CN113429461B - 一种聚集诱导发光多肽胶束型诊断试剂及其在近红外区域生物成像中的应用 - Google Patents
一种聚集诱导发光多肽胶束型诊断试剂及其在近红外区域生物成像中的应用 Download PDFInfo
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Abstract
本发明属于抗肿瘤药物制备技术领域,具体公开了一种具有聚集诱导发光(AIE)性质的多肽胶束型诊断试剂AGPR及其在近红外区域生物成像中的应用。所述诊断试剂AGPR是由两亲性小分子功能肽ACBT‑GGFLG‑PEGn‑R8GD,4≤n≤20,在水溶液中自组装形成的球型多肽胶束。本发明设计合成的新型AIEgen化合物ACBT是线性D‑A‑A分子,其发射峰值位于近红外一区,本发明进一步用多功能嵌合肽GGFLG‑PEG8‑R8GD对该ACBT分子进行改性,实现AIEgen分子(ACBT)高效富集于肿瘤部位,并在富含Cathepsin B的细胞溶酶体/内涵体中具有明显增强的荧光强度,成像效果优异,因此有望应用于肿瘤细胞特异性示踪。
Description
技术领域
本发明属于抗肿瘤药物制备技术领域,具体涉及一种具有聚集诱导发光(AIE) 性质的多肽胶束型诊断试剂AGPR及其在近红外区域生物成像中的应用。
背景技术
一直以来,荧光材料在生物成像、分子识别与示踪、环境检测与保护及医学诊断等领域提供重要的应用价值。现有的荧光示踪试剂(例如荧光蛋白、量子点、荧光素酶及有机荧光分子染料等)具有不少问题,其中荧光素酶和荧光蛋白需要基因转染,导致细胞的正常生理功能会受到干扰;量子点具有重金属毒性,并且在细胞分化过程中其荧光强度会急剧下降;传统的有机荧光分子在高浓度溶液或聚集状态下往往会出现荧光猝灭现象(ACQ),因而在生物荧光成像和信号追踪等领域的应用受到了严重限制。聚集诱导发光分子(AIEgens)作为一种特殊的有机发光分子,在稀溶液中分子内部可以自由旋转和振动,导致能量的散失以非辐射跃迁为主,从而呈现极弱的荧光发射或非发射状态。但是在聚集态和固态中,AIEgens的分子内转动受到限制,能量散失从非辐射跃迁变为辐射跃迁,从而表现出强烈的荧光发射,从根本上解决有机荧光染料的ACQ问题,对发展新型的荧光示踪试剂具有重要意义。
由于AIEgen分子本身具有一定的疏水性,高毒性甚至致癌性,而生物体系的所有生命活动均处于水环境中,因此制备水溶性的AIEgen衍生物材料有助于实现其生物医学应用。目前较多的解决方案是利用物理包埋法将其载入聚合物纳米颗粒内部,或者通过化学反应将其接枝到聚合物链段上,从而增强AIEgen的水溶性。但是,AIEgen分子与聚合物反应的合成过程复杂,易引入有机试剂,降低其生物安全性,且分子本身缺乏肿瘤靶向性,不能进行长期高效、低毒示踪。因此,在尽可能保持AIEgen的发光性能的基础上,探索简单高效低毒的合成方法以提高AIEgen衍生物材料水溶性和靶向生物成像性能具有重要意义。
发明内容
针对上述现有技术的不足,本发明的首要目的在于提供一种水溶性好,具有良好的生物相容性的聚集诱导发光多肽胶束型诊断试剂。
本发明的另一目的在于提供一种反应条件温和,简单可控的聚集诱导发光多肽胶束型诊断试剂的制备方法。
本发明的再一目的在于提供上述的聚集诱导发光多肽胶束型诊断试剂在制备肿瘤细胞示踪药物中的应用。
本发明的构思:功能嵌合肽(GGFLG-PEG8-R8GD)因链段含有丰富的氨基 (-NH2)和羧基(-COOH),能够与含有羧基的AIEgen分子(ACBT)发生酰胺缩合反应,从而对AIEgen分子(ACBT)进行改性,因功能嵌合肽 (GGFLG-PEG8-R8GD)的功能多样性,从而能够有效抑制AIEgen分子(ACBT) 的体内蛋白质非特异性吸附,延长其在体内的长血液循环,实现AIEgen分子 (ACBT)高效富集于肿瘤部位,并在富含Cathepsin B的细胞溶酶体/内涵体中具有明显增强的荧光强度,成像效果优异,且发射峰值位于近红外一区,因此有望应用于肿瘤细胞特异性示踪。
