CN114617978B - 一种荧光共振能量转移纳米探针及其制备方法和应用 - Google Patents
一种荧光共振能量转移纳米探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种荧光共振能量转移纳米探针及其制备方法和应用,属于靶向纳米材料技术领域。本发明包括负载组分和包封所述负载组分的包封组分,所述负载组分包括载体和包覆在所述载体内的阿霉素,所述载体为羧基改性的空心介孔二氧化硅,所述包封组分包括RVRR肽和PAMAM/TPE。基于羧基改性的空心介孔二氧化硅(HMSN)的羧基的缩合特性,本发明利用弗林蛋白酶(Furin)特异性响应的RVRR肽和PAMAM/TPE表面修饰的氨基与HMSN表面的羧基反应,将RVRR肽和PAMAM/TPE小分子聚合物包裹在HMSN外表面,实现对阿霉素(DOX)的有效包封。
Description
技术领域
本发明属于靶向纳米材料技术领域,具体涉及一种荧光共振能量转移纳米探针及其制备方法和应用。
背景技术
荧光生物传感器是分析和检测活细胞生物分子的重要工具,目前已经开发出许多用于生物检测的荧光探针。但是,大多数荧光团在水溶液中的溶解度非常低,尽管许多具有荧光“开/关”效应的有机染料被设计为离子形式以增加在水溶液中的溶解度,但它们仍然容易发生聚集引起的猝灭(ACQ)。本世纪初,研究人员发现聚集诱导发射(AIE)和ACQ的作用机制是相反的。当AIE荧光分子簇分散良好时,分子内运动的动态旋转和振动会降低激发能,进而降低荧光强度。当AIE荧光分子簇聚集时,分子内的运动会受到限制,阻止激发能量的耗散,从而增加荧光强度。现有研究已经证明了使用AIE分子与阿霉素(DOX)所组成的荧光共振能量转移(FRET)对在生物医学应用(如细胞内成像和药物递送)中表现出巨大潜力的可行性([1]TiantianWang,Qichun Wei,Zhentao Zhang,Jianqing Gao et.al,AIE/FRET-based versatile PEG-Pep-TPE/DOX nanoparticles for cancer therapy andreal-time drug release monitoring,Biomater.Sci.,2020,8,118-124.[2]XiongqiHan,De-E Liu,Qixian Chen,Hui Gao et.al,Aggregation-Induced-Emissive MoleculeIncorporated into Polymeric Nanoparticulate as FRET Donor for ObservingDoxorubicin Delivery,ACS Appl.Mater.Interfaces 2015,7,23760-23766.)。
目前的研究中,随着DOX在肿瘤细胞中的释放,使得TPE/DOX分离,FRET消失,只能通过FRET信号的减弱进行相关分析,具有局限性。
发明内容
有鉴于此,本发明的目的在于提供一种荧光共振能量转移纳米探针及其制备方法和应用。本发明采用逆向手段,通过细胞内自组装,实现了细胞内FRET由无到有的过程,为实现细胞内Furin的定量检测提供了一种新的策略。
本发明提供了一种荧光共振能量转移纳米探针,包括负载组分和包封所述负载组分的包封组分,所述负载组分包括载体和包覆在所述载体内的阿霉素,所述载体为羧基改性的空心介孔二氧化硅,所述包封组分包括RVRR肽和PAMAM/TPE。
优选的,所述羧基改性的空心介孔二氧化硅和阿霉素的质量比为(1~2):(1~2)。
本发明还提供了上述方案所述的荧光共振能量转移纳米探针的制备方法,包括以下步骤:
1)将羧基改性的空心介孔二氧化硅溶液和阿霉素溶液混合,得到HMSN/DOX溶液;
