CN108635595B - 基于光响应聚合的超小氧化铁纳米探针及其制备和应用 - Google Patents
基于光响应聚合的超小氧化铁纳米探针及其制备和应用 Download PDFInfo
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- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
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Abstract
本发明涉及一种基于光响应聚合的超小氧化铁纳米探针及其制备和应用。该纳米探针由表面修饰了NH2‑PEG‑(DA)‑FA的超小氧化铁纳米颗粒组成。制备方法包括:Fe3O4‑COOH NPs制备,DA制备,活化的NHS‑DA制备,NH2‑PEG‑(DA)‑FA制备,Fe3O4‑PEG‑(DA)‑FA NPs制备。该纳米探针具有优异的生物相容性,长的血液循环时间和特异性靶向炎症功能。制备方法简单,成本低,具有产业化实施的前景。
Description
技术领域
本发明属于T1/T2双模态MR成像探针及其制备和应用领域,特别涉及一种基于光响应聚合的超小氧化铁纳米探针及其制备方法和应用。
背景技术
精准医学的前提是对疾病的精准诊断并指导个性化治疗来提高治疗的效果。对于致命的炎症感染疾病而言,非侵入式的磁共振成像(MRI)技术由于高空间分辨率可以实现对炎症的精准成像诊断,从而提高炎症感染的治疗效果。MRI的造影剂分为T1阳性造影剂(顺磁性材料)和T2阴性造影剂(超顺磁性材料)两种。T1/T2双模态MRI有利于克服两种成像各自的不足和联合两种成像的优势来提高诊断的准确率。目前,T1/T2双模态MRI造影剂的设计通常是将两种或两种以上不同成像功能的MRI造影剂整合到同一个纳米器件中,如将T1阳性造影剂-钆(Gd)或锰(Mn)试剂与T2阴性造影剂-超顺磁性氧化铁等整合到一个纳米系统中用于T1/T2双模态MRI。然而,这种整合通常是复杂的,而且Gd-和Mn-试剂对人体也是具有较大的毒性。近年来的研究发现粒径小于3nm的超小氧化铁具有优异的T1MRI效果,而且超小氧化铁由于小尺寸而具有避免快速肾清除的能力和容易从伤口渗透过血管进入病灶(Luo Y.et al.Nanoscale.2015,7:14538-14546.)。然而,小尺寸也导致超小氧化铁更容易反进入血液循环,从而使其无法有效地在病灶处聚集。另外,许多研究结果表明,大尺寸的纳米颗粒可以限制其从病灶溢出重新进入血液循环而增加病灶处的聚集,以及叶酸分子(FA)的修饰具有特异性靶向炎症的功能。因此,我们希望能通过一种新的策略来实现超小氧化铁聚合为大尺寸氧化铁而增强其在关节炎处的聚集以及实现对关节炎的T1/T2双模态MRI。
目前虽然已经发展了少量具有聚合功能的纳米颗粒,如超小氧化铁自组装成大尺寸团簇(Wang,L.et al.ACS Nano.2017,11:4582-4592.)和巯基化金纳米颗粒的自组装聚合(Deng,H.et al.Adv.Mater.2015,27:3645-3653.),但这几种纳米颗粒和自组装方法都完全不适合用于体内的精准可控的聚合。此外,一些具有pH敏感聚合的金纳米颗粒或酶敏感聚合的氧化铁在肿瘤微环境能实现响应聚合用于肿瘤的诊疗。然而,这些微环境响应聚合颗粒由于在复杂的生物系统(血液核酸酶的敏感和诱导免疫应答)的影响而导致不规则或无效的聚合。而且许多炎症环境也不具备这种pH微环境或特定的酶环境,不能实现pH或酶响应聚合。光响应聚合技术能克服上述问题而实现纳米颗粒在炎症处的精准控制聚合。在过去的几年中,许多光响应聚合纳米颗粒基于光响应分子的修饰在体外的应用已被研究。然后,光响应聚合的超小氧化铁在体内的自组装及精准诊断应用从未实现过,以及光响应开关的合成通常很复杂。因此,发展一种新的策略利用超时空精准可控的光响应聚合超小氧化铁来增加其关节炎处聚集和用于关节炎的T1/T2双模态MRI是非常有意义的。
