CN113813406A - 一种mri和化学激发光动力治疗动脉粥样硬化的纳米颗粒 - Google Patents

一种mri和化学激发光动力治疗动脉粥样硬化的纳米颗粒 Download PDF

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CN113813406A
CN113813406A CN202111163702.8A CN202111163702A CN113813406A CN 113813406 A CN113813406 A CN 113813406A CN 202111163702 A CN202111163702 A CN 202111163702A CN 113813406 A CN113813406 A CN 113813406A
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牡丹
张冰
徐标
谢峻
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Nanjing Drum Tower Hospital
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Abstract

本发明属于靶向动脉粥样硬化斑块的诊疗一体化纳米颗粒制备技术领域，提供一种结合MRI和过氧化氢激发光动力治疗动脉粥样硬化的纳米颗粒，所述纳米颗粒为Fe3+‑邻苯二酚交联的双草酸酯负载的mPEG‑Plys‑(DA‑Ce6)纳米颗粒FeCNPs，FeCNPs可以有效地聚集到动脉粥样硬化斑块中；FeCNPs在pH7.4和pH6.5条件下都表现出较钆双胺高的T1磁共振成像效果；在炎性巨噬细胞或泡沫巨噬细胞内过表达的高浓度过氧化氢的作用下，CPPO转化为高能中间体，该中间体将能量转移给光敏剂二氢卟吩e6（Ce6），受激发的光敏剂将氧转变为单线态氧，从而引起细胞死亡，实现针对动脉粥样硬化的化学激发光动力治疗。