2)将所述HMSN/DOX溶液、RVRR肽和PAMAM/TPE进行混合,得到荧光共振能量转移纳米探针。
优选的,步骤1)和步骤2)中,所述混合的温度独立为20~30℃;所述混合的时间独立为20~24h;所述混合避光进行。
本发明还提供了上述方案所述的荧光共振能量转移纳米探针或者所述的制备方法制备得到的荧光共振能量转移纳米探针在弗林蛋白酶检测中的应用。
优选的,所述弗林蛋白酶包括细胞内弗林蛋白酶。
优选的,所述检测包括定量检测。
本发明还提供了上述方案所述的荧光共振能量转移纳米探针或者所述的制备方法制备得到的荧光共振能量转移纳米探针在制备抗肿瘤药物中的应用。
优选的,所述抗肿瘤药物包括抗肿瘤靶向药物。
本发明提供了一种荧光共振能量转移纳米探针,包括负载组分和包封所述负载组分的包封组分,所述负载组分包括载体和包覆在所述载体内的阿霉素,所述载体为羧基改性的空心介孔二氧化硅,所述包封组分包括RVRR肽和PAMAM/TPE。基于羧基改性的空心介孔二氧化硅(HMSN)的羧基的缩合特性,本发明利用弗林蛋白酶(Furin)特异性响应的RVRR肽和PAMAM/TPE表面修饰的氨基与HMSN表面的羧基反应,将RVRR肽和PAMAM/TPE小分子聚合物包裹在HMSN外表面,实现对阿霉素(DOX)的有效包封。当本发明的荧光共振能量转移纳米探针进入肿瘤细胞后,肿瘤细胞中过表达的Furin会切断封堵在HMSN纳米孔处的特异性肽序列,使HMSN内部的DOX得以释放,由于PAMAM/TPE具有黏附作用,PAMAM/TPE会阻碍DOX的快速释放,且释放的DOX与负载在HMSN外表面的PAMAM/TPE紧密缀合,形成TPE/DOX,TPE/DOX可以实现灵敏的FRET荧光“开启”效应,具体过程是:TPE/DOX具有独特的荧光特性:TPE的荧光发射光谱与DOX的吸收光谱具有明显的光谱重叠,可以发生FRET效应,当330nm的激发光被TPE吸收,TPE所发射的光又同时激发DOX荧光发射,从而检测出DOX的荧光信号,即580nm信号峰,通过产生的FRET荧光信号定量检测细胞内Furin。Furin作为一种肿瘤标志物,其含量可以作为肿瘤诊断的依据,通过对细胞内Furin的定量检测,可以实现肿瘤的早期诊断。
先前的研究中,随着DOX在肿瘤细胞中的释放,使得TPE/DOX分离,FRET消失,只能通过FRET信号的减弱进行相关分析。不同于先前研究的由细胞外到细胞内FRET由有到无的过程,本发明采用逆向手段,通过细胞内自组装,实现了细胞内FRET由无到有的过程,为实现细胞内Furin的定量检测提供了一种新的策略。细胞内FRET荧光信号的产生,除了能够特异性分析肿瘤细胞中的过表达Furin的含量外,随着DOX的不断释放,还能够杀伤肿瘤细胞。
附图说明
图1为HMSN纳米颗粒的透射电镜图;
图2为HMSN纳米颗粒的粒径分布图;
图3为HMSN纳米颗粒的N2吸附曲线与孔径分析,B曲线和A曲线分别代表氮气吸附和解吸等温线;
图4为PAMAM(G=1.5)纳米颗粒的透射电镜图;
图5为PAMAM(G=1.5)纳米颗粒的粒径分布图;
图6为HMSN/DOX/RVRR/PAMAM/TPE纳米探针的透射电镜图;
图7为HMSN/DOX/RVRR/PAMAM/TPE纳米探针的粒径分布图;
图8为HMSN纳米颗粒、PAMAM纳米颗粒、DOX、TPE和HMSN/DOX/RVRR/PAMAM/TPE的Zeta电位图;
图9为PAMAM-TPE和DOX的吸收和发射光谱(a:PAMAM/TPE(Abs),b:DOX(Abs),c:PAMAM/TPE(FL Intensity),d:DOX(FL Intensity));
图10为(a)PAMAM-TPE和(b)PAMAM-TPE-DOX在相同浓度(330nm激发)下的荧光光谱;
