CN115068633A - 一种特异性靶向细胞内脂滴的长荧光寿命纳米探针 - Google Patents
一种特异性靶向细胞内脂滴的长荧光寿命纳米探针 Download PDFInfo
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Abstract
本发明属于医药技术领域，公开了一种特异性靶向细胞内脂滴的长荧光寿命纳米探针，具体涉及一种热激活延迟荧光(TADF)纳米探针材料的细胞脂滴荧光成像。
Description
技术领域
本发明属于医药技术领域，具体涉及一种热激活延迟荧光(thermally activateddelayed fluorescence,TADF)纳米探针材料的细胞脂滴荧光成像。
背景技术
脂滴(lipid droplet,LDs)是用于存储中性脂质的动态亚细胞细胞器，主要存在于脂肪细胞、肝细胞、肾上腺皮质和肌细胞中，并在多种生物过程，包括脂质储存和代谢、膜形成和维持、蛋白降解、信号转导和细胞凋亡等[1,2]。据报道，细胞中LDs的异常与多种疾病密切相关，包括脂肪肝、高脂血症、肥胖症、II型糖尿病、动脉粥样硬化、炎症、病毒感染，甚至是阿尔茨海默氏病[3-5]。最近的研究进一步揭示，LDs在细胞中的异常行为应被视为癌症的关键标志物[6,7]。为了研究LDs的结构及其多种多样的功能，开发了一系列用于LD成像和跟踪的染料，其中苏丹Ⅲ、苏丹Ⅳ、锇酸、油红O等是普通光学显微镜观测中较为常用的染料，但这些染料操作步骤繁琐，并且难以在活细胞中进行追踪观测，不能进行定量研究。近年来，发展了荧光探针染料用于细胞中脂滴的观测，其中较为广泛应用的有两个商业化的脂滴探针：尼罗红(Nile Red)和氟硼二吡咯(BODIPY)。但是这两种探针又有各自的不足，Nile Red选择性差，染色脂滴的同时也会把细胞中其他疏水性结构一同染色，导致信噪比较低；BODIPY相对于Nile Red选择性有所增加，但是由于其具有较小的斯托克斯位移，导致激发和发射光谱有重叠。因此，迫切的需要一种具有高选择性和大斯托克斯位移的荧光探针，能够特异性的识别LDs，并实现高质量的LDs成像。
热激活延迟荧光(TADF)材料就是一类具有大斯托克斯位移的长荧光寿命材料，具有荧光效率高和荧光寿命长等优点。自从Adachi等人首次报道了用于高效有机发光二极管(oganic light emitting diodes,OLEDs)的TADF材料以来，众多新型分子结构的TADF材料的设计和合成被相继报道[8-10]。理论上，TADF材料需要在最低激发单重态(S1)和最低激发三重态(T1)之间有足够小的能极差(ΔEST<0.3eV)，来实现有效的反系间穿越(reverseintersystem crossing,RISC)过程。RISC过程使TADF基团具有发光效率高和荧光寿命长的延迟荧光特性[11,12]。如今，荧光寿命探针通过时间分辨成像技术可以有效避免生物体内自发荧光的干扰，在荧光寿命生物成像中已经得到了广泛的应用[13-18]。
本发明通过将TADF荧光团AI-Cz封装到两亲共聚物DSPE-mPEG2000聚合物中而一步制成用于脂滴特异性成像的新型TADF胶束纳米探针AI-Cz-M。与其他已知的细胞器不同，LDs主要由覆盖有磷脂单层的中性脂质(三酰基甘油，胆固醇酯)簇组成，这意味着LDs具有特定的两亲结构，包含完全无水的核心。考虑到AI-Cz分子的亲脂性和LDs内的疏水环境，我们推测TADF胶束纳米探针可能会由于“相似相溶”相互作用而选择性地聚集在LDs的疏水核中。胶束纳米探针AI-Cz-M在水溶液中分散良好、细胞毒性低、细胞膜穿透性高以及成像操作简单，具有有效的LDs靶向能力，可以进行活细胞脂滴追踪检测。同时，由于封装，在哺乳动物细胞的LDs(143μs)中观察到了AI-Cz-M的长荧光寿命，这是目前为止TRFI基于TADF材料获得的最长荧光寿命。AI-Cz-M在脂滴荧光成像技术，特别是在脂滴时间分辨荧光成像(time resolved fluorescence imaging，TRFI)研究中具有巨大的临床应用潜力和研究开发价值，适合脂滴靶向荧光成像技术的应用和普及。