检索国内外有关关节炎的T1/T2双模态MRI纳米探针方面的文献和专利结果表明,目前还没有发现基于光响应聚合的超小氧化铁纳米探针及其制备和靶向关节炎T1/T2双模态MRI诊断应用方面的报道。
发明内容
本发明所要解决的技术问题是提供一种基于光响应聚合的超小氧化铁纳米探针及其制备方法和应用,以克服现有技术纳米颗粒不能在炎症处精准控制聚合的缺陷。
本发明的一种基于光响应聚合的超小氧化铁纳米探针,该纳米探针由表面修饰了3-甲基-3H-双吖丙啶-3-丙酸和叶酸化聚乙二醇混合物NH2-PEG-(DA)-FA的超小氧化铁纳米颗粒组成,其中NH2-PEG-(DA)-FA和超小氧化铁纳米颗粒的质量比为7-8:56-60。
本发明的一种基于光响应聚合的超小氧化铁纳米探针的制备方法,包括:
(1)将无水氯化铁溶解在溶剂中,超声震荡,加入二水合柠檬酸钠,搅拌至溶液澄清,加入无水醋酸钠,继续搅拌至溶液澄清,将溶液转移至高压反应釜中溶剂热反应,冷却,离心,弃上清液,回溶,烘干,得到表面富含羧基的超小四氧化三铁纳米颗粒Fe3O4-COOHNPs,其中无水氯化铁、溶剂、二水合柠檬酸钠和无水醋酸钠的比例为4-5mmol:40-45mL:1.6-2mmol:12-15mmol;
(2)在氮气保护下将乙酰丙酸加入氨甲醇溶液中,搅拌,加入羟胺磺酸甲醇溶液,继续搅拌,提纯,将提纯得到的液体溶解于溶剂中,依次加入三乙胺和碘甲醇溶液,搅拌反应,稀释,冲洗,干燥,纯化,得到淡黄色油状3-甲基-3H-双吖丙啶-3-丙酸DA,其中乙酰丙酸、氨甲醇溶液、羟胺磺酸甲醇溶液、三乙胺和碘甲醇溶液的比例为2.0-2.5g:25-30mL:3.0-3.5g:4.3-5.0mL:2.1-2.4g;
(3)将步骤(2)中DA溶于溶剂中,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和N-羟基琥珀酰亚胺NHS活化,稀释,冲洗,干燥,纯化,得到活化的NHS-DA,其中DA、EDC和NHS的质量比为0.35-0.40:0.57-0.60:0.34-0.40;
(4)将步骤(3)中活化的NHS-DA、氨基端聚乙二醇NH2-PEG-NHBOC与活化的叶酸NHS-FA分别溶解在溶剂中,避光混合搅拌,加入稀盐酸,继续避光搅拌反应,透析冻干,得到DA和FA修饰的聚乙二醇混合物NH2-PEG-(DA)-FA,其中NHS-DA、NH2-PEG-NHBOC、NHS-FA和稀盐酸的比例为5.6-6.0g:50-55g:13.7-14.5g:50-55mL;
(5)将步骤(1)中Fe3O4-COOH NPs、步骤(4)中NH2-PEG-(DA)-FA分别溶于溶剂中,得到Fe3O4-COOH NPs溶液和NH2-PEG-(DA)-FA溶液,将Fe3O4-COOH NPs溶液经EDC溶液和NHS溶液活化,加入NH2-PEG-(DA)-FA溶液避光搅拌反应,透析冻干,得到超小氧化铁的靶向T1/T2双模态MRI纳米探针Fe3O4-PEG-(DA)-FA NPs,其中Fe3O4-COOH NPs、NH2-PEG-(DA)-FA、EDC和NHS的质量比为56-60:7-8:144-150:70-75。
所述步骤(1)中溶剂为二甘醇。
所述步骤(1)中搅拌是在加热至80-90℃条件下进行的。
所述步骤(1)中溶剂热反应温度为190-200℃,溶剂热反应时间为5-6h。