图11为HMSN/DOX/RVRR/PAMAM/TPE荧光探针在pH=5.0和1.5U/mLFurin存在下在特定时间间隔的荧光光谱(a~f:0h,0.5h,1h,1.5h,2h,4h);
图12为HMSN/DOX/RVRR/PAMAM/TPE荧光探针在pH=5.0和不同浓度Furin存在下在共孵育2h的荧光光谱(a~e:0U/mL,0.5U/mL,1U/mL,1.5U/mL,2U/mL);
图13为在Furin刺激下HMSN/DOX/RVRR/PAMAM/TPE荧光探针的荧光强度比与Furin浓度之间的关系,RSD为3.5%;
图14为HMSN/DOX/RVRR/PAMAM/TPE荧光探针在不同底物存在下的荧光强度;
图15为MDA-MB-468细胞与HMSN/DOX/RVRR/PAMAM/TPE以10μg/mL的剂量孵育不同时间的CLSM图像,比例尺为20μm;
图16为经0.05mM Furin蛋白酶抑制剂I预处理的MDA-MB-468细胞与HMSN/DOX/RVRR/PAMAM/TPE以10μg/mL的剂量孵育不同时间的CLSM图像,比例尺为20μm。
具体实施方式
本发明提供了一种荧光共振能量转移纳米探针,包括负载组分和包封所述负载组分的包封组分,所述负载组分包括载体和包覆在所述载体内的阿霉素,所述载体为羧基改性的空心介孔二氧化硅,所述包封组分包括RVRR肽和PAMAM/TPE。
在本发明中,所述荧光共振能量转移纳米探针的粒径优选为150~190nm,更优选为160~180nm。
在本发明中,所述负载组分与包封组分的质量比优选为1:2;所述RVRR肽和PAMAM/TPE的质量比优选为1:2。
在本发明中,所述羧基改性的空心介孔二氧化硅和阿霉素的质量比优选为(1~2):(1~2),更优选为1:1。
在本发明中,所述羧基改性的空心介孔二氧化硅能够与其他小分子相互作用,RVRR肽和PAMAM/TPE表面修饰的氨基与HMSN表面的羧基反应,将RVRR肽和PAMAM/TPE小分子聚合物包裹在HMSN外表面,实现对阿霉素(DOX)的有效包封。同时,HMSN外表面的PAMAM/TPE具有一定的黏附作用,同时所具备的树枝状结构可以共同实现PAMAM/TPE与DOX的缀合,从而为TPE/DOX实现FRET创造条件。
在本发明中,所述羧基改性的空心介孔二氧化硅优选的利用TESPSA在HMSN表面进行羧基修饰;所述TESPSA在pH=1的条件下与HMSN缩合,所生成的酸酐基团在用水洗涤期间随着pH值增加(pH=6~7)被转化为-COOH;本发明对所述利用TESPSA在HMSN表面进行羧基修饰的方法没有特殊限制,采用本领域的常规方法即可。
本发明对所述HMSN的制备方法没有特殊限制,采用本领域的常规方法即可。本发明具体实施过程中,所述HMSN优选的通过模板法合成。
在本发明中,所述PAMAM/TPE(四苯基乙烯树枝形聚合物)优选的采用以下方法制备得到:
将TPE-COOH溶液、EDC溶液和NHS溶液进行混合,实现羧基的活化,将活化后的TPE-COOH和PAMAM溶液混合,得到PAMAM/TPE。在本发明中,所述TPE-COOH溶液的溶剂优选为DMSO,所述TPE-COOH溶液的摩尔浓度优选为10mM;所述EDC溶液的摩尔浓度优选为0.5mM;所述NHS溶液的摩尔浓度优选为0.5mM;所述活化的温度优选为25℃;所述活化的时间优选为30min;所述PAMAM溶液的浓度优选为4mg/mL;所述混合的温度优选为25℃;所述混合优选为震荡混合;所述混合的时间优选为12h。
本发明还提供了上述方案所述的荧光共振能量转移纳米探针的制备方法,包括以下步骤:
1)将羧基改性的空心介孔二氧化硅溶液和阿霉素溶液混合,得到HMSN/DOX溶液;
2)将所述HMSN/DOX溶液、RVRR肽和PAMAM/TPE进行混合,得到荧光共振能量转移纳米探针。
