CN115192544B - 一种诱导铁死亡的铁螯合物纳米颗粒及其制备和应用 - Google Patents
一种诱导铁死亡的铁螯合物纳米颗粒及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种诱导铁死亡的铁螯合物纳米颗粒及其制备和应用。该铁螯合物纳米颗粒是生物可降解的脂质递送系统,包括含三价铁离子的铁螯合物内核、磷脂双分子层外壳和包载于磷脂双分子层之间的铁死亡引发剂。该纳米颗粒经静脉给药后,可以定向分布至肿瘤组织或其他靶部位,在溶酶体偏酸性环境中缓慢降解,释放出铁死亡引发剂、铁离子和鞣酸,通过自循环的方式产生活性氧增强铁死亡效应,从而发挥抗肿瘤作用。此外,该纳米颗粒具备良好的光热效应,对铁死亡有促进作用,同时具备T1MRI成像功能,可对肿瘤起到示踪作用。
Description
技术领域
本发明涉及一种用于肿瘤治疗的能够诱导铁死亡的铁螯合物纳米颗粒(Ferroptosis-triggering Iron-chelating Nanoparticle,FIN)及其制备方法,属于纳米制剂领域。
背景技术
化疗最常见的治疗癌症方法之一,目前临床上绝大多数的化疗药物都是通过直接作用和干扰与凋亡相关蛋白,激活细胞凋亡通路来抑制肿瘤细胞的生长。但在临床实践中,靶蛋白经常发生突变,导致凋亡诱导剂产生多重耐药。此外,在许多情况下,凋亡治疗引起恶性肿瘤过度表达某些蛋白抑制分子或激活新的信号通路来对抗凋亡诱导剂的抗肿瘤作用。因此,迫切需要开发一些新的非凋亡策略来有效治疗癌症。
近年来,铁死亡(Ferroptosis)的发现为恶性肿瘤的治疗提供了新的思路。铁死亡是一种新的非凋亡调控的细胞死亡形式。大量的癌症相关基因和信号通路被发现调节铁死亡。系统Xc—(System Xc—)的抑制和铁代谢导致GPX4的抑制或GSH的耗尽,活性氧(ROS)在细胞质中积累并产生不受抑制的脂质过氧化。这些被氧化的磷脂进一步导致细胞膜破裂和细胞裂解,最终导致细胞死亡。由于铁死亡细胞裂解而非凋亡过程中调控靶蛋白的特点,诱导肿瘤细胞铁死亡的治疗策略比传统的凋亡治疗具有显著优势。首先,诱导缺铁治疗能够避免靶蛋白的突变和多重耐药。其次,癌症特有的代谢特征,如铁过载、高H2O2水平和铁死亡防御系统的不平衡等,使得诱导铁死亡的治疗对恶性肿瘤具有特异性。目前,已有大量的小分子如索拉非尼、Erastin、柳氮磺胺嘧啶等被用于诱导多种癌症的铁死亡,包括非小细胞肺癌、乳腺癌、白血病等。总的来说,诱导铁死亡的治疗策略在癌症治疗中具有巨大的潜力。
然而,铁离子直接在体内用于诱导铁死亡仍存在以下问题:其在体内给药后存在安全性的问题并无法实现特异性的分布。在脑部,FeCl3会导致癫痫的发生。一部分铁在结合成为铁蛋白后,剩余的游离铁会导致神经元的损伤。铁与活性氧中间物(如过氧化氢、超氧阴离子等)通过芬顿反应或哈伯-韦斯反应产生高反应的自由基,如羟自由基,引起细胞的死亡,进而产生癫痫。此外,外源铁离子进入细胞主要通过浓度梯度扩散的方式,无法特异性地进入靶细胞而不进入非靶细胞。诱导铁死亡的药物同样存在非特异性分布的问题,且诱导效率有待进一步提高。
发明内容
针对铁离子用于抗肿瘤治疗存在的非特异性分布和毒性等问题,以及铁死亡诱导剂效率不足的问题,本发明拟构建一种铁螯合物纳米颗粒脂质体,该脂质体经静脉给药后,可以定向分布至肿瘤组织或其他靶部位,在溶酶体偏酸性环境中缓慢降解,释放出铁死亡引发剂(Trigger)、铁离子和鞣酸(TA),通过自循环的方式产生活性氧增强铁死亡效应,从而发挥抗肿瘤作用。
在本发明中,我们设计并优化了一种颗粒小、稳定性好、特异性分布的能够诱导铁死亡的铁螯合物纳米颗粒(FIN)(结构如图1中A所示,作用机制如图1中B所示)。在该纳米颗粒被细胞吸收并释放有效载荷后,Trigger起始了铁死亡。鞣酸将Fe3+还原为Fe2+,然后在肿瘤细胞中通过高水平的H2O2再氧化Fe3+。这一自循环过程产生了大量的ROS,从而增强了脂质过氧化的作用和随后的铁死亡。此外,在近红外(NIR)激光照射下,FIN表现出光热效应。温度的轻微升高进一步提高了芬顿反应的效率,对铁死亡有促进作用。
本发明的铁螯合物纳米颗粒为诱导铁死亡的生物可降解的脂质递送系统,包括铁螯合物内核、磷脂双分子层外壳和铁死亡引发剂,其中,含三价铁离子的铁螯合物被包裹在磷脂双分子层形成的外壳内,铁死亡引发剂包载在磷脂双分子层之间。