为了实现上述目的,本发明采用的技术方案为:
一种聚集诱导发光多肽胶束型诊断试剂AGPR,所述诊断试剂AGPR由两亲性小分子功能肽ACBT-GGFLG-PEGn-R8GD(4≤n≤20)在水溶液中自组装形成的球型多肽胶束,疏水性AIEgen化合物ACBT构成胶束的疏水性内核,亲水性功能肽GGFLG-PEGn-R8GD构成胶束的亲水性外壳。所述两亲性小分子功能肽 ACBT-GGFLG-PEGn-R8GD(4≤n≤20,优选n=8)由聚集诱导发光化合物AIEgen (ACBT)与功能嵌合肽GGFLG-PEGn-R8GD通过酰胺缩合反应连接。
所述ACBT分子的结构式为:
分子式:C34H22N4O2S;
分子量:550.63;
所述AIEgen分子(ACBT)采取以下合成工艺路线进行合成:
所述AIEgen分子(ACBT)的合成方法为:
(1)在四-(三苯基膦)钯催化下,4-硼酸三苯胺和7-溴苯并[c][1,2,5]噻二唑 -4-甲醛以2:1的摩尔比在甲苯/水(体积比为20:3)的混合溶液中,于110℃的无氧条件下发生Suzuki偶联反应,合成得到化合物1(ABT);
(2)将化合物1(ABT)和4-(氰甲基)苯甲酸甲酯以1:1.2的摩尔比在含有哌啶的乙醇溶液中,于80℃的无氧条件下发生加成反应,生成化合物2(ECBT);
(3)向含有化合物2(ECBT)的四氢呋喃溶液中加入1mol/L氢氧化钠的水溶液,得到化合物2(ECBT)的钠盐;随后,用二氯甲烷(DCM)萃取出上层水相,并加入1mol/L HCl的水溶液(40mL),搅拌24h,再次用二氯甲烷(DCM) 萃取出有机相液体,通过旋转蒸发仪旋干以上溶液,最终得到紫红色化合物3,即ACBT。
所述功能嵌合肽GGFLG-PEGn-R8GD,4≤n≤20;其中PEGn指n个乙二醇分子组成的线性聚合物,其结构式如下:
优选的,所述功能嵌合肽GGFLG-PEGn-R8GD,n=8;
所述功能嵌合肽GGFLG-PEG8-R8GD,其结构式如下:
分子量:2826.45;
所述功能嵌合肽GGFLG-PEG8-R8GD是由四种功能性多肽序列构成,分别为能与肿瘤细胞发生特异性粘附的肿瘤靶向肽序列RGD,能快速跨膜进入细胞的细胞穿膜肽序列R8,容易被溶酶体中组织蛋白酶(Cathepsin B)水解断裂的酶敏感性多肽序列GGFLG,及亲水性聚乙二醇(PEG),赋予了聚集诱导发光多肽胶束型诊断试剂较好的水溶性、主动靶向肿瘤细胞、快速穿膜进入细胞以及酶刺激响应性四种不同功能。
所述两亲性小分子功能肽ACBT-GGFLG-PEGn-R8GD,其化学结构式如下:
所述两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD,其化学结构式如下:
所述两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD的合成方法:利用多肽固相合成-Fmoc法依次连接天冬氨酸Asp(D),甘氨酸Gly(G),精氨酸 Arg(R)×8,氨基-八聚乙二醇-羧基(NH2-PEG8-COOH),甘氨酸Gly(G),亮氨酸Leu(L),苯丙氨酸Phe(F),甘氨酸Gly(G),甘氨酸Gly(G)和 AIEgen分子(ACBT),用茚三酮的甲醇溶液(10mg/mL)验证树脂上的氨基酸之间酰胺缩合反应是否彻底完成,若验色溶液为亮黄色或者未变色则证明酰胺缩合反应成功。通过脱除氨基保护基团(FMOC)以后,继续下一步酰胺缩合反应,直至合成目标产物ACBT-GGFLG-PEG8-R8GD。其分子量由基质辅助激光解析- 飞行时间质谱(MALDI-TOF MS)测得。
聚集诱导发光多肽胶束型诊断试剂AGPR的制备方法:将两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD溶于水,然后将所得溶液加入透析袋,于超纯水中透析除去杂质,两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD在水溶液中自组装形成核壳型胶束。其中,疏水性AIEgen化合物ACBT构成胶束的疏水性内核,亲水性功能肽GGFLG-PEG8-R8GD构成胶束的亲水性外壳。透析完成后,将透析袋内的水溶液冷冻干燥成粉末,即得聚集诱导发光多肽胶束型诊断试剂AGPR。