本发明首先将羧基改性的空心介孔二氧化硅溶液和阿霉素溶液混合,得到HMSN/DOX溶液。
在本发明中,所述羧基改性的空心介孔二氧化硅溶液中羧基改性的空心介孔二氧化硅的质量浓度优选为2~3mg/mL,更优选为2.5mg/mL。在本发明中,所述阿霉素溶液中阿霉素的质量浓度优选为2~3mg/mL,更优选为2.5mg/mL。在本发明中,所述混合的温度优选为20~30℃,更优选为25℃;所述混合的时间优选为20~24h;所述混合避光进行。
本发明在得到HMSN/DOX溶液后,优选的还包括对HMSN/DOX溶液进行离心后,得到沉淀,采用PBS缓冲液对所述沉淀进行洗涤后,将洗涤后的沉淀分散于DMSO。在本发明中,所述离心的转速预选为10000rpm;所述离心的时间优选为10min;所述PBS缓冲液的pH值优选为7.4;所述PBS缓冲液的浓度优选为10mM;所述洗涤的次数优选为3次。
得到HMSN/DOX溶液后,本发明将所述HMSN/DOX溶液、RVRR肽和PAMAM/TPE进行混合,得到荧光共振能量转移纳米探针。
在本发明中,所述混合的温度优选为20~30℃,更优选为25℃;所述混合的时间优选为20~24h;所述混合避光进行。
本发明还提供了上述方案所述的荧光共振能量转移纳米探针或者所述的制备方法制备得到的荧光共振能量转移纳米探针在制备弗林蛋白酶检测试剂中的应用。
在本发明中,所述弗林蛋白酶优选的包括细胞内弗林蛋白酶。
在本发明中,所述检测优选的包括定量检测。本发明通过拟合标准曲线,得到荧光信号强度与弗林蛋白酶浓度的定量关系,即Y=0.396X+0.00952(Y代表DOX的荧光强度I2/TPE的荧光强度I1,X代表Furin浓度,U mL-1,R=0.998)。
本发明还提供了上述方案所述的荧光共振能量转移纳米探针或者所述的制备方法制备得到的荧光共振能量转移纳米探针在制备抗肿瘤药物中的应用。抗肿瘤药物为阿霉素(DOX),将其包封在HMSN孔中。鉴于荧光共振能量转移纳米探针具有合适的粒径,可通过ERP效应进入肿瘤细胞。
在本发明中,所述抗肿瘤药物优选的包括抗肿瘤靶向药物。
下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述。
实施例1
1.1试剂和材料
NH2-PAMAM购自西安瑞熙生物科技有限公司,1-(4-羧基苯基)-1,2,2-三苯基乙烯(TPE-COOH)购自上海迪宝生物科技有限公司,3-三乙氧基甲硅烷基丙基琥珀酸酐(TESPSA)、十六烷基三甲基溴化铵(CTAB)、N-(3-二甲基氨基丙基)-N-乙基氨基丙基二酰亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)购自上海阿拉丁生物化学技术有限公司,KRVRRC多肽链由生工(上海)有限公司合成,盐酸多柔比星(DOX)由生工(上海)有限公司提供,Furin和Furin抑制剂购自艾美捷科技有限公司(湖北)。
1.2仪器
在Zeta-size纳米测试仪(ZEN 3600,Malvern Instruments Ltd.)上进行尺寸和Zeta电位测定。透射电子显微镜图像(TEM)由透射电子显微镜(JEM-2100,JEOL)测定。F-4600荧光分光光度计(日本)用于测量荧光;尼康激光共焦显微镜(尼康,日本)用于获得细胞的共聚焦荧光图像。
1.3制备PAMAM/TPE(四苯基乙烯树枝形聚合物)
首先,使用DMSO溶解TPE-COOH(10mM),将其与10μL EDC(0.5mM),10μLNHS(0.5mM)溶液混合,并在25℃下摇动30分钟以活化羧基,然后将活化后的TPE-COOH转移到含有4mgmL-1PAMAM的离心管中并均匀混合,在25℃下震荡反应12小时。
1.4介孔二氧化硅纳米粒子(HMSN)的制备