所述磷脂双分子层外壳由磷脂材料和胆固醇组成,磷脂材料可选自以下材料中的一种或几种:DOPA(二油酰磷脂酸)、DOTAP((2,3-二油氧基丙基)三甲基氯化铵)、DSPE-PEG(二硬脂酰基磷脂酰乙醇胺-聚乙二醇)等。胆固醇与磷脂材料的用量摩尔比为1:10~1:1之间。磷脂双分子层外壳带有负电荷,进入细胞后在偏酸性的环境中,脂质体缓慢降解并释放铁螯合物内核,解离出铁离子。
所述Trigger可选自以下材料中的一种:索拉非尼(Sor)、Erastin、RSL3等。
优选的,本发明所述铁螯合物纳米颗粒是粒径为20-100nm的纳米颗粒,能有效富集于肿瘤部位。该脂质体包含的铁螯合物内核为生物可降解内核,该脂质体易经内吞方式进入细胞,可在溶酶体pH环境中被解离。所述铁螯合物内核优选为鞣酸铁,转变为三价铁离子和鞣酸,从而在细胞中发挥作用。
本发明所述的铁螯合物纳米颗粒可应用于抗肿瘤药物或肿瘤诊断材料。
所述肿瘤包括但不限于乳腺癌、肾癌、卵巢癌、肝癌、肺癌、胃癌、胰腺癌、皮肤癌、恶性黑色素瘤、头颈癌、肉瘤、胆管癌、结直肠癌、胎盘绒毛膜癌、宫颈癌、睾丸癌、子宫癌和白血病。
本发明所述FIN因其纳米级尺寸可有效富集至肿瘤部位,并响应肿瘤细胞中高水平的H2O2,引发高效的铁死亡,同时减少游离铁离子的毒性。
本发明所述铁螯合物纳米颗粒主要通过以下反向微乳限阈沉淀法(前三步)和薄膜分散法(后两步)联合制备得到:
(1)将含鞣酸根的化合物配制成水溶液,在超声情况下逐滴滴入一种油溶液中,并继续超声,使水滴被裂解并均匀分散在油相中,水相与油相的体积比为1:10~1:1000之间;
(2)将含三价铁离子的化合物配制成水溶液,在超声情况下逐滴滴加进入步骤(1)所得体系中,并继续超声,使水滴被裂解并均匀分散在油相中,水相与油相的体积比为1:10-1:1000之间;
(3)向上述溶液中加入辅助成核的阴离子磷脂溶液(所述阴离子磷脂优选为DOPA,阴离子磷脂与加入的Fe3+的摩尔比为1:5~1:1之间),超声混匀后静置10-60分钟,使铁离子与鞣酸根反应形成铁螯合物微沉淀,然后加入与油溶液体积相当的第一有机溶剂破坏油相,离心得到铁螯合物微沉淀,将铁螯合物微沉淀用第二有机溶剂超声分散,其中所述第一有机溶剂能够溶解油相但不溶解铁螯合物微沉淀,而第二有机溶剂为非极性有机溶剂;
(4)在含有铁螯合物微沉淀的非极性有机溶剂中加入磷脂材料、胆固醇、Trigger,转移至茄形瓶中,通过旋转蒸发仪蒸除有机溶剂,形成一层膜状物;
(5)加入缓冲液或去离子水,30-60℃下超声10-60分钟,确保所有膜状物均超声至溶液中,用滤膜(优选孔径为0.45μm)过滤,即得诱导铁死亡的铁螯合物纳米颗粒(FIN)。
上述步骤(1)中,所述含鞣酸根的化合物能与三价铁离子形成螯合物沉淀,将此化合物配制成浓度为1~200mmol/L的水溶液。
上述步骤(1)中,所述油溶液是指与水不相容的溶液,可以选自下列油性溶剂中的一种或多种:环己烷、环戊烷、石油醚、苯等。在油溶液中添加一定量的非离子型表面活性剂以促进乳化作用,帮助水相均匀分散于油相中。所述非离子型表面活性剂例如壬苯醇醚、烷基酚聚氧乙烯醚、辛基酚聚氧乙烯醚、月桂醇聚氧乙烯醚等。优选的,所述油溶液与非离子型表面活性剂的体积比为1~7。
上述步骤(2)中,所述含三价铁离子的化合物可溶于水,可选自下列化合物中的一种或多种:氯化铁、硝酸铁、硫酸铁等。优选的,将含三价铁离子的化合物配制成浓度为1~200mmol/L的水溶液,滴入步骤(1)所得的体系中。
上述步骤(3)中,所述第一有机溶剂优选为醇类或醚类有机溶剂,例如甲醇、乙醇、丙醇和乙醚等,优先选择乙醇。所述第二有机溶剂优选为易挥发的非极性有机溶剂,例如二氯甲烷、氯仿、四氯化碳、溴乙烷、苯、环己烷、己烷、乙醚、异丙醚和乙酸乙酯等,优先选择二氯甲烷。
本发明所提供的FIN具有以下优点:
(1)通过分子螯合制备出适合颗粒大小的FIN,有利于体内给药。
(2)FIN能够实现Fe3+和鞣酸的高效跨膜传递到肿瘤细胞,且能利用肿瘤细胞高H2O2水平的特征,具有特异性。
(3)FIN在细胞内释放有效载荷,形成自循环的芬顿反应体系,产生大量ROS,有效诱导铁死亡。
(4)FIN具备良好的光热效应,能够对铁死亡起到放大效果,同时具备T1 MRI成像功能,可对肿瘤起到示踪作用。
附图说明
图1.本发明诱导铁死亡的铁螯合物纳米颗粒(FIN)的结构示意图(A)和作用机制示意图(B)。
图2.实施例1和实施例2制备的IN(A)和FIN(B)的透射电镜图(比例尺:100nm)。
图3.实施例1和实施例2制备的IN和FIN的粒径分布图(A)和表面电位的测定结果(B)。