进一步,所述透析袋的截留分子量为500-3500Da。
进一步,所述透析过程至少为24h。
所述聚集诱导发光多肽胶束型诊断试剂AGPR在良性溶剂(N,N-二甲基甲酰胺)中的荧光强度极弱,这是因为AIEgen分子在溶解状态能够自由运动,通过非辐射衰变消耗能量。随着N,N-二甲基甲酰胺/水混合溶液中水的体积比 (Water Fraction,指水与DMF的体积比)增加到80%时,AGPR在606nm处的荧光强度突然显著增强,主要因为AIEgen在不良溶剂中的聚集会限制分子内的运动,这时候荧光分子通过辐射跃迁的形式消耗能量,激活了AIE性能。同时根据发射光谱显示,AIEgen(ACBT)的发射光峰值在660nm,属于近红外一区(650-900nm),有助于生物成像。ACBT是一类线性三苯胺类给体-苯并噻二唑受体-氰基受体(D-A-A)推拉分子,通过在吸电子氰基附近增加一个苯环有利于分子吸收光谱的红移。
上述聚集诱导发光多肽胶束型诊断试剂AGPR在制备肿瘤细胞特异性示踪药物中的应用,所述肿瘤细胞为整合素蛋白过表达的肿瘤细胞。
由上述制得的聚集诱导发光多肽胶束型诊断试剂AGPR具有很好的组织蛋白酶(Cathepsin B)响应性和水溶性,与传统的小分子荧光探针相比,不仅具有光稳定性好,灵敏度高,荧光光谱不漂移和高浓度时无猝灭等优点,在富含 Cathepsin B的细胞溶酶体/内涵体中具有明显增强的荧光强度,成像效果优异,有望应用于肿瘤细胞特异性示踪,尤其适用于整合素蛋白过表达的肿瘤细胞或肿瘤内皮血管,包括人宫颈癌细胞(HeLa)、人神经胶质瘤细胞(U87)、乳腺癌细胞(4T1)、人源三阴性乳腺癌细胞(MDA-MB-231)、人肝癌细胞(HEPG-2) 和人脐静脉内皮细胞(ECV304)中的示踪,以及药物代谢检测和细胞代谢检测等领域。
与现有技术相比,本发明的技术方案具有以下优点和有益效果:
1、本发明设计和合成的新型AIEgen化合物ACBT具有合成简单、产率高和NIR(在第一近红外NIR-I窗口,650-900nm)荧光发射性能的优点,有利于其在生物医用领域的应用。
2、本发明设计和合成的聚集诱导发光多肽胶束型诊断试剂AGPR具有肿瘤细胞靶向性(RGD),细胞穿膜性(R8),组织蛋白酶敏感性(GGFLG)和自组装性能,在水溶液中能够自组装形成核-壳型纳米胶束。该两亲性小分子功能肽的临界胶束浓度值(CMC)为8.5mg/L,远远小于传统的两亲性多肽的CMC 值,表明该两亲性小分子功能肽在极低的浓度下能够自组装形成胶束,且纳米胶束的结构更稳定。
3、聚集诱导发光多肽胶束型诊断试剂AGPR在水溶液中自组装形成尺寸在 40nm±1nm的球形纳米颗粒,具有EPR(高渗透长滞留)效应,在药物载体的制备和药物可控释放等生物领域具有巨大的应用潜力。
4、相比常用的聚合物胶束,多肽胶束具有无毒,生物相容性好、生物可降解性及功能多样化等优势。材料的结构组成简单,能有效避免组成结构复杂的治疗系统中效率低、系统副作用大的不良后果,为精准的可视化肿瘤诊疗提供新的研究思路。
附图说明
图1为实施例一合成的ABT的核磁共振氢谱(1H NMR);
图2为实施例一合成的ECBT的核磁共振氢谱(1H NMR);
图3为ACBT的MALDI-TOF MS图谱;
图4为AGPR的MALDI-TOF MS图谱;
图5为AGPR在含有组织蛋白酶Cathepsin B的环境下24h以后的 MALDI-TOF MS图谱;
图6为实施例三中AGPR(ACBT-GGFLG-PEG8-R8GD)的CMC图;
图7为实施例三中AGPR的TEM图谱;
图8为实施例四中ACBT的AIE性能测试实验结果图;
图9为实施例四中AGPR的AIE性能测试实验结果图;