通常,二氧化硅(sSiO2)首先通过方法合成。简而言之,将100mL乙醇和8mL水与4mL氨溶液(37%~38%)混合,然后加入3mLTEOS。在30℃下搅拌6小时后,得到sSiO2。随后,通过表面活性剂模板溶胶-凝胶的方法将介孔二氧化硅壳包覆在二氧化硅核上,使用表面活性剂CTAB作为模板形成核/壳结构,我们将制备的sSiO2悬浮液倒入含有220mL水、10mL乙醇和1200mg CTAB的溶液中,并将混合物搅拌30分钟。加入1.075mL TEOS并将分散体搅拌过夜。通过离心收集沉淀物并重新分散在水中,获得sSiO2@mSiO2核/壳纳米粒子。随后,我们采用选择性蚀刻方法来制备HMSN。通过离心收集制备的sSiO2@mSiO2核/壳纳米粒子并重新分散在150mL Na2CO3(0.4M)水溶液中,并将混合物在50℃下搅拌2h去除固体二氧化硅核。其中CTAB是加速sSiO2蚀刻的助剂和保护硅酸盐免受碱性蚀刻的稳定剂。这种CTA+促进的蚀刻再沉积形成是使用Na2CO3蚀刻机制对sSiO2@mSiO2进行选择性蚀刻。为了消除阳离子CTAB对细胞的潜在毒害作用,在超声处理的辅助下,通过HCl/乙醇混合物去除HMSN中的CTAB。在去除介孔壳中的CTAB胶束后,通过用浓HCl/乙醇(v/v=1:10)和水反复洗涤3次,得到HMSN。
1.5用于改性羧基HMSN(HMSN/COOH)的制备
为了便于实现HMSN与其他小分子相互作用,使用TESPSA在HMSN表面进行羧基修饰,它可以在较低的pH值(pH=1)下与HMSN缩合。所生成的酸酐基团在用水洗涤期间随着pH值增加(pH=6~7)被转化为-COOH。即我们将0.1mL TESPSA加入含50mL HCl(0.1M)的HMSN悬浮液中,并将混合物在50℃下搅拌5小时。通过离心收集所得羧基修饰的HMSN,(表示为HMSN,用于所有实验,除非另有说明)并用去离子水洗涤3次。
1.6负载DOX的HMSN(HMSN/DOX)的制备
首先,将2.5mg mL-1HMSN与2.5mg mL-1DOX在PBS缓冲液(pH=7.4,10mM)中混合。将该混合物在室温下避光搅拌24h,确保DOX在较大程度上被捕获到HMSN中。然后,将所得HMSN/DOX溶液离心(10000rpm,10分钟),并用PBS缓冲液(pH=7.4,10mM)洗涤三次,弃去上清液。随后将其分散在1mL DMSO中以进行下一步使用。
1.7 HMSN/DOX/RVRR/PAMAM/TPE探针的制备
首先,将1mL溶解于DMSO中的HMSN/DOX与10μL EDC(0.5mM)溶液和10μLNHS(0.5mM)溶液混合,并在25℃下摇动30分钟以完成活化。然后,加入2.5mg RVRR肽。同时,将500μLPAMAM/TPE添加到反应体系。并在室温下避光搅拌24小时,以促进药物的肽纳米级包封与PAMAM/TPE的缀合。最后将混合物使用PBS缓冲液洗涤三次,通过离心(10000g,10分钟)收集所得的HMSN/DOX/RVRR/PAMAM/TPE,并在PBS(pH=7.4)中重悬,储存在4℃。
2结果与讨论
2.1材料基本表征
首先,对所合成的HMSN纳米粒子用透射电子显微镜(TEM)和动态光散射(DLS)进行表征。通过TEM分析,可以观察到由此产生的HMSN约120nm的粒径和约30~40nm的壳厚度(图1)。同时DLS所测得的粒径分布主要在140nm左右(图2)。实验数据相互吻合。同时,对其进行了氮气吸附与脱附实验,实验结果表明其孔径为2.5nm左右(图3),这与文献报道的CTAB胶束的粒径相近。为了增加HMSN的反应位点,我们使用TESPSA在HMSN表面进行羧基修饰,修饰后的Zeta电位向更负的电位移动了,这意味着我们实现了HMSN的羧基修饰。接下来,通过TEM表征PAMAM(G=1.5)树枝状聚合物的粒径大小。如图4所示,PAMAM的粒径约为5nm,且分散性良好。