图4.FIN在水、DMEM培养基和PBS中两周时间内粒径变化。
图5.实施例6中FIN(Fe:450μmol/L)在pH=4、pH=7、pH=4+3%H2O2条件下与o-Phenanthroline(1mmol/L)反应显色(A),以及三种体系在450-550nm的紫外-可见光吸收值(B)。
图6.实施例7中4T1细胞在不同时间点对FIN-DiD的摄取实验的共聚焦激光扫描显微镜照片(比例尺:30μm)。
图7.实施例8的细胞毒性实验结果,其中:A显示了FIN和对应浓度Sor对3T3细胞的毒性;B显示了FIN和对应浓度Sor对4T1细胞的毒性;C显示了Fer-1和DFO对Sor、IN和FIN处理后的对4T1细胞的挽救作用;D显示了FIN与FIN+H2O2(10μmol/L)对NIH3T3细胞的毒性。
图8.实施例9中C11 BIODIPY 581/591染色后激光扫描共聚焦成像照片(比例尺:30μm)。
图9.实施例9中C11 BIODIPY 581/591染色后流式细胞术分析结果,用FITC通道检测绿色荧光、用PE通道检测红色荧光,Q1区域为目标区域。
图10.实施例10中DCFH-DA染色后荧光显微镜成像照片(比例尺:50μm)。
图11.实施例11中JC-1染色后荧光显微镜成像照片(比例尺:50μm)。
图12.实施例11中JC-1染色后就是流式细胞术分析结果,用FITC通道检测绿色荧光、用PE通道检测红色荧光,Q1区域为目标区域。
图13.实施例12中经相应刺激24h后透射电镜观察线粒体形态变化(比例尺:200nm)。
图14.实施例13中FerroOrange染色后荧光显微镜成像照片(比例尺:50μm)。
图15.实施例14中经相应刺激24h后GPX4 Western blot结果。
图16.实施例15中相应刺激24h后GSH相对量。
图17.实施例16中不同条件下MB与·OH的反应后的颜色变化(A)和三种体系在450-800nm的紫外-可见光吸收值(B)。
图18.实施例17中IN与对应浓度的FeCl3、TA接受808nm NIR照射时3min之内的温度变化成像(A),以及不同浓度FIN接受808nm NIR照射时3min之内的温度变化统计(B)。
图19.实施例17中不同浓度FIN接受808nm NIR照射时3min之内的温度变化成像(A),以及不同浓度FIN接受808nm NIR照射3min之内的温度变化统计(B)。
图20.实施例17中FIN接受808nm NIR照射后对细胞的杀伤有增强的效果。
图21.实施例18中小鼠尾静脉注射Free DiD和FIN-DiD后24h内的活体成像。
图22.实施例18中小鼠尾静脉注射Free DiD和FIN-DiD后24h后心、肝、脾、肺、肾、肿瘤成像。
图23.实施例19中治疗完成后各处理组小鼠肿瘤大小(A),以及治疗过程中各处理组小鼠肿瘤体积变化(B)。
图24.实施例19中FIN+Laser组小鼠每次接受光热治疗时肿瘤温度随时间的变化(A),以及FIN+Laser组小鼠每次接受光热治疗时肿瘤温度随时间变化的统计图(B)。
图25.实施例20中各处理组小鼠肝功能和肾功能评价的血生化指标浓度。
图26.实施例20中经H&E染色后各处理组的心、肝、脾、肺、肾切片(比例尺:50μm)。
图27.实施例21中FIN(Fe:225μmol/L)在96孔板中于pH=6.5反应1h、4h、12h的成像效果(A),不同浓度FIN的成像效果(B),以及注射FIN后3h、6h、24h的成像效果(C)。
具体实施方式
以下通过实施例进一步说明和解释本发明,但不作为对本发明进行的限制。
实施例1.铁螯合物纳米颗粒(Iron-chelating Nanoparticle,IN)的制备
(1)配制油相:按照体积比71:29配制环己烷/壬苯醇醚(Cyclohexane/Igepal CO-520)混合溶剂作为油相。
(2)制备内核:将150μL TA (50mmol/L)水溶液在水浴超声下滴加至15mL油相中,边滴加边振摇离心管,然后在水浴超声下加入150μL FeCl3(25mmol/L)水溶液,确保管底所有水液滴全部分散至油相中。然后加入100μL DOPA溶液(20mg/mL),超声10-15s后,静置20min。
(3)破乳:每管中加入15mL无水乙醇溶解5min;在15000g条件下离心20min后,弃掉上清油相/乙醇混合溶剂,再加入无水乙醇至离心管中洗涤2次,去除残留油相溶剂。稍微风干后,加入2mL二氯甲烷至离心管中,吹打,并在水浴超声中稍加分散,转移至25mL茄形瓶中。