图10为实施例五中AGPR的细胞靶向能力测试实验结果图;
图11为实施例五中AGPR的细胞AIE成像能力测试实验结果图;
图12为实施例六中AGPR的的细胞毒性实验结果图,其中,*:p<0.05,**: p<0.01。
具体实施方式
下面申请人将结合具体的实施例和附图对本发明的技术方案做详细说明,便于本领域技术人员清楚地理解本发明。但是以下实施例不应以任何方式被解释为对本发明权利要求书请求保护的范围的限制。
以下实施例中所用试剂均为常见市售商品,纯度级别均为分析纯。
实施例一、ACBT的合成
按以下合成路线合成ACBT:
ACBT的合成步骤为:
将7-溴苯并[c][1,2,5]噻二唑-4-甲醛(简称为ABB)(1mmol),4-硼酸三苯胺(2mmol),无水碳酸钠(1.06g,10mmol),四(三苯基膦)钯(50mg,0.04mmol)放入三口烧瓶中,通入氮气以除尽烧瓶中的氧气。随后在氮气氛围下取20mL甲苯和3mL水注入烧瓶中,将烧瓶置于110℃的油浴锅内回流反应24h。反应结束后,加入40mL二氯甲烷以及4.0g硅胶粉,利用旋转蒸发仪旋成粉末。化合物 1(简称为ABT)通过硅胶柱色谱法纯化,以二氯甲烷与石油醚体积比为3:1的混合溶剂作为洗脱剂,最终纯化得到红色固体,其结构通过核磁氢谱验证(见图1)。 ABT的结构通过1H NMR(400MHz,CDCl3)得到,δ7.12(m,2H),7.21(m,6H), 7.32(m,4H),7.85(d,1H),7.94(m,2H),8.29(d,1H),10.75(s,1H)。表明我们成功合成了化合物ABT。
将化合物1(1mmol),4-(氰甲基)苯甲酸甲酯(1.2mmol))放入三口烧瓶中,通入氮气以除尽烧瓶中的氧气。在氮气氛围下取20mL无水乙醇注入烧瓶中,并将装置移入80℃的油锅中回流搅拌,此时取1mL哌啶缓慢滴加至烧瓶,12h后停止反应。向烧瓶内加入40ml二氯甲烷和4.0g硅胶粉,利用旋转蒸发仪旋成粉末。化合物2(ECBT)通过硅胶柱色谱法纯化,以二氯甲烷与乙酸乙酯体积比为 4:1的混合溶剂作为洗脱剂,最终纯化得到红色固体,其结构通过核磁氢谱1H NMR(400MHz,CDCl3)验证(见图2)。δ3.82(s,3H),3.96(s,1H),7.10(td,1H),7.20(m,4H),7.31(dd,2H),7.41(m,4H),7.89(m,1H),8.06(m,2H), 8.15(dd,4H),8.64(s,1H),8.75(d,1H)。表明我们成功合成了AIE荧光分子化合物ECBT。
向溶有化合物2(1mmol)的四氢呋喃(20mL)溶液中加入1mol/L氢氧化钠水溶液(20mL),搅拌24h。待反应结束后,将液体加入到分液漏斗中,用二氯甲烷进行萃取,取上层水相倒入烧瓶,加入1mol/L HCl的水溶液(40mL),搅拌 24h。待反应结束后,将液体加入分液漏斗中,用二氯甲烷萃取,得到的有机相用旋转蒸发仪旋干,最终得到化合物3(ACBT),为紫红色固体。利用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)检测其分子量(见图3),理论分子量为551.15,实际测分子量得为551,与理论值符合,再结合其中间产物的核磁数据,证明成功合成得到羧基修饰的ACBT。
实施例二、聚集诱导发光多肽胶束型诊断试剂AGPR的合成
1、采用标准的Fmoc固相多肽合成法合成功能嵌合肽GGFLG-PEG8-R8GD:称取2-氯-三苯甲基氯树脂(0.8g,0.97mmol/g)加入多肽固相合成柱中,加入20 mLN,N-二甲基甲酰胺(DMF)浸泡树脂30分钟,使其充分溶胀后抽滤除去DMF。准备接入第一个氨基酸,进入氨基酸缩合反应。随后向多肽固相合成柱中加入溶有Fmoc-Asp(OtBu)-OH(相当于树脂取代度的3倍量)和DIEA(相当于树脂取代度的6当量)的DMF溶液,反应2h。反应结束后抽走滤液,并用DMF洗涤4次。得到第一个被FMOC保护的氨基酸缩合后的肽树脂。