发明内容
本发明解决的技术问题是提供了一种已知的荧光纳米探针在细胞或活体水平对细胞中脂滴特异性靶向的应用。该探针本文记载为AI-Cz-M，其结构特征为两亲性共聚物DSPE-PEG2000包裹疏水性TADF分子AI-Cz制备成胶束。本发明特异性靶向细胞内脂滴的荧光纳米探针的用途，是作为检测试剂在特异性标记细胞内脂滴时使用。
为解决本发明的技术问题，本发明提供如下技术方案：
本发明技术方案是胶束AI-Cz-M在制备靶向脂滴的荧光探针中的应用，所述胶束AI-Cz-M是由两亲性共聚物DSPE-PEG2000包裹疏水性热激活延迟荧光分子制备而成。提供纳米探针AI-Cz-M在细胞或活体水平对细胞中脂滴进行荧光成像和荧光寿命成像的应用，其中所述的细胞选自HeLa、HepG2、3T3-L1或WT1细胞。
有益技术效果
利用AI-Cz-M纳米探针分子，对细胞中脂滴进行了荧光成像研究。
1、与市售脂滴染料相比，对脂滴准确靶向，并且成像信噪比高。
2、具有长荧光寿命，极大的提高了脂滴的成像灵敏度，更有利于探针分子降低脂滴荧光成像的背景噪音、提高成像信噪比。
3、脂滴染色操作简单，避免了繁琐的处理过程。
4、具有大斯托克斯位移，降低自吸收效应和内滤效应，减少了发射光和散射光之间的检测错误。
5、可用于活细胞脂滴成像和追踪应用，为实时监测细胞内脂滴的生理活动奠定了基础。
6、可以对细胞中脂滴进行定量研究。
附图说明
图1为纳米探针分子与BODIPY 493/503在细胞中共染色成像，标尺＝10μm。
图2为纳米探针分子在不同诱导天数的3T3-L1细胞中共聚焦成像，标尺＝25μm。
图3为纳米探针分子在不同诱导天数的3T3-L1细胞中共聚焦成像中的平均荧光强度小提琴图，ns:P>0.05，无显著性差异；***：P<0.01，有显著性差异。
图4为纳米探针分子在不同诱导天数的3T3-L1细胞中荧光强度测量。
图5为纳米探针分子在HeLa细胞中荧光寿命成像，标尺＝10μm。
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。本发明所用胶束AI-Cz-M为发明人课题组制备，制备方法同已报道发明专利申请号：CN201811325113.3[19]。
实验例1：纳米探针分子与BODIPY 493/503在细胞中共染色实验
(1)首先待HeLa或HepG 2细胞生长至80％时，将HeLa或HepG 2细胞(300μL,1.2×104细胞/mL)接种在激光共聚焦用8孔腔室盖玻片(Thermo Fisher,Nunc LAB-Tek TMⅡ)中，过夜贴壁培养。
(2)用配制好的油酸(50μM)培养基溶液在37℃诱导6h。
(2)用磷酸缓冲盐溶液(phosphate buffer saline,PBS)缓冲液洗涤两次，然后加入配制好的AI-Cz-M(终浓度为10μM)培养基溶液分别在37℃培养2h。
(3)用PBS缓冲液洗涤，以除去未结合的探针，然后加入配制好的BODIPY493/503(终浓度为3.8μM)培养基溶液在37℃培养15min。
(4)用PBS缓冲液洗涤，以除去未结合的探针。进行共聚焦成像。