(4)成膜:向茄形瓶中分别加入300μL DOPA溶液(20mg/mL)、300μL DOTAP/Cholesterol(10mmol/L,1:1)二氯甲烷溶液、300μL DSPE-PEG(3mmol/L)二氯甲烷溶液,用旋转蒸发仪37℃减压蒸除有机溶剂后成膜。
(5)水化:加入3mL超纯水水浴超声5min,确保所有膜均超声至溶液中,用孔径为0.45μm的滤膜过滤,制备的溶液即为IN。
所制备的IN透射电镜成像如图2中A所示,激光粒径仪测得的粒径和表面电位如图3所示。
实施例2.诱导铁死亡的铁螯合物纳米颗粒(Ferroptosis-triggering Iron-chelating Nanoparticle,FIN)的制备
(1)配制油相:按照体积比71:29配制环己烷/壬苯醇醚(Cyclohexane/Igepal CO-520)混合溶剂作为油相。
(2)制备Fe3+-TA内核:将150μL TA(50mmol/L)水溶液在水浴超声下滴加至15mL油相中,边滴加边振摇离心管,然后在水浴超声下加入150μL FeCl3(25mmol/L)水溶液,确保管底所有水液滴全部分散至油相中。然后加入100μL DOPA溶液(20mg/mL),超声10-15s后,静置20min。
(3)破乳:每管中加入15mL无水乙醇溶解5min;在15000g条件下离心20min后,弃掉上清油相/乙醇混合溶剂,再加入无水乙醇至离心管中洗涤2次,去除残留油相溶剂。稍微风干后,加入2mL二氯甲烷至离心管中,吹打,并在水浴超声中稍加分散,转移至25mL茄形瓶中。
(4)成膜:向茄形瓶中分别加入300μL DOPA溶液(20mg/mL)、300μL DOTAP/Cholesterol(10mmol/L,1:1)二氯甲烷溶液、300μL DSPE-PEG(3mmol/L)二氯甲烷溶液、450μL浓度为3.33mg/mL的索拉非尼甲醇溶液,用旋转蒸发仪37℃减压蒸除有机溶剂后成膜。
(5)水化:加入3mL超纯水水浴超声5min,确保所有膜均超声至溶液中,用孔径为0.45μm的滤膜过滤,制备的溶液即为FIN。
所制备的FIN表现为均匀的深蓝黑色液体。透射电镜成像如图2中B所示,激光粒径仪测得的粒径和表面电位如图3所示。经ICP测试,FIN中Fe元素的浓度约为900μmol/L。
实施例3.IN、FIN的透射电镜图
将实施例1、实施例2中制备的IN、FIN稀释4倍后滴加在230目普通碳支撑膜上,用醋酸铀溶液负染。使用JEM-1200EX观察,FIN粒径约为30-40nm,略大于IN的粒径,参见图2。
实施例4.IN、FIN的粒径和表面电位的测定
取实施例1、实施例2中制备的IN、FIN,用激光粒度仪(Zetasizer,美国PSS)分别用粒径杯和电位杯测定25℃时的粒径和电位。每次装入样品池后测量前平衡1分钟。测定结果如图3所示,FIN粒径均值为37.85nm,略大于IN的粒径,电位约-2.04mV。
实施例5.FIN的稳定性检测
为了检测FIN的稳定性,将FIN分别用水、DMEM培养基和PBS稀释,在两周时间内每隔两天测定其粒径分布。如图4所示,粒径均没有明显变化。
实施例6.FIN引起芬顿反应的检测
为了检测FIN引起的芬顿反应,将o-Phenanthroline(邻菲罗啉)配置成浓度为10mmol/L的DMSO溶液,并设置三种不同的体系:(1)250μL FIN、200μL H2O,调节pH=4,加入50μL o-Phenanthroline;(2)250μL FIN、200μL H2O,调节pH=7,加入50μL o-Phenanthroline;(3)250μL FIN、150μL H2O、50μL 30%H2O2,调节pH=4,加入50μL o-Phenanthroline。扫描三种体系在450nm-550nm的紫外-可见光吸收光谱。如图5所示,管1呈现深红色,表明有Fe2+的生成,管2颜色变化不明显,表明可能只有少量的Fe2+存在于体系中,管3呈现橙黄色表明Fe2+的含量低于管1。三种体系在450nm-550nm的紫外可见吸收光谱管1吸光度最大,管3次之,管2最低。结果表明TA可还原Fe3+为Fe2+,H2O2可将Fe2+氧化为Fe3+。
实施例7.FIN的细胞摄取实验