所述步骤(1)中离心速率为8000-9000rpm,离心时间为15-30min。
所述步骤(1)中回溶为:用无水乙醇回溶,重复操作2-4次。
所述步骤(1)中烘干温度为60-70℃。
所述步骤(2)中加入氨甲醇溶液、依次加入三乙胺和碘甲醇溶液均是在0℃下进行的。
所述步骤(2)中搅拌时间为2-4h;继续搅拌为:室温搅拌过夜。
所述步骤(2)中搅拌反应为:搅拌反应直至出现深褐色后,继续搅拌10-20min;氨甲醇溶液的当量浓度为7N;溶剂为无水甲醇。
所述步骤(2)中提纯和将提纯得到的液体溶解于溶剂中具体为:在真空减压浓缩下得到残渣再分散于无水甲醇中,之后过滤除去白色沉淀物,滤液通过减压蒸馏进行浓缩并重新溶解在无水甲醇中。
所述步骤(2)中稀释是用乙酸乙酯;冲洗是用稀盐酸和硫代硫酸钠饱和水溶液连续冲洗。
所述稀盐酸的当量浓度为1N。
所述步骤(2)中纯化是用真空蒸发和硅胶层析纯化。
所述步骤(3)中溶剂为二氯甲烷;加入EDC和NHS是在0℃下进行的。
所述步骤(3)中活化温度为室温,活化时间为2-3h。
所述步骤(3)中稀释是用DCM;冲洗是用纯水冲洗3-4次和盐水冲洗1-2次。
所述纯水体积为75-100mL,盐水体积为25-50mL。
所述步骤(3)中纯化是用真空浓缩和硅胶层析纯化。
所述步骤(4)中溶剂为二甲基亚砜;稀盐酸浓度为2-4M。
所述步骤(4)中避光混合搅拌时间为2-4d;继续避光搅拌反应时间为1-2h;透析为:用透析膜在蒸馏水中透析60-80h,透析膜为纤维素透析膜MWCO=2000。
所述步骤(5)中溶剂为DMSO;经EDC溶液和NHS溶液活化具体为:加入EDC溶液避光混合搅拌20-40min,之后加入NHS溶液,继续避光搅拌反应2-4h。
所述步骤(5)中避光搅拌反应时间为2-4d;透析为:用透析膜在蒸馏水中透析60-80h,透析膜为纤维素透析膜MWCO=8000-14000。
本发明的一种基于光响应聚合的超小氧化铁纳米探针的应用。包括用于增加关节炎处的聚合及靶向关节炎的T1/T2双模态MR成像。
本发明利用一步溶剂热法合成具有高r1弛豫率的超小氧化铁纳米颗粒,再合成具有光响应交联功能的NHS-DA,用稀盐酸脱去-BOC基团得到含氨基端的NH2-PEG-(DA)-FA,然后NH2-PEG-(DA)-FA修饰到超小氧化铁上能提高超小氧化铁的生物相容性,延长其血液循环时间,赋予光响应聚合和炎症特异性靶向功能,该纳米探针具有优异的体内精准可控光响应聚合,能增加其在关节炎处的聚集和靶向T1/T2双模态MRI效果,实现关节炎的精准诊断,为T1/T2双模态MRI纳米探针的开发提供了一种新方法。
本发明使用透射电子显微镜(TEM)、核磁共振氢谱和碳谱(1H,13C NMR)、热重分析(TGA)、电势粒径(zeta potential and DLShydodynamic diameter)、细胞活力分析(CCK8测试)以及体内靶向T1/T2双模态MRI表征本发明制备的基于光响应聚合的超小氧化铁的靶向T1/T2双模态MRI纳米探针的应用前景。
有益效果
(1)本发明通过合成方法的改良,利用一步溶剂热法合成具有高r1弛豫率(r1=3.83mM-1s-1)的超小氧化铁纳米颗粒,高的r1弛豫率为实现T1/T2双模态MR成像提供了可能,以及纳米探针的制备方法简单,成本较低,具有产业化实施的前景;
(2)本发明制备的纳米探针在体内具有优异的生物相容性,长的血液循环时间和特异性靶向炎症功能;
(3)本发明制备的纳米探针在体内能实现精准可控的光响应聚合以及增加其在关节炎处的聚集;
(4)本发明制备的纳米探针仅以一种铁基纳米颗粒实现了同时具有靶向关节炎T1/T2双模态MR成像效果,具有很好的应用前景。
附图说明
图1为本发明纳米探针的合成及应用示意图;