Description

一种MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒
技术领域
本发明属于靶向动脉粥样硬化斑块的诊疗一体化纳米颗粒制备技术领域，具体涉及一种MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒及其制备方法。
背景技术
光动力疗法(PDT)已成为一种新兴的肿瘤治疗方法。光动力疗法具有创伤小，副作用小，选择性和适用性好等优势，它的功能需要三个要素：光敏剂、用于激活光敏剂的光源和氧。在激发光照射下，光敏剂将细胞内的氧转化为单线态氧，从而杀死目标细胞。而巨噬细胞在动脉粥样硬化斑块中大量富集，且对疾病的进展发挥重要作用，已经证明清除斑块内的巨噬细胞是治疗动脉粥样硬化的有效方法。因此，光动力疗法在治疗动脉粥样硬化上具有极大潜力。然而，近红外光的组织穿透深度只有几毫米，不能满足动脉粥样硬化的治疗要求，导致光动力疗法治疗动脉粥样硬化的试验大多停留在细胞层面。
为了克服光的组织穿透能力差的问题，当前多使用插入主动脉的光纤导管实现局部给药并激发光敏剂。但插入导管的方式也增加了感染的风险，这可能会促进动脉粥样硬化的进展。另一种方法是使用低强度超声（更强的组织穿透能力）激发光敏剂，也称为声动力疗法。尽管这两种方法在克服光穿透性差方面表现出明显的优势，但它们在斑块诊断和定位方面表现不理想，这对于提高治疗效果和减少副作用至关重要。
动脉粥样硬化是一种慢性炎症疾病，斑块内炎性巨噬细胞和泡沫巨噬细胞中高表达过氧化氢。化学激发光动力疗法是利用过氧化氢和高能化合物之间的化学反应来原位激发附近的光敏剂，摆脱了外部光源的限制，且提高了对板块中巨噬细胞的选择性杀伤。双草酸酯（CPPO）是常用的高能化合物，它可以和过氧化氢（H2O2）反应，产生化学能激活光敏剂。然而迄今为止，还没有化学激发光动力疗法治疗动脉粥样硬化的案例。
发明内容
为解决上述问题，本发明公开了一种结合磁共振成像（MRI）和化学激发光动力治疗动脉粥样硬化的纳米颗粒，克服了光动力疗法治疗疾病（如动脉粥样硬化）过程中外部光组织穿透能力差的缺陷，实现了炎症部位特定条件（高浓度过氧化氢）激发的光动力治疗。
为了达到上述目的，本发明提供如下技术方案：
本发明提供一种MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒，所述纳米颗粒为mPEG-Plys-(DA-Ce6)、双草酸酯、Fe3+的复合材料FeCNPs。
进一步地，所述mPEG-Plys-(DA-Ce6)的结构式如式Ⅰ所示：
Figure DEST_PATH_IMAGE002AA
；
其中，a为3~9的整数；b为2~5的整数；q为40~50的整数。
本发明还提供一种MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒的制备方法，包括以下步骤：
（1）将双草酸酯和mPEG-Plys-(DA-Ce6)溶于溶剂中，用纯水透析得到悬浮液；
（2）将悬浮液通过0.45μm孔径的微孔膜过滤获得负载双草酸酯的mPEG-Plys-(DA-Ce6)纳米颗粒；
（3）将FeCl3加入步骤（2）得到的负载双草酸酯的mPEG-Plys-(DA-Ce6)纳米颗粒中，避光搅拌，得到Fe3+-邻苯二酚交联的双草酸酯负载的mPEG-Plys-(DA-Ce6)纳米颗粒FeCNPs。
进一步地，所述步骤（1）中双草酸酯与mPEG-Plys-(DA-Ce6)的质量比为1:8~12；所述双草酸酯（mg）与溶剂（mL）的质量体积比为1：0.2~0.5。
进一步地，所述溶剂为二甲基亚砜、N, N-二甲基甲酰胺中的一种或多种。
进一步地，所述步骤（3）中FeCl3与负载双草酸酯的mPEG-Plys-(DA-Ce6)的邻苯二酚基团的质量比为1:0.6~1.5。
进一步地，所述mPEG-Plys-(DA-Ce6)的制备方法，包括以下步骤：
（1）将单甲醚聚乙二醇胺和N6-苄氧羰基-L-赖氨酸环内酸酐溶解在无水DMF中，氮气气氛下，35~45℃下反应48~72小时，减压除去溶剂，残余物溶于二氯甲烷后滴加于冰乙醚中，形成沉淀，得到聚乙二醇-聚苄氧羰基赖氨酸；
（2）将聚乙二醇-聚苄氧羰基赖氨酸溶解于三氟乙酸中，0~5℃下，滴加浓度为33wt%的溴化氢乙酸溶液，反应30~60分钟，升温至20~30℃继续反应1~2小时，反应液滴加至冰乙醚中，分离油状沉淀，纯化、冷冻干燥得到聚乙二醇-聚赖氨酸；
（3）将聚乙二醇-聚赖氨酸与活化的二氢卟吩e6混合，20~30℃搅拌反应24~48小时，加入活化的3,4-二羟基苯乙酸，继续搅拌反应24~48小时，过滤、纯化得到mPEG-Plys-(DA-Ce6)。
进一步地，所述步骤（1）中单甲醚聚乙二醇胺与N6-苄氧羰基-L-赖氨酸环内酸酐的质量比为1:1~2；所述单单甲醚聚乙二醇胺（g）与DMF（mL）的质量体积比为1:1~20；所述二氯甲烷与DMF的体积比为1:1~2。
进一步地，所述步骤（2）中聚乙二醇-聚苄氧羰基赖氨酸（g）与三氟乙酸（mL）的质量体积比为1:5~10；所述三氟乙酸与溴化氢乙酸溶液的体积比为1:1~2。
进一步地，所述步骤（3）中所述二氢卟吩e6与聚乙二醇-聚赖氨酸的质量比为1:2~3；所述3,4-二羟基苯乙酸与聚乙二醇-聚赖氨酸的质量比为1:2~3。
与现有技术相比，本发明具有如下优点和有益效果：
本发明提供的一种Fe3+-邻苯二酚交联的CPPO负载的mPEG-Plys-(DA-Ce6)纳米颗粒子FeCNPs，FeCNPs可以有效地聚集到动脉粥样硬化斑块中；FeCNPs在pH7.4和pH6.5条件下都表现出较钆双胺高的T1磁共振成像效果；在炎性巨噬细胞或泡沫巨噬细胞内过表达的高浓度过氧化氢的作用下，CPPO转化为高能中间体，该中间体将能量转移给光敏剂二氢卟吩e6（Ce6），受激发的光敏剂将氧转变为单线态氧，从而引起细胞死亡，实现针对动脉粥样硬化的化学激发光动力治疗。