为了观察FIN的细胞摄取情况,在制备载药鞣酸铁纳米颗粒FIN成膜步骤前,于茄型瓶中加入150μL浓度为1mg/mL的DiD甲醇溶液,旋转减压蒸发成膜水化后即得到FIN-DiD。我们将4T1细胞接种到共聚焦小皿中,每孔1×105个细胞。孵育24h后,弃去培养基。在用DMEM稀释FIN-DiD(Sor=5μmol/L)后,加入1mL到共聚焦小皿中,分别孵育贴壁生长的4T1细胞1h、3h、12h。PBS洗涤细胞,使用含有Hochest 33342和Lyso-tracker Green(终浓度60nmol/L)的DMEM在37℃下孵育20min。用PBS洗涤细胞后,更换新鲜无血清DMEM,使用共聚焦激光扫描显微镜观察。如图6所示,细胞内DiD荧光随着时间逐渐增强,溶酶体的绿色荧光与脂质体的紫色荧光出现共定位。随着时间进程的发展,细胞内DiD的紫色荧光变强,表明FIN被细胞不断地摄取。
实施例8.FIN的细胞毒作用与铁死亡抑制剂的挽救
为了评价FIN的细胞毒性,将4T1细胞、NIH3T3细胞接种到96孔板中,每孔5×103个细胞。孵育24h后,弃去96孔板中的培养基。然后将索拉非尼(用20%SBE-β-CD溶液助溶)、IN、FIN、Fer-1、DFO等按相应比例稀释于新鲜培养基中,与细胞共孵育24h,然后用CellCounting Kit-8(CCK-8)检测细胞毒作用和铁死亡抑制剂的挽救效果。如图7所示,当Sor浓度为5μmol/L时,索拉非尼对NIH3T3和4T1以及FIN对NIH3T3细胞毒性不强,但FIN对4T1细胞表现出现明显的细胞毒性,细胞存活率<50%。铁死亡抑制剂Fer-1和DFO可以起到挽救效果。在NIH3T3细胞培养基中加入H2O2以模拟肿瘤的高H2O2水平,CCK-8结果表明,FIN对NIH3T3细胞的杀伤比不加H2O2时更强。
实施例9.细胞脂质ROS(Lipid ROS)检测
为观察细胞脂质ROS的变化,将4T1细胞接种到共聚焦小皿中,每孔1×105个细胞。孵育24h后,弃去培养基。配制含不同成分(Control、Sor、IN、FIN、FIN+Fer-1)的DMEM,与细胞孵育24h后,弃去培养基,用C11 BIODIPY 581/591在37℃下染色20min。用PBS洗涤细胞后,更换新鲜无血清DMEM,使用共聚焦激光扫描显微镜(CLSM)观察。如图8所示,FIN处理的4T1细胞比IN或Sor处理的细胞在细胞膜上表现出更强的绿色荧光,表明有更多的脂质ROS生成,有在加入铁死亡抑制剂Fer-1后,绿色荧光强度降低。
同时,对4T1细胞的脂质ROS水平用流式细胞术进行了检测。将细胞以3×105/孔的密度接种于6孔板上培养24h。给药和染色操作同上。在用PBS洗涤细胞后,消化离心收集细胞,用1mL HBSS重悬后流式检测。如图9所示,FIN处理后Q1区域细胞数量增加,表明绿色荧光增强,与CLSM观察结果相一致。
实施例10.细胞总体ROS检测
为观察4T1细胞总体ROS水平,我们将细胞以3×105/孔的密度接种于6孔板上培养24h。配制含不同成分(Control、Sor、IN、FIN、FIN+Fer-1)的DMEM,与细胞孵育24h后,用PBS洗涤细胞两次。用2,7-二氯二氢荧光素二醋酸酯(DCFH-DA)(工作浓度为10μmol/L)在37℃下染色20min。在用无血清细胞培养液洗涤细胞三次后,更换新鲜无血清培养基,在荧光显微镜下观察。如图10所示,在FIN处理后4T1细胞中检测到明显的绿色荧光,表明有更多的ROS生成。FIN与Fer-1共同处理细胞后,绿色荧光减弱,而Sor组、IN组均未发现明显的绿色荧光。
实施例11.线粒体电位检测
为了检测4T1细胞线粒体电位变化,将细胞以3×105/孔的密度接种于6孔板,培养24h,弃去培养基。配制含不同成分(Control、Sor、IN、FIN、FIN+Fer-1)的DMEM,与细胞孵育24h后,弃去培养基,用增强型线粒体膜电位检测试剂盒(JC-1)检测细细胞线粒体电位。我们按试剂盒说明将JC-1配置成染色工作液,每一孔用PBS洗涤细胞两次后,加入1mL细胞培养和1mL JC-1染色工作液,充分混匀,于37℃孵育20min。孵育结束后,吸除上清,用JC-1染色缓冲液洗涤2次,加入1mL无血清培养基,荧光显微镜下观察。如图11所示,FIN处理后的4T1细胞绿色荧光明显增强,强于Sor组和IN组,表明线粒体电位降低。在用Fer-1与FIN共同处理细胞后,绿色荧光减弱。
同时,对4T1细胞的线粒体电位用流式细胞术进行了检测。给药及染色操作方法同上。染色结束用JC-1染色缓冲液洗涤2次后,消化并离心收集细胞,用1mL HBSS重悬后流式检测。如图12所示,在用FIN处理后,Q1区细胞较Sor和IN处理的更多,在用Fer-1与FIN共同处理细胞后,Q1区细胞减少,与CLSM观察结果相一致。