结果如图1所示，从图中可以得出，AI-Cz-M探针与商售脂滴染料BODIPY493/503荧光成像区域高度重叠，并且通过image J计算得到探针在HepG2和Hela细胞中共定位系数分别为0.83和0.741，表明探针对于脂滴靶向的准确性。并且与BODIPY 493/503相比，AI-Cz-M染色的图像背景信号较低，表明AI-Cz-M特异性靶向脂滴效果优于BODIPY 493/503。
实验例2：纳米探针分子在细胞中共聚焦成像
(1)将3T3-L1分别以30万/皿的密度在10％CBS(小牛血清)的DMEM(Dulbecco’smodified Eagle’s medium)培养基中培养。
(2)细胞生长到汇合后(约2天)，换成含10％FBS(胎牛血清)的DMEM高葡萄糖(这称为-2天)。
(3)换用诱导培养基培养2天(称为第0天)。
(4)将培养基更换为胰岛素/T3培养基分别培养2天(第2天)、4天(第4天)、6天(第六天)。
(5)将培养基更换为500μL/皿含有10μM AI-Cz-M的培养基。37℃、5％CO2培养箱培养2h后，PBS洗3次，进行共聚焦成像。
诱导培养基：胰岛素50nM，T3 100nM，吲哚美辛0.125mM，地塞米松2ug/ml，IBMX(3-异丁基-1-甲基黄嘌呤)0.5mM(吲哚美辛必须在75℃加热才能溶解)
胰岛素/T3培养基：胰岛素50nM，T3 1nM
结果如图2所示，从图中可以得出，AI-Cz-M可以在3T3-L1细胞中进行脂滴成像，随着诱导时间的增加，细胞中脂滴数量增多，体积变大，荧光强度增强。并对图2的脂滴成像结果进行平均荧光强度分析，结果如图3所示，进一步说明该纳米探针可以对脂滴进行定量研究。
实验例3：纳米探针分子在细胞中荧光强度测定
将实验例2中诱导6天和-2天的3T3-L1细胞，分别用10μM纳米探针培养2h，PBS洗3次，进行荧光强度测量。
结果如图4所示，从图中可以得出，AI-Cz-M可以在3T3-L1细胞中进行脂滴定量测定，荧光强度和脂滴数量成正比，可以简单、快速、定量检测脂滴。
实验例4：纳米探针分子在细胞中荧光寿命测定
(1)待HeLa细胞生长至80％时，将细胞转至底部放有细胞爬片的24孔板(1mL,3.5×104细胞/mL)，过夜贴壁培养。
(2)用配制好的油酸(50μM)培养基溶液在37℃诱导6h。
(3)用PBS缓冲液洗涤一次，然后加入AI-Cz-M(终浓度为10μM)培养基溶液在37℃温育2h。
(4)用PBS缓冲液洗涤，以除去未结合的探针。每孔加入500μL 4％多聚甲醛，室温固定20min。固定后，每孔用PBS漂洗三次，取出细胞爬片，将其倒置于滴有10μL抗荧光衰减封片剂的干净载玻片上，轻轻压盖玻片，用滤纸小心吸除爬片周围多余液体，室温放置数分钟，用透明指甲油小心封片。进行长荧光寿命成像。
结果如图5所示，从图中可以得出，长荧光寿命纳米探针AI-Cz-M可以在HeLa细胞中进行脂滴成像，AI-Cz-M在HeLa细胞中具有较长的荧光寿命，经单指数拟合分析，其平均荧光寿命为143μs，是目前基于TADF材料的TRFI获得的最长荧光寿命。
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Claims (3)
1.胶束AI-Cz-M在制备靶向脂滴的荧光探针中的应用，其特征在于，所述胶束AI-Cz-M是由两亲性共聚物DSPE-PEG2000包裹疏水性热激活延迟荧光分子制备而成
2.根据权利要求1的应用，其特征在于，所述靶向脂滴是在细胞或活体水平细胞内进行的。
3.根据权利要求2的应用，其特征在于，所述的细胞选自HepG2、HeLa、3T3-L1或WT1。
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