图2为实施例1中Fe3O4-COOH NPs的TEM图,其中插图为Fe3O4-COOH NPs的TEM的放大图;
图3为实施例1中NHS-DA的1H NMR图(a)和13C NMR图(b);
图4为实施例1中NH2-PEG-(DA)-FA的1H NMR图;
图5为实施例1中Fe3O4-PEG-(DA)-FA NPs的TGA图;
图6为实施例2中Fe3O4-PEG-(DA)-FA NPs在不同时间的激光照射下的TEM图(a)和相对应的水合粒径(b);
图7为实施例3中Fe3O4-PEG-(DA)-FA NPs在不同时间的激光照射下的T1弛豫率图(a)、T2弛豫率图(b)和相对应的T1加权MR成像(c)、T2加权MR成像(d);
图8为实施例4中Fe3O4-PEG-(DA)-FA NPs在不同浓度下的细胞毒性试验结果;
图9为实施例5中Fe3O4-PEG-(DA)-FA NPs分别与Raw264.7细胞和FA阻断的Raw264.7细胞共培养6h后细胞中的铁元素的吞噬量;
图10为实施例6中Fe3O4-PEG-(DA)-FA NPs生理盐水溶液(200μL,[Fe]=30mM)通过尾静脉注射到裸鼠体内(a:不做处理的裸鼠,b:注射FA的裸鼠)取不同时间点的裸鼠关节炎处T1加权MR成像图;
图11为实施例7中Fe3O4-PEG-(DA)-FA NPs生理盐水溶液(200μL,[Fe]=30mM)通过尾静脉注射到裸鼠体内,取对照组、光照射前后的裸鼠关节炎处T1(a)和T2(b)加权MR成像图。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
无水氯化铁、二水合柠檬酸钠从国药集团化学试剂有限公司购买,氨基端聚乙二醇NH2-PEG-NHBOC从上海永颐生物科技有限公司购买,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和N-羟基琥珀酰亚胺NHS从百灵威化学技术有限公司购买,活化的叶酸NHS-FA和其他所有试剂均从Sigma-Aldrich中国公司购买。
实施例1
(1)将4mmol无水氯化铁溶解在40mL二甘醇中,超声震荡,加入1.6mmol二水合柠檬酸钠,水浴加热至80℃搅拌至溶液澄清,待溶液冷却后加入12mmol无水醋酸钠,持续搅拌至溶液澄清,将溶液转移至50mL高压反应釜中于200℃反应5.5h;自然冷却至室温,8500rpm离心15min,弃上清液,用无水乙醇回溶,重复操作3次,将沉淀物于60℃烘干,得到表面富含羧基的超小四氧化三铁纳米颗粒Fe3O4-COOH NPs。Fe3O4-COOH NPs的TEM图如图2所示,结果表明:合成的超小Fe3O4-COOH NPs的直径为2.8nm。
(2)将2g乙酰丙酸冷却至0℃并在氮气保护下缓慢加入7N氨甲醇溶液(25mL),搅拌3h,再滴加入3.2g羟胺磺酸甲醇溶液(其中羟胺磺酸的质量是3.1g),室温搅拌过夜,在真空减压浓缩下得到残渣再分散于无水甲醇中,之后过滤除去白色沉淀物,滤液通过减压蒸馏进行浓缩并重新溶解在50mL无水甲醇中,冷却至0℃,依次加入4.3mL三乙胺和2.1g碘甲醇溶液(其中碘的质量是2.0g),搅拌反应直至出现深褐色后,继续搅拌10min,将溶液用乙酸乙酯稀释,随后用1N稀盐酸和硫代硫酸钠饱和水溶液连续冲洗,接着有机相用无水硫酸镁MgSO4干燥并过滤,真空蒸发和硅胶层析纯化,得到淡黄色油状3-甲基-3H-双吖丙啶-3-丙酸DA。