实施例12.线粒体形态观察
为了观察4T1细胞线粒体形态的变化,将Film用紫外照30min后,置于24孔板中,浸泡于培养基过夜。以1×105/孔的密度接种4T1细胞,培养24h过夜。配制含不同成分的(Control、Sor、IN、FIN、FIN+Fer-1)DMEM,与细胞孵育24h后,弃去上清液,并用PBS洗涤1次。配制固定液:2.5%戊二醛,0.1mol/L PB,pH=7.4。37℃预热后,先用固定液细胞培养基1:1混合,漂洗一次,再用纯固定液在室温下固定1h。之后交于北京大学生命科学学院公共仪器中心进行后续制样,并使用JEM-1400观察。如图13所示,FIN处理的细胞线粒体明显萎缩变小,嵴数量减少,部分外膜呈现断裂状态,符合铁死亡特征。
实施例13.细胞内Fe2+检测
为观察4T1细胞中Fe2+的含量,将细胞以3×105/孔的密度接种于6孔板上培养24h。配制含不同成分的(Control、Sor、IN、FIN、FIN+DFO)DMEM,与细胞孵育24h后,弃去上清液,并用HBSS洗涤细胞3次。用HBSS配制1μmol/L的FerroOrange工作液,每孔加入1mL。在37℃,5%CO2培养箱中染色30min后,直接用荧光显微镜观察。如图14所示,FIN处理的4T1细胞表现出最强的荧光,IN处理的细胞和Sor处理的细胞与对照组相比荧光强度也有所增强,表明Fe2+的含量增加。在加入铁死亡抑制剂DFO后,荧光强度显著降低。
实施例14.GPX4免疫印迹实验(Western blot)
(1)提前制备好10%SDS-聚丙烯酰胺凝胶,将凝胶置于电泳装置中,加入电泳缓冲液(1×running buffer)后,每孔加入等量的蛋白样品(总蛋白含量30μg/孔)并加入蛋白Marker(2.5μL)。首先,80V电泳使蛋白跑至分离胶后(约20min)改为120V至电泳结束(约90min)。
(2)将PVDF膜泡于甲醇中活化,然后取出凝胶,切去浓缩胶部分,组装转印夹层,并置于转膜缓冲液中,120V恒压的条件冰浴转膜120min。
(3)转膜完成后,将PVDF膜置于5%脱脂牛奶中于室温条件下在摇床上缓慢摇动2h,以封闭蛋白位点。
(4)将膜从封闭液中取出,用1‰PBST清洗3次,每次10min。
(5)用1‰PBST配制一抗溶液(1:1000),将蛋白对应位置的膜剪下并置于一抗溶液中,于4℃摇床上缓慢摇动,孵育过夜。
(6)将膜从一抗溶液中取出,用1‰PBST清洗3次,每次10min。
(7)用1‰PBST配制二抗溶液(1:4000),将膜转移至二抗中,摇床上缓慢摇动,室温孵育1h。
(8)将膜取出,用1‰PBST清洗3次,每次10min。
(9)加入ECL PLUS发光液(A液:B液=1:1),室温孵育2min。采用Beyolmager 600进行曝光和图像采集。
如图15所示,与单独用Sor或IN处理相比,FIN处理后更有效地下调了4T1细胞中的GPX4水平。在加入铁离子抑制剂Fer-1后现象有所缓解。
实施例15.GSH水平检测
为了检测细胞GSH水平,将4T1细胞以3×105/孔的密度接种于6孔板中,培养24h。经过各种处理后,收集细胞,用PBS洗涤三次。然后,用500μL的0.1%Triton-X-100裂解液裂解细胞,离心后收集裂解液,将100μL的裂解液与100μL的DTNB溶液(400μmol/L)混合。共孵育30min后,在412nm波长下测定混合物的吸收度,并与未处理细胞的GSH含量比较,得到GSH的相对含量。如图16所示,GSH水平也能被FIN下调,在加入铁离子抑制剂Fer-1后起到了挽救效果。
实施例16.温度对·OH产生效率的探究
为了检测温度对芬顿反应的影响,以亚甲基蓝(MB)为指示剂检测·OH的生成。配制三个反应体系:(1)3%H2O2+10μg/mL MB;(2)FIN(Fe:180μmol/L)+3%H2O2+10μg/mL MB,pH=4;(3)FIN(Fe:180μmol/L)+3%H2O2+10μg/mL MB,pH=4,45℃水浴。混合均匀,反应1h后,扫描波长为550-800nm的紫外/可见吸收光谱(比较其在665nm处的吸光度)。如图17所示,45℃水浴后,体系在665nm处的吸光度有了明显的下降,表明温度升高促进了大量·OH的产生,提高了芬顿反应效率。
实施例17.光热效应评价