DLS测得粒径分布在8nm左右(图5)。鉴于PAMAM表面存在大量氨基,其Zeta电位为9.83mV,而DOX和TPE-COOH的Zeta电位分别为24.3和-8.6mV(图8)。同时,为了防止DOX的提前泄露,利用RVRR肽和PAMAM/TPE实现DOX的封装。为了验证HMSN/DOX成功实现了包封,对其进行了TEM表征。如图6所示,可以清楚的看到HMSN表面有一个厚度均匀的透明薄层结构,其粒径大约为150nm,证明HMSN/DOX/RVRR/PAMAM/TPE探针的成功合成。通过DLS和Zeta电位表征,得到其粒径分布在160nm左右(图7),Zeta电位为3.25mV(图8),这些结果证实了RVRR/PAMAM/TPE已经成功包裹在HMSN表面。
2.2可行性研究
研究TPE/DOX实现FRET的可行性。经过荧光实验测得,PAMAM/TPE分子的最大发射波长位于480nm,与DOX的最大吸收波长存在较明显重叠,表明TPE和DOX可以组成FRET对以实现细胞中Furin的特异性定量检测(图9)。
在330nm紫外光激发下,比较PAMAM/TPE与PAMAM/TPE/DOX的荧光光谱,发现:480nm处,PAMAM/TPE/DOX的荧光发射强度远低于PAMAM/TPE的荧光强度;而在580nm与600nm处,PAMAM/TPE/DOX的荧光发射强度却大大增强(图10)。该结果说明FRET可以发生在TPE、DOX之间。为了进一步验证HMSN/DOX/RVRR/PAMAM/TPE的FRET效应并同时监测DOX分子的释放,我们利用了pH/Furin触发的肽链断裂反应监测在pH=5.0和1.5U mL-1 Furin处理HMSN/DOX/RVRR/PAMAM/TPE并测量在不同时间点的荧光强度变化。我们可以发现随着时间的延长,TPE分子的荧光强度逐渐减弱,而DOX在580nm与600nm处发射的荧光强度大大增强,在2h之后逐渐趋于稳定(图11)。这反映了在Furin的作用下,RVRR肽的断裂触发了DOX的释放,同时被PAMAM/TPE所捕获,从而发生TPE和DOX之间的FRET效应,可实现Furin的定性分析。
为了研究本方法对Furin的定量检测能力,使用不同浓度的Furin溶液作为检测样品,将孵育后的溶液进行荧光检测。图12显示了不同浓度的Furin(0、0.5、1、1.5和2U mL-1)孵育后两种荧光信号(TPE的荧光强度I1和DOX的荧光强度I2)的变化曲线。如图所示,随着Furin浓度的增加,I1逐渐降低,而I2随之增强,说明FRET的产生具有一定的浓度依赖性。据此拟合线性方程为Y=0.396X+0.00952(Y代表I2/I1,X代表Furin浓度,U mL-1 ,R=0.998),RSD为3.5%(图13)。通过工作曲线得到1.0×106个MDA-MB-468细胞中的Furin浓度为1.15±0.08ng mL-1,与文献报道一致。
为了检测本方法对Furin的选择性,我们评估了不同底物反应后的荧光信号变化情况。如图14所示,分别用Furin、GSH、Cys、BSA和葡萄糖处理,发现Furin处理的I2/I1荧光信号明显高于其他物质,这是因为Furin可以诱导RVRR肽裂解,导致DOX分子释放,并与TPE组成FRET对,使得TPE的荧光信号I1逐渐降低,而DOX的荧光信号I2随之增强。因此本方法具有令人满意的选择性。
2.3材料的细胞内化能力与细胞内荧光检测