(3)将步骤(2)中所得产品0.35g DA溶于10mL无水二氯甲烷DCM中,冷却至0℃,搅拌加入0.57g 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC、0.34g N-羟基琥珀酰亚胺NHS,之后室温下继续搅拌2h,将混合液用DCM稀释,并用75mL纯水冲洗3次和25mL盐水冲洗1次,有机相用无水MgSO4干燥并过滤,真空浓缩和硅胶层析纯化,得到活化的NHS-DA。1H NMR和12C NMR测试结果表明:NHS-DA结构中的所有非活性H的化学位移峰均能在图中一一对应,因此成功制备了NHS-DA(图3)。
(4)将步骤(3)中所得产品5.6mg NHS-DA溶于4mL二甲基亚砜DMSO中,依次加入50mg氨基端聚乙二醇NH2-PEG-NHBOC的二甲基亚砜溶液(5mL)和13.7mg活化的叶酸NHS-FA的二甲基亚砜溶液(5mL),避光混合搅拌3d,加入50μL的稀盐酸(3M),避光搅拌反应2h,用纤维素透析膜(MWCO=2000)对反应液进行透析(蒸馏水透析80h),之后冻干得到DA和FA修饰的聚乙二醇混合物NH2-PEG-(DA)-FA。1H NMR测试结果表明:谱图中出现在0.5-2.0ppm、3.6ppm和6.5-9.0ppm的化学位移峰分别对应为DA、聚乙二醇和FA的特征峰,由积分面积计算可得,每个聚乙二醇表面修饰了约0.7个DA和0.3个FA(图4)。
(5)将步骤(1)中所得产品56mg超小Fe3O4-COOH NPs溶解在5mL DMSO中,加入144mgEDC的DMSO溶液(2mL),避光混合搅拌30min,再加入70mg NHS的DMSO溶液(1mL),继续避光搅拌反应3h,之后加入步骤(4)中所得产品7mg NH2-PEG-(DA)-FA溶于3mL DMSO中,继续避光搅拌反应3d,用纤维素透析膜(MWCO=8000-14000)对反应液进行透析(蒸馏水透析80h),之后冻干得到基于光响应聚合的超小氧化铁的靶向T1/T2双模态MRI纳米探针Fe3O4-PEG-(DA)-FANPs。TGA测试结果表明:超小氧化铁表面修饰的聚乙二醇的含量约为14.3%(图5)。
实施例2
取实施例1中产品Fe3O4-PEG-(DA)-FA NPs先用ICP-OES测试法测得其中Fe元素的含量,再用超纯水配置成Fe浓度为30mM的溶液,分别取100μL放置于200μL PE管中,用405nm激光(功率为1.0W cm-2)分别照射0、2、5、8和12min,之后用TEM和DLS分别表征不同时间的激光照射下Fe3O4-PEG-(DA)-FA NPs的光响应聚合形态和相对应的水合粒径。测试结果表明:随着激光照射时间的增加,Fe3O4-PEG-(DA)-FA NPs的聚合程度越高(图6a),其对应的水合粒径也越大,未激光照射前的Fe3O4-PEG-(DA)-FA NPs的水合粒径约60nm,而经激光照射12min后的水合粒径达到了约800nm(图6b),远大于炎症处血管组织间隙(约20-300nm)。结果说明,本发明制备的Fe3O4-PEG-(DA)-FA NPs具有可控的光响应聚合效果,且聚合的Fe3O4-PEG-(DA)-FA NPs可以增加其在关节炎处的聚集量,限制其反进入血液循环。
实施例3
取实施例1中产品Fe3O4-PEG-(DA)-FA NPs用超纯水配置成Fe浓度为30mM的母液,之后梯度稀释出1.6、0.8、0.4、0.2和0.1mM的样品,共5组样品,之后对每组的样品用405nm激光(功率为1.0W cm-2)分别照射0、2、5、8和12min,之后测定每组样品的不同Fe浓度下的T1弛豫时间、T2弛豫时间和相对应的T1加权MR成像、T2加权MR成像。弛豫率测试结果表明:随着激光照射时间的增加,Fe3O4-PEG-(DA)-FA NPs的r1弛豫率从3.83mM-1s-1降到了1.61mM-1s-1(图7a),r2弛豫率从9.04mM-1s-1提高到了31.6mM-1s-1(图7b)。