为了探究IN中具有光热效应的组分,我们将IN和对应浓度的Fe、TA和Blankliposome用808nm NIR照射3min,并记录升温过程。如图18所示,Fe3+、TA和Blank liposome在用808nm NIR照射3min之后温度没有明显的变化,而IN温度有显著上升,达到约70℃(图4.2),表明Fe3+、TA和Blank liposome没有光热效应,只有形成鞣酸铁(Fe3+-TA)时,才具有光热效应,且具有时间梯度响应。
为了考察浓度对光热效应的影响,将不同浓度FIN用808nm NIR照射3min的光热效应。如图19所示,FIN与IN光热效应没有明显差异,且光热效应会随着浓度的降低而减弱。
为了检测光热效应对肿瘤细胞的杀伤作用,我们将FIN用PBS稀释后,分别用808nmNIR照射0s、15s、30s、60s、120s、240s,之后用CCK-8检测细胞活性,与未经照射的对照组进行对比。如图20所示,在相同浓度FIN的作用下,接受808nm NIR照射60秒后,体现出对细胞的杀伤的增强效果。
实施例18.体内分布观察实验
为了观察FIN的体内分布观察,将Free DiD(游离DiD,DiD:2mg/kg,用生理盐水稀释DiD的甲醇溶液)和FIN-DiD(索拉非尼:2.5mg/kg,Fe:252μg/kg,DiD:2mg/kg,减压蒸除二氯甲烷前加入DiD的甲醇溶液)以尾静脉注射的形式给药,并在设定的时间点将两组小鼠麻醉后进行活体成像。24小时后,取出心、肝、脾、肺、肾和肿瘤成像。如图21所示,FIN组肿瘤部位荧光信号在注射后逐渐增强,到6h达到最高值,游离DiD组在注射后荧光信号没有明显的增强。在给药24h后,处死小鼠,取出心、肝、脾、肺、肾和肿瘤,观察FIN的组织分布和在肿瘤的富集程度。如图22所示,FIN主要分布在肿瘤部位,荧光信号最强,肝脏和脾脏也有荧光信号,但强度较弱。
实施例19.药效评价
为了评价FIN的抗肿瘤效果,当肿瘤体积达到约100mm3时,用4T1荷瘤小鼠进行体内抗肿瘤实验。将小鼠随机分为6组(每组5只):(1)PBS;(2)Sor(索拉非尼用20%SBE-β-CD溶液助溶);(3)IN;(4)FIN(索拉非尼:2.5mg/kg,Fe:252μg/kg);(5)IN+Laser;(6)FIN+Laser。于第1、2、3、5、6、7、9、10、11天,所有小鼠均以尾静脉注射形式给药,并于第4、8、12天,对IN+Laser和FIN+Laser组小鼠进行光热治疗,使用808nm NIR照射4min。药效评价实验开始后,每隔两天测量小鼠体重、肿瘤的宽度与长度,根据公式计算肿瘤体积:肿瘤体积=(长度×宽度2)/2。治疗完成后,于第14天处死小鼠,进行后续实验。如图23所示,FIN组相较于IN组和索拉非尼组治疗效果有了明显的提高,抑瘤率达到了55.6%(按体积计算)。结合光热治疗后,IN和FIN的治疗效果均有显著提升,抑瘤率分别为65.7%和75.3%(按体积计算)。各组小鼠的体重在治疗过程中保持相对稳定。
在治疗过程中,对FIN+Laser组接受808nm NIR照射时肿瘤部位的温度进行了记录。如图24所示,照射处温度会随着照射时间而上升,且每一轮照射都可以比前一轮达到相对更高的温度。
实施例20.FIN的安全性评价
为了评价FIN的安全性,小鼠治疗结束以后,将小鼠气麻后摘眼球取血,获得的血样于室温静置60min,然后以3000r/min离心10min后缓慢吸取上层血清,于-80℃冰箱中保存。使用OLYMPUS AU400全自动生化分析仪检测血清中血清碱性磷酸酶(Alkalinephosphatase,ALP)、天冬氨酸转移酶(Aspartic acid transferase,AST)、丙氨酸氨基转移酶(Alanine aminotransferase,ALT)、尿素(UREA)、肌酐(CREA)5个血生化指标。如图25所示,FIN组与对照组血液生化指标无明显变化,说明肝肾功能正常。FIN+Laser组与FIN组的血液生化指标无明显变化,说明808nm NIR照射不影响肝肾功能。
此外,我们对小鼠的主要器官心、肝、脾、肺、肾进行了H&E染色。如图26所示,未发现明显损伤。结果表明,808nm NIR照射不会影响FIN的生物相容性。
实施例21.MRI T1加权造影