附图说明
图1A为本发明纳米颗粒子FeCNPs的结构示意图，B为FeCNPs工作原理示意图；
图2为本发明纳米颗粒子FeCNPs的透射电镜图；
图3为本发明FeCNPs和钆双胺在pH7.4和6.5下的MRI图片和弛豫率，其中，左图为MRI图片，右图为弛豫率；
图4为本发明FeCNPs产生单线态氧随时间的变化图；
图5为FeCNPs与RAW264.7共孵育不同时间的共聚焦图像；
图6为本发明FeCNPs、CPPO和未载药纳米颗粒的细胞毒性，其中，A为未载药纳米颗粒、B为CPPO、C为FeCNPs；
图7为不同时间点钆双胺和FeCPNs对动脉粥样硬化斑块的磁共振成像图。
具体实施方式
以下将结合具体实施例对本发明提供的技术方案进行详细说明，应理解下述具体实施方式仅用于说明本发明而不用于限制本发明的范围。
实施例1
聚合物mPEG-Plys-(DA-Ce6)的合成
将1g单甲醚聚乙二醇胺（Mw2000）和1gN6-苄氧羰基-L-赖氨酸环内酸酐溶解在10mL无水DMF中。在氮气气氛下于35℃搅拌3天。随后减压除去溶剂，将残余物溶解在10 mL二氯甲烷中，逐滴加入冰乙醚中，形成沉淀。重复沉淀操作3次，获得聚乙二醇-聚苄氧羰基赖氨酸。
取1g聚乙二醇-聚苄氧羰基赖氨酸溶于5 mL三氟乙酸中，0oC条件下，滴加5 mL溴化氢乙酸溶液(33wt％)。0oC条件下搅拌30分钟后，将反应在25oC下再搅拌1小时。随后将反应液滴加到冰乙醚中，分离出油状沉淀，并用纯水透析(MWCO 5000 Da)进一步纯化，最后冷冻干燥得到聚乙二醇-聚赖氨酸。
取0.56g聚乙二醇-聚赖氨酸，溶解在5 mL DMSO中，加入0.21 g NHS/DCC活化的二氢卟吩e6，25oC搅拌24小时，再加入0.25 g NHS/DCC活化的3,4-二羟基苯乙酸，25oC下再搅拌24小时。之后，将反应液过滤并用纯水透析纯化，得到聚合物mPEG-Plys-(DA-Ce6)。mPEG-Plys-(DA-Ce6)的结构如式Ⅰ所示：
Figure DEST_PATH_IMAGE003
；
其中，a为8；b为3；q为44。
实施例2
FeCNPs纳米颗粒的制备
5mg CPPO和50 mg mPEG-Plys-(DA-Ce6)溶解在2mL DMSO中，然后用纯水透析(MWCO 3500)24小时。之后，将悬浮液通过0.45μm孔径的微孔膜过滤获得负载CPPO的mPEG-Plys-(DA-Ce6)纳米颗粒53.6mg；加入3.6 mgFeCl3，避光搅拌30min，得到FeCNPs，其结构示意图如图1所示，透射电镜图如图2所示。
实施例3
体外MRI成像实验
钆双胺：欧乃影®通用电气药业（上海）；
FeCNPs采用上述方法制得。
分别用磷酸缓冲溶液配置pH为7.4和6.5的钆双胺和FeCNPs，其中Gd或Fe的摩尔浓度分别0.1、0.2、0.4、0.6和0.8 mM。体外分别测试其MRI信号值，拟合曲线，计算弛豫率，结果如图3所示。
尽管当pH从7.4变为6.5后，FeCNPs的弛豫率减小了5.6%，而钆双胺的弛豫率增大了54%，但是FeCNPs的弛豫率是钆双胺的2.86倍以上。以上结果表明，FeCNPs的MRI效果优于临床使用的钆双胺。
实施例4
使用单线态氧特异性分子探针9,10-蒽基-双(亚甲基)二丙二酸（ABDA）研究了在过氧化氢存在下由FeCNPs产生的单线态氧。将ABDA添加到FeCNPs悬浮液中，FeCNPs和ABDA的最终浓度分别为0.5mg/mL和100μM。将混合物分成五等份装在小瓶中，并在37oC下避光孵育。同时将过氧化氢添加到每个小瓶中至终浓度为10mM，每两分钟测量一个小瓶的紫外吸收光谱光谱；FeCNPs产生单线态氧随时间的变化如图4所示，随着孵育时间的延长，ABDA的吸收强度逐渐降低，表明单线态氧的逐渐生成。
实施例5
将小鼠巨噬细胞RAW264.7接种在玻璃底板中并孵育12小时。细胞与磷酸缓冲液配制的FeNPs (0.2 mg/mL)共培养1小时、3小时和6小时，然后吸取上清液，用4%多聚甲醛固定并用DAPI标记。通过共聚焦激光扫描显微镜（CLSM，Leica SP5）拍摄细胞的荧光图像，如图5所示，随着培养时间的延长，细胞内观察到的荧光越来越强，表明更多的FeCNPs被细胞摄取。
实施例6
取经LPS刺激小鼠巨噬细胞RAW264.7和经oxLDL刺激的RAW264.7，分别接种在96孔板中，用不同浓度的未载CPPO的纳米颗粒（FeNPs：Fe3+-邻苯二酚交联的mPEG-Plys-(DA-Ce6)纳米粒），CPPO和FeCNPs处理细胞48h，MTT法计算细胞存活率。测试结果如图6所示，在测试浓度下，未载药纳米颗粒和CPPO几乎没有细胞毒性，而FeCNPs表现出明显的浓度依赖性细胞毒性，且RAW264.7细胞经刺激后，FeCNPs的细胞毒性更强。
实施例7
取2只经过4周高脂饮食的ApoE-/-小鼠，分别尾静脉注射0.2 mL gadodiamide（50mM）和FeCNPs（3.5 mg/mL），立即进行磁共振成像，并且每隔半个小时再次成像。如图7所示，与gadodiamide相比，FeCNPs可以有效地聚集到动脉粥样硬化斑块中，增强斑块的T1磁共振造影，观察到明显的动脉粥样硬化斑块。
综上所述，FeCNPs可以有效地被巨噬细胞摄取，例如，动脉粥样硬化斑块，利用炎症部位的氧化应激，实现诊断-治疗一体的MRI成像和光动力治疗。
本发明方案所公开的技术手段不仅限于上述实施方式所公开的技术手段，还包括由以上技术特征任意组合所组成的技术方案。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。