同时T1加权成像显示随着Fe浓度的提高,图像亮度变亮,然而随着激光照射时间的增加,相同浓度下的图像亮度相对变暗,但激光照射12min后仍保持图像信号增强的效果(图7c);T2加权成像显示未经激光照射的Fe3O4-PEG-(DA)-FA NPs不能使图像完全变暗,然而随着激光照射时间的增加,相同浓度下的图像亮度一直变暗,尤其是激光照射12min后图像信号完全减弱(图7d)。结果说明,本发明制备的Fe3O4-PEG-(DA)-FA NPs具有高的r1弛豫率,具有T1MR成像效果;而经激光照射12min后的聚合Fe3O4-PEG-(DA)-FA NPs不仅具有T2MR成像效果,同时也保留了T1MR成像效果。
实施例4
取实施例1中产品Fe3O4-PEG-(DA)-FA NPs用无菌PBS配置成Fe浓度为30mM的母液,之后梯度稀释为30、20、10、5和2mM的样品。取培养好的Raw264.7细胞种于96孔板中,按照1万细胞/孔的密度接种,每孔体积100μL。培养过夜后,加入上述各稀释梯度的样品,与细胞共培养24h。每个梯度用培养液稀释10倍,即每孔终浓度分别为3、2、1、0.5和0.2mM。每个梯度做5个平行孔,以PBS作为空白对照。培养结束后用100μL PBS清洗3次,每孔加100μL无血清培养基和10μL CCK8溶液,37℃孵化3h,用酶标仪检测450nm处吸光度值。CCK8法检测细胞活力测试结果表明:Fe3O4-PEG-(DA)-FA NPs在此范围内不显示出细胞毒性,表现出良好的细胞相容性(图8)。结果说明,本发明制备的Fe3O4-PEG-(DA)-FA NPs可以安全地用于生物体内MR成像。
实施例5
取实施例1中产品Fe3O4-PEG-(DA)-FA NPs分别用无菌PBS配制成Fe浓度为30mM的母液,之后梯度稀释为30、20、10、5和2mM的样品。取培养好的Raw264.7细胞种于96孔板中,按照20万细胞/孔的密度接种,每孔体积为1mL。培养过夜后,将细胞分为两组,一组为正常Raw264.7细胞,另一组Raw264.7细胞加入2nM FA共培养8h(定义为FA阻断的Raw264.7细胞)。接着,加入上述各稀释梯度的样品,与细胞共培养6h。每个梯度用培养液稀释10倍,即每孔终浓度分别为3、2、1、0.5和0.2mM。每个梯度做5个平行孔,以PBS作为空白对照。培养结束后用PBS清洗3次,再胰酶消化离心后收集细胞,加入2mL王水消化24h,然后通过ICP-OES检测细胞中Fe元素的吞噬量。特异性靶向测试结果表明:在研究浓度范围内,正常Raw264.7细胞对Fe3O4-PEG-(DA)-FA NPs的吞噬量明显高于FA阻断的Raw264.7细胞(图9)。结果说明,本发明制备的Fe3O4-PEG-(DA)-FA NPs由于靶向分子FA的修饰使得其对正常Raw264.7细胞有特异性靶向效果。
实施例6
取实施例1中产品Fe3O4-PEG-(DA)-FA NPs用无菌生理盐水配制成Fe浓度为30mM的母液。通过外科手术切断裸鼠右后腿关节处韧带,1周后形成关节炎,将裸鼠分为两组,一组不做处理,另一组注射100μL FA(6mM)。取200μL母液通过尾静脉注射到裸鼠体内,用MR成像仪分别扫描裸鼠在材料注射前和注射后15、30、45和60min的关节炎部位的T1MR成像图。小鼠体内T1MR成像测试结果显示:不做处理的裸鼠组的T1MR图像(图10a)的亮度明显高于注射FA的裸鼠组(图10b),而且不做处理的裸鼠组在注射后30min的T1MR图像的亮度最大。结果说明,本发明制备的Fe3O4-PEG-(DA)-FA NPs具有优异的靶向关节炎的T1MR成像效果。
实施例7