为了观察FIN的体外MRI成像能力,以时间梯度和浓度梯度对FIN进行MRI成像。将FIN用PBS稀释后(pH=6.5)置于96孔板中。在经过指定的时间(0h、1h、4h、12h)后,用MRI系统(SIEMENS MAGNETOM Trio Tim system)拍摄样品的T1加权图像。此外,我们将0.2mL不同浓度的FIN(Fe:900μmol/L、450μmol/L、225μmol/L、112.5μmol/L、62.25μmol/L、0μmol/L)置于96孔板中,12h后,观察浓度对MRI成像的影响。如图27中A所示,随着孵育时间的增加,孔板中的样品变得更亮,因为鞣酸铁纳米颗粒在酸性环境下倾向于解离并释放铁离子。此外FIN的成像能力与浓度有关,如图27中B所示,磁共振成像随着FIN浓度的增加而增强。
为了观察FIN的体内MRI成像能力,瘤内注射50μL的FIN(Fe:252μg/kg)至荷瘤小鼠。然后,在指定的时间进行MRI成像(0h、3h、6h、24h)。如图27中C所示,注射3h后可以清晰的观察和记录肿瘤部位,6h后成像效果更佳。24h后,成像效果减弱,但肿瘤区域仍有部分成像功能。
Claims (7)
1.一种铁螯合物纳米颗粒,为诱导铁死亡的生物可降解的脂质递送系统,包括铁螯合物内核、磷脂双分子层外壳和铁死亡引发剂,其中,含三价铁离子的铁螯合物被包裹在磷脂双分子层形成的外壳内,铁死亡引发剂包载在磷脂双分子层之间;所述铁螯合物内核为鞣酸铁;该铁螯合物纳米颗粒通过下述方法制备得到:
1)将含鞣酸根的化合物配制成水溶液,在超声情况下逐滴滴入一种油溶液中,并继续超声,使水滴被裂解并均匀分散在油相中,水相与油相的体积比为1:10~ 1:1000之间;所述油溶液选自下列油性溶剂中的一种或多种:环己烷、环戊烷、石油醚、苯;在所述油溶液中添加有非离子型表面活性剂;
2)将含三价铁离子的化合物配制成水溶液,在超声情况下逐滴滴入步骤1)所得体系中,并继续超声,使水滴被裂解并均匀分散在油相中,水相与油相的体积比为1:10 - 1:1000之间;
3)向步骤2)所得溶液中加入辅助成核的阴离子磷脂溶液,超声混匀后静置10-60分钟,使铁离子与鞣酸根反应形成铁螯合物微沉淀,然后加入与油溶液体积相当的第一有机溶剂破坏油相,离心得到铁螯合物微沉淀,将铁螯合物微沉淀用第二有机溶剂超声分散;其中,所述第一有机溶剂选自甲醇、乙醇、丙醇和乙醚,第二有机溶剂选自二氯甲烷、氯仿、四氯化碳、溴乙烷、苯、环己烷、己烷、乙醚、异丙醚和乙酸乙酯;
4)在含有铁螯合物微沉淀的第二有机溶剂中加入磷脂材料、胆固醇、铁死亡引发剂,转移至茄形瓶中,通过旋转蒸发仪蒸除有机溶剂,形成一层膜状物;
5)加入缓冲液或去离子水,30-60℃下超声10-60分钟,确保所有膜状物均超声至溶液中,用滤膜过滤,即得诱导铁死亡的铁螯合物纳米颗粒。
2.如权利要求1所述的铁螯合物纳米颗粒,其特征在于,其粒径为20-100 nm。
3.如权利要求1所述的铁螯合物纳米颗粒,其特征在于,所述磷脂双分子层外壳由磷脂材料和胆固醇组成,其中磷脂材料选自以下材料中的一种或多种:DOPA、DOTAP、DSPE-PEG。
4.如权利要求1所述的铁螯合物纳米颗粒,其特征在于,所述铁死亡引发剂选自以下材料中的一种:索拉非尼、Erastin、RSL3。
5.如权利要求1所述的铁螯合物纳米颗粒,其特征在于,步骤1)中所述含鞣酸根的化合物选自鞣酸;步骤2)中所述含三价铁离子的化合物选自下列化合物中的一种或多种:氯化铁、硝酸铁、硫酸铁。
6.权利要求1~5任一所述的铁螯合物纳米颗粒在制备抗肿瘤药物或肿瘤诊断材料中的应用。
7.如权利要求6所述的应用,其特征在于,所述肿瘤包括但不限于乳腺癌、卵巢癌、肺癌、胃癌、肝癌、胰腺癌、胆管癌、肾癌、结直肠癌、宫颈癌、睾丸癌、子宫癌。
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CN112402453A (zh) * | 2020-11-18 | 2021-02-26 | 中国药科大学 | 一种酶和难溶性药物共载脂质体及其制备方法和应用 |
CN112603999A (zh) * | 2020-12-30 | 2021-04-06 | 重庆医科大学 | 一种基于仿生工程的肿瘤微环境响应型纳米粒及其制备方法和应用 |
CN112791185A (zh) * | 2021-01-20 | 2021-05-14 | 广州医科大学 | 用于肿瘤光热联合铁剂治疗纳米药物及其制备方法 |
CN113521098A (zh) * | 2021-07-29 | 2021-10-22 | 山东大学齐鲁医院 | 铂(Ⅳ)及cRGD修饰的GA/Fe纳米颗粒搭载多柔比星及其靶向治疗肿瘤的方法 |
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