Claims (10)

1.一种MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒，其特征在于，所述纳米颗粒为包括mPEG-Plys-(DA-Ce6)、双草酸酯、Fe3+的复合材料FeCNPs。
2.根据权利要求1所述的一种MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒，其特征在于，所述mPEG-Plys-(DA-Ce6)的结构式如式Ⅰ所示：
Figure 300808DEST_PATH_IMAGE002
；
其中，a为3~9的整数；b为2~5的整数；q为40~50的整数。
3.一种如权利要求1-2任一项所述的MRI和化学激发光动力治疗动脉粥样硬化的纳米颗粒的制备方法，其特征在于，包括以下步骤：
（1）将双草酸酯和mPEG-Plys-(DA-Ce6)溶于溶剂中，用纯水透析得到悬浮液；
（2）将悬浮液通过0.45μm孔径的微孔膜过滤获得负载双草酸酯的mPEG-Plys-(DA-Ce6)纳米颗粒；
（3）将FeCl3加入步骤（2）得到的负载双草酸酯的mPEG-Plys-(DA-Ce6)纳米颗粒中，避光搅拌，得到FeCNPs。
4.根据权利要求3所述的制备方法，其特征在于，所述步骤（1）中双草酸酯与mPEG-Plys-(DA-Ce6)的质量比为1:8~12；所述双草酸酯与溶剂的质量体积比为1：0.2~0.5。
5.根据权利要求4所述的制备方法，其特征在于，所述溶剂为二甲基亚砜、N, N-二甲基甲酰胺中的一种或多种。
6.根据权利要求3所述的制备方法，其特征在于，所述步骤（3）中FeCl3与负载双草酸酯的mPEG-Plys-(DA-Ce6)的质量比为1: 7~18。
7.根据权利要求3所述的制备方法，其特征在于，所述mPEG-Plys-(DA-Ce6)的制备方法，包括以下步骤：
（1）将单甲醚聚乙二醇胺和N6-苄氧羰基-L-赖氨酸环内酸酐溶解在无水DMF中，氮气气氛下，35~45℃下反应48~72小时，减压除去溶剂，残余物溶于二氯甲烷后滴加于冰乙醚中，形成沉淀，得到聚乙二醇-聚苄氧羰基赖氨酸；
（2）将聚乙二醇-聚苄氧羰基赖氨酸溶解于三氟乙酸中，0~5℃下，滴加浓度为33wt%的溴化氢乙酸溶液，反应30~60分钟，升温至20~30℃继续反应1~2小时，反应液滴加至冰乙醚中，分离油状沉淀，纯化、冷冻干燥得到聚乙二醇-聚赖氨酸；
（3）将聚乙二醇-聚赖氨酸与活化的二氢卟吩e6混合，20~30℃搅拌反应24~48小时，加入活化的3,4-二羟基苯乙酸，继续搅拌反应24~48小时，过滤、纯化得到mPEG-Plys-(DA-Ce6)。
8.根据权利要求7所述的制备方法，其特征在于，所述步骤（1）中单甲醚聚乙二醇胺与N6-苄氧羰基-L-赖氨酸环内酸酐的质量比为1:1~2；所述单甲醚聚乙二醇胺与DMF的质量体积比为1:1~20；所述二氯甲烷与DMF的体积比为1:1~2。
9.根据权利要求7所述的制备方法，其特征在于，所述步骤（2）中聚乙二醇-聚苄氧羰基赖氨酸与三氟乙酸的质量体积比为1:5~10；所述三氟乙酸与溴化氢乙酸溶液的体积比为1:1~2。
10.根据权利要求7所述的制备方法，其特征在于，所述步骤（3）中所述二氢卟吩e6与聚乙二醇-聚赖氨酸的质量比为1:2~3；所述3,4-二羟基苯乙酸与聚乙二醇-聚赖氨酸的质量比为1:2~3。
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