取实施例1中产品Fe3O4-PEG-(DA)-FA NPs用无菌生理盐水配制成Fe浓度为30mM的母液。通过外科手术切断裸鼠左右两个后腿关节处韧带,1周后形成关节炎。取200μL母液通过尾静脉注射到裸鼠体内,材料注射后30min,用405nm激光(功率为1.0W cm-2)对左后腿关节炎部位进行照射12min,以不进行激光照射右后腿关节炎部位作为对比,用MR成像仪分别扫描裸鼠注射前(对照组)和激光照射前后的关节炎部位的T1/T2MR成像图。小鼠体内T1/T2MR成像测试结果显示:在T1MR图像中,在注射材料前的关节炎部位呈现为低信号状态,注射材料后(光照射前)的关节炎部位呈现为明显的高信号状态,经光照射后的关节炎部位的信号虽有所下降,但呈现高信号状态(图11a);在T2MR图像中,在注射材料前的关节炎部位呈现为高信号状态,注射材料后(光照射前)的关节炎部位的信号强度几乎没有改变,仍为高信号状态,但经激光照射后的左后腿关节炎部位的信号明显下降,呈现低信号状态。而未经激光照射的右后腿关节炎处仍为高信号,与对照组相比,几乎没有变化(图11b)。结果说明,本发明制备的Fe3O4-PEG-(DA)-FA NPs具有体内精准可控的光响应聚合功能,经激光照射后的材料具有优异的T1/T2双模态MR成像效果。
上述结果表明,本发明制备的超小氧化铁作为纳米探针可实现体内精准可控的光响应聚合的靶向关节炎的T1/T2双模态MRI。
Claims (6)
1.一种基于光响应聚合的超小氧化铁纳米探针,其特征在于,该纳米探针由表面修饰了3-甲基-3H-双吖丙啶-3-丙酸和叶酸化聚乙二醇混合物NH2-PEG-(DA)-FA的超小氧化铁纳米颗粒组成,其中NH2-PEG-(DA)-FA和超小氧化铁纳米颗粒的质量比为7-8:56-60;
所述纳米探针的制备方法包括:
(1)将无水氯化铁溶解在二甘醇中,超声震荡,加入二水合柠檬酸钠,加热至80℃搅拌至溶液澄清,加入无水醋酸钠,继续搅拌至溶液澄清,200℃溶剂热反应5.5h,冷却,离心,回溶,烘干,得到表面富含羧基的超小四氧化三铁纳米颗粒Fe3O4-COOH NPs,其中无水氯化铁、二甘醇、二水合柠檬酸钠和无水醋酸钠的比例为4mmol:40mL:1.6mmol:12mmol;
(2)在氮气保护下0℃将乙酰丙酸加入氨甲醇溶液中,搅拌3h,加入羟胺磺酸甲醇溶液,室温搅拌过夜,提纯,将提纯得到的液体溶解于无水甲醇中,0℃下依次加入三乙胺和碘甲醇溶液,搅拌反应直至深褐色后,继续搅拌10min,稀释,冲洗,干燥,纯化,得到3-甲基-3H-双吖丙啶-3-丙酸DA,其中乙酰丙酸、氨甲醇溶液、羟胺磺酸甲醇溶液、三乙胺和碘甲醇溶液的比例为2.0g:25mL:3.2g:4.3mL:2.1g,氨甲醇溶液的当量浓度为7N;
(3)将步骤(2)中DA溶于二氯甲烷中,0℃下加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和N-羟基琥珀酰亚胺NHS,然后室温活化2h活化,稀释,冲洗,干燥,纯化,得到活化的NHS-DA,其中DA、EDC和NHS的质量比为0.35:0.57:0.34;
(4)将步骤(3)中活化的NHS-DA、氨基端聚乙二醇NH2-PEG-NHBOC与活化的叶酸NHS-FA分别溶解在二甲基亚砜中,避光混合搅拌3d,加入稀盐酸,继续避光搅拌反应2h,透析冻干,得到DA和FA修饰的聚乙二醇混合物NH2-PEG-(DA)-FA,其中NHS-DA、NH2-PEG-NHBOC、NHS-FA和稀盐酸的比例为5.6g:50g:13.7g:50mL,稀盐酸浓度为2-4M;
