液的紫外可见光谱图,如图1所示。高锰酸钾溶液在315nm、525nm和545nm有特征吸收峰,随时间推移高锰酸钾的三个特征峰逐渐消失,与此同时在360nm左右出现一个新的特征吸收峰表明了多糖杂化二氧化锰纳米粒子的生成。随反应时间增强,该吸收峰的强度也不断升高,这是因为,刚开始高锰酸钾还未完全反应完全,随时间进行,反应生成二氧化锰的量也不断增加,同时也导致了吸收峰的红移。反应进行了13分钟之后,峰型不再有变化,这是因为此时高锰酸钾已经完全消耗完,反应终止。所得多糖杂化二氧化锰纳米粒子均匀稳定地分散在水溶液中。
实施例2
将1g的透明质酸钠(HA,分子量10~30KDa)在37℃下温和溶解于40mL水中,将0.10g高锰酸钾溶于水10mL水中,将多糖水溶液与高锰酸盐溶液在37摄氏度下混合均匀,所得的混合物继续在37摄氏度下进行反应2h,用纯水透析后冻干成粉末,通过X射线光电子能谱考察产物的价态,在高分辨谱中可以明显看到四价锰Mn(IV)的2p轨道的特征吸收峰,进一步证实了二氧化锰杂化型纳米粒子的生成。
实施例3
将0.01g的透明质酸钠(HA,分子量5~10KDa)在室温下溶解配置多糖质量分数为0.05%的溶液,与此同时将0.01g高锰酸钾溶于水10mL水中,将多糖水溶液与高锰酸盐溶液在20摄氏度下混合均匀,所得的混合物继续在90摄氏度下进行反应12分钟,用纯水透析并稀释后,取10μL滴于200目表面涂有碳膜的铜网上,1min后,用滤纸吸干多余液体,晾干后利用JEM100CX透射式电子显微镜(日本电子公司,日本)进行观察,放大倍数为50K,通过透射电镜观察其微观形貌,大小约80nm左右,与动态光散射测定结果大约一致。
实施例4
将0.25g的透明质酸(HA,分子量6~9KDa)在30℃下温和溶解于50mL水中,将0.01g一水合高锰酸钠溶于水50mL水中,将多糖水溶液与高锰酸钠溶液在30摄氏度下混合均匀,所得的混合物继续在30摄氏度下进行水热反应0.5h,用pH=7.4的缓冲溶液透析后,通过BI-200SM型光散射仪(Brookhaven Instruments,USA)对其粒径及粒径分布进行表征,激发光源波长为532nm,散射角为90度。所有测试均在25摄氏度下进行,并重复五次。得到粒径分布图,如图3所示,平均粒径为80nm左右。取10μL滴于200目表面涂有碳膜的铜网上,1min后,用滤纸吸干多余液体,晾干后利用JEM100CX透射式电子显微镜(日本电子公司,日本)进行观察,电镜照片如图3所示。采用CCK8试剂盒测定样品的体外细胞(大鼠胶质瘤C6细胞)毒性,结果如图4所示,在最终锰浓度固定为5~50μg/mL的条件下,多糖杂化二氧化锰纳米粒子与C6细胞共同孵育24h后的细胞存活率仍然达到90%以上,说明该多糖杂化二氧化锰纳米粒子在该浓度下表现出较低的细胞毒性。
实施例5
将1g的透明质酸钠(HA,分子量(8~10KDa)在37℃下温和溶解于40mL水中,将0.10g高锰酸钾溶于水10mL水中,将多糖水溶液与高锰酸盐溶液在37摄氏度下混合均匀,所得的混合物继续在30摄氏度下进行水热反应0.5h,用pH7.4的缓冲溶液透析后得到了透明质酸杂化二氧化锰纳米粒子分散液。将分散液稀释成0.02~0.5mmol/L不同浓度,置于96孔细胞板内,在Intera型1.5T磁共振成像仪上(Philips,the Netherlands)进行体外MRI扫描,具体操作为:吸取100微升锰浓度为1mmol/L透明质酸多糖杂化二氧化锰纳米粒子分散液置于96孔板内,其内分别依次加入100微升PBS溶液、pH值为5的酸性溶液、pH值为5的酸性溶液及过氧化氢(H2O2,肿瘤细胞中含量偏高)、pH值为5的谷胱甘肽溶液(GSH,肿瘤细胞中含量偏高),使得每孔最终液体量为200毫升,按照1:2的浓度梯度分别进行稀释后行MRI扫描。