鉴于HMSN/DOX/RVRR/PAMAM/TPE在细胞外对Furin的优异检测特性,我们将其用于活细胞中Furin引发的TPE/DOX中产生的FRET荧光信号的变化,这可以直观地反映药物的释放情况,同时实现细胞中Furin的分析。为了实现自体供应Furin,我们选择了Furin过表达的乳腺癌细胞MDA-MB-468细胞作为研究对象。首先将MDA-MB-468细胞与10μg mL-1HMSN/DOX/RVRR/PAMAM/TPE在37℃下孵育不同时间以进行细胞成像。如图15所示,当细胞与HMSN/DOX/RVRR/PAMAM/TPE荧光探针孵育2h后,可直接观察到蓝色和绿色荧光,表明HMSN/DOX/RVRR/PAMAM/TPE荧光探针在2h左右可以实现细胞内化,并且主要集中在细胞质中。此时,DOX的荧光很弱,甚至处于关闭状态,说明DOX分子主要被包裹在纳米粒子的核心。随着孵育时间的不断延长,HMSN/DOX/RVRR/PAMAM/TPE孵育的MDA-MB-468细胞的荧光成像显示逐渐减弱的TPE蓝色荧光与逐渐增强的DOX绿色荧光,与细胞外FRET荧光信号的变化趋势一致。此外,如图16所示,在预孵育0.05mM Furin蛋白酶抑制剂I的MDA-MB-468细胞中只是观察到了TPE具有的蓝色荧光,而无明显绿色荧光的产生。这些结果进一步证实了MDA-MB-468细胞中过表达的Furin与HMSN/DOX/RVRR/PAMAM/TPE中的特异性多肽链响应,实现特异性切割,从而促使DOX的释放。鉴于HMSN外部紧密排列PAMAM/TPE可以将释放出的DOX扦插于PAMAM内部,从而减小TPE/DOX的分子间距,实现细胞内FRET效应由无到有的过程。从而通过细胞内FRET荧光信号的产生对细胞内Furin进行特异性分析。
3总结
综上所述,本发明提供了一种能够在细胞内发生FRET效应的复合纳米体系,实现了FRET在细胞内由“无”到“有”的过程。一方面,在Furin作用下,HMSN/DOX/RVRR/PAMAM/TPE探针中的RVRR肽被特异性识别切割,DOX得以释放,形成TPE/DOX的FRET复合体,从而产生FRET荧光信号。借助这种荧光信号的产生,将其应用于肿瘤细胞中Furin定量检测。另一方面,在FRET荧光信号产生的同时,完成DOX在肿瘤细胞内的精准释放,从而减小对正常细胞的损伤。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (9)
1.一种荧光共振能量转移纳米探针,其特征在于,包括负载组分和包封所述负载组分的包封组分,所述负载组分包括载体和包覆在所述载体内的阿霉素,所述载体为羧基改性的空心介孔二氧化硅,所述包封组分为RVRR肽和PAMAM-TPE。
2.根据权利要求1所述的荧光共振能量转移纳米探针,其特征在于,所述羧基改性的空心介孔二氧化硅和阿霉素的质量比为(1~2):(1~2)。
3.权利要求1或2所述的荧光共振能量转移纳米探针的制备方法,包括以下步骤:
1)将羧基改性的空心介孔二氧化硅溶液和阿霉素溶液混合,得到HMSN/DOX溶液;
2)将所述HMSN/DOX溶液、RVRR肽和PAMAM-TPE进行混合,得到荧光共振能量转移纳米探针。
4.根据权利要求3所述的制备方法,其特征在于,步骤1)和步骤2)中,所述混合的温度独立为20~30℃;所述混合的时间独立为20~24h;所述混合避光进行。
5.权利要求1或2所述的荧光共振能量转移纳米探针或者权利要求3或4所述的制备方法制备得到的荧光共振能量转移纳米探针在弗林蛋白酶检测中的应用;所述应用为非诊断目的。
6.根据权利要求5所述的应用,其特征在于,所述弗林蛋白酶包括细胞内弗林蛋白酶。
7.根据权利要求5或6所述的应用,其特征在于,所述检测包括定量检测。
8.权利要求1或2所述的荧光共振能量转移纳米探针,权利要求3或4所述的制备方法制备得到的荧光共振能量转移纳米探针在制备抗肿瘤药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述抗肿瘤药物包括抗肿瘤靶向药物。
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