(5)将步骤(1)中Fe3O4-COOH NPs、步骤(4)中NH2-PEG-(DA)-FA分别溶于DMSO中,得到Fe3O4-COOH NPs溶液和NH2-PEG-(DA)-FA溶液,将Fe3O4-COOH NPs溶液加入EDC溶液避光混合搅拌30min,之后加入NHS溶液,继续避光搅拌反应3h,加入NH2-PEG-(DA)-FA溶液避光搅拌反应3d,透析冻干,得到超小氧化铁纳米探针Fe3O4-PEG-(DA)-FA NPs,其中Fe3O4-COOHNPs、NH2-PEG-(DA)-FA、EDC和NHS的质量比为56:7:144:70。
2.一种基于光响应聚合的超小氧化铁纳米探针的制备方法,包括:
(1)将无水氯化铁溶解在二甘醇中,超声震荡,加入二水合柠檬酸钠,加热至80℃搅拌至溶液澄清,加入无水醋酸钠,继续搅拌至溶液澄清,200℃溶剂热反应5.5h,冷却,离心,回溶,烘干,得到表面富含羧基的超小四氧化三铁纳米颗粒Fe3O4-COOH NPs,其中无水氯化铁、二甘醇、二水合柠檬酸钠和无水醋酸钠的比例为4mmol:40mL:1.6mmol:12mmol;
(2)在氮气保护下0℃将乙酰丙酸加入氨甲醇溶液中,搅拌3h,加入羟胺磺酸甲醇溶液,室温搅拌过夜,提纯,将提纯得到的液体溶解于无水甲醇中,0℃下依次加入三乙胺和碘甲醇溶液,搅拌反应直至深褐色后,继续搅拌10min,稀释,冲洗,干燥,纯化,得到3-甲基-3H-双吖丙啶-3-丙酸DA,其中乙酰丙酸、氨甲醇溶液、羟胺磺酸甲醇溶液、三乙胺和碘甲醇溶液的比例为2.0g:25mL:3.2g:4.3mL:2.1g,氨甲醇溶液的当量浓度为7N;
(3)将步骤(2)中DA溶于二氯甲烷中,0℃下加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐EDC和N-羟基琥珀酰亚胺NHS,然后室温活化2h活化,稀释,冲洗,干燥,纯化,得到活化的NHS-DA,其中DA、EDC和NHS的质量比为0.35:0.57:0.34;
(4)将步骤(3)中活化的NHS-DA、氨基端聚乙二醇NH2-PEG-NHBOC与活化的叶酸NHS-FA分别溶解在二甲基亚砜中,避光混合搅拌3d,加入稀盐酸,继续避光搅拌反应2h,透析冻干,得到DA和FA修饰的聚乙二醇混合物NH2-PEG-(DA)-FA,其中NHS-DA、NH2-PEG-NHBOC、NHS-FA和稀盐酸的比例为5.6g:50g:13.7g:50mL,稀盐酸浓度为2-4M;
(5)将步骤(1)中Fe3O4-COOH NPs、步骤(4)中NH2-PEG-(DA)-FA分别溶于DMSO中,得到Fe3O4-COOH NPs溶液和NH2-PEG-(DA)-FA溶液,将Fe3O4-COOH NPs溶液加入EDC溶液避光混合搅拌30min,之后加入NHS溶液,继续避光搅拌反应3h,加入NH2-PEG-(DA)-FA溶液避光搅拌反应3d,透析冻干,得到超小氧化铁纳米探针Fe3O4-PEG-(DA)-FA NPs,其中Fe3O4-COOHNPs、NH2-PEG-(DA)-FA、EDC和NHS的质量比为56:7:144:70。
3.根据权利要求2所述的制备方法,其特征在于,所述步骤(1)中离心速率为8000-9000rpm,离心时间为15-30min。
4.根据权利要求2所述的制备方法,其特征在于,所述步骤(4)中透析为:用透析膜在蒸馏水中透析60-80h,透析膜为纤维素透析膜MWCO=2000。
5.根据权利要求2所述的制备方法,其特征在于,所述步骤(5)中透析为:用透析膜在蒸馏水中透析60-80h,透析膜为纤维素透析膜MWCO=8000-14000。
6.一种如权利要求1所述的纳米探针在制备靶向关节炎的T1/T2双模态MR成像药物中的应用。
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