MRI检查使用1.5T磁共振成像仪,成像的序列包括:T1加权成像、T2加权成像、T1图序列及T2图序列。主要的成像参数如下:T1加权成像:TR/TE=500/15ms,层厚/层距=1.5/0mm,矩阵=256×256,视野=90×60mm,NSA=3次。T2加权成像:TR/TE=2600/100ms,层厚/层距=1.5/0mm,矩阵=256×256,视野=90×60mm,NSA=3次。T1图成像使用SE及反转恢复(IR)序列交替的混合(Mix)序列测量T1驰豫时间,SE序列的TR/TE为3500/20ms,IR序列4000ms/20ms,TI:400ms,8个回波,层厚/层距为1.5/0mm,视野90×60mm,矩阵256×256,NSA为1次,翻转角90°。T2图成像采用单层面多SE序列测量T2驰豫时间,TR/TE=2000ms/20–160ms,8个回波,层厚=1.5mm,矩阵=256×256,视野=90×60mm,NSA=3次。在工作站上,利用感兴趣区技术测量不同条件下各浓度梯度的对比剂的T1及T2弛豫时间。计算r1与r2弛豫率时,以锰浓度(mM/L)为横坐标(X),所对应T1或T2值的倒数为纵坐标(Y)作图6,所得直线的斜率即弛豫率。可以看出在肿瘤细胞高表达的过氧化氢和谷胱甘肽存在的酸性环境下,溶液的弛豫率显著增强,因此,该多糖杂化二氧化锰纳米粒子特别适用于肿瘤组织微环境(偏酸、高含量谷胱甘肽)条件下的磁共振成像。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法,其特征在于,包括以下步骤:将透明质酸或透明质酸钠水溶液与高锰酸盐溶液按照比例充分混合均匀;然后进行水热反应;反应完成后,将反应生成的分散液透析洗涤,即得到多糖杂化二氧化锰纳米粒子;
所述透明质酸或透明质酸钠水溶液中的透明质酸或透明质酸钠与高锰酸盐溶液中的高锰酸盐的质量比为1:0.01~1;
所述水热反应的温度为20~90℃,时间为0.2~3h。
2.根据权利要求1所述的一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法,其特征在于,所述透明质酸或透明质酸钠水溶液质量分数为0.05%~10%。
3.根据权利要求1所述的一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法,其特征在于,所述透明质酸或透明质酸钠的分子量为5~500KDa。
4.根据权利要求1所述的一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法,其特征在于,所述高锰酸盐溶液质量分数为0.01%~5%。
5.根据权利要求1所述的一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法,其特征在于,所述高锰酸盐溶液中的高锰酸盐为高锰酸钠或高锰酸钾。
6.一种用于磁共振显影的多糖杂化二氧化锰纳米粒子,其特征在于,其由权利要求1至5任一项所述的一种用于磁共振显影成像的多糖杂化二氧化锰纳米粒子的制备方法制得。
7.权利要求6所述的用于磁共振显影的多糖杂化二氧化锰纳米粒子的应用,其特征在于,所述用于磁共振显影的多糖杂化二氧化锰纳米粒子应用于肿瘤组织部位的磁共振成像,或是通过所用透明质酸或透明质酸钠组分化学偶联抗肿瘤药物实现诊疗一体化。
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