然后将20%的哌啶(piperidine)/DMF(v/v)溶液加到树脂中,脱除氨基酸N端的氨基保护基FMOC基团,反应半小时(15min×2)后除去反应溶液,并用DMF洗涤树脂三次。此后每一次在树脂上连接氨基酸残基时,均将溶解有被FMOC保护的氨基酸(相当于树脂取代度的4倍量)、1-羟基苯并三唑(HOBt,相当于树脂取代度的4倍量)、苯并三氮唑-N,N,N’N’-四甲基脲六氟磷酸盐(HBTU,相当于树脂取代度的4倍量)和 N,N-二异丙基乙胺(DIEA,相当于树脂取代度的6倍量)的DMF加入到树脂中进行缩合反应。反应1.5h后,用茚三酮的甲醇溶液(10mg/mL)验证树脂上的氨基酸之间酰胺缩合反应是否彻底完成,若验色溶液为亮黄色或者未变色则证明酰胺缩合反应成功。酰胺缩合反应成功后,脱除FMOC基团以后,继续进行酰胺缩合反应,依次与FMOC-Gly-OH,FMOC-Arg(Pbf)-OH(与FMOC-Arg(Pbf)-OH 重复八次),FMOC-NH-PEG8-COOH(其中原料氨基-八聚乙二醇-羧酸购自aladdin 试剂有限公司,其N端也被FMOC基团保护),FMOC-Gly-OH,FMOC-Leu-OH, FMOC-Phe-OH,FMOC-Gly-OH,FMOC-Gly-OH进行酰胺缩合反应,直至合成目标氨基酸序列GGFLG-PEG8-R8GD。
2、待多肽序列合成完毕,脱除FMOC基团以后,接着向固相合成柱中加入实施例一合成的化合物ACBT(4当量),PyBOP(4.8当量),HOBt(4.8当量)和 DiEA(2mL)的DMF溶液,反应24小时后抽滤,依次用DMF、甲醇、二氯甲烷洗涤树脂3次,并将树脂进行真空干燥。干燥后,向反应器中加入切落剂(体积占比为95%TFA、2.5%H2O、2.5%TIS)反应100分钟后,收集滤液,经过旋蒸浓缩后,用冷乙醚沉淀后离心,真空干燥24h,得到两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD。两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD的分子量通过MALDI-TOF MS测得(见图4)。其中ACBT-GGFLG-PEG8-R8GD 的理论分子量为2826.45,实际测得的主峰分子量为2826,证明 ACBT-GGFLG-PEG8-R8GD的成功合成。
3、将两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD溶于水中得溶液,然后将所得溶液加入透析袋(MWCO 3500Da),于超纯水中透析24h除去小分子杂质,每隔4h换一次超纯水。透析完后,将透析袋内的水溶液冻成固体,然后置于冻干机冷冻干燥成粉末。两亲性小分子功能肽ACBT-GGFLG-PEG8-R8GD 在水溶液中自组装形成核壳型聚集诱导发光多肽胶束型诊断试剂AGPR。利用透射电子显微镜(TEM)表征实施例二步骤3制备的多肽胶束型诊断试剂AGPR 形成胶束的形貌和尺寸,如图7所示。从图7TEM中观察到胶束呈球形,并具有较好的分散性,胶束的干态粒径为40nm±1nm,证明了功能嵌合肽在水溶液中具有较好的自组装性能。
此外,利用MALDI-TOF MS对聚集诱导发光多肽胶束型诊断试剂AGPR与组织蛋白酶Cathepsin B(20units/mg)以10:1的质量比共孵育酶解24h后的分子量进行检测。与AGPR酶解前的分子量分布相比,酶解后的MALDI-TOF MS 图谱上出现了分子量为770、1074和1229的三个峰,这与AGPR中的多肽片段 LG-PEG8-R、FLG-PEG8-RR和FLG-PEG8-RRR分子量相吻合(见图5),表明了AGPR能够被组织蛋白酶Cathepsin B有效瓦解。
实施例三、聚集诱导发光多肽胶束型诊断试剂AGPR的自组装性能和形貌表征
以芘为疏水性荧光探针,利用荧光分光光度计测AGPR的临界胶束浓度 (CMC)值,评估AGPR的自组装性能和稳定性。精确称量9.7mg疏水性芘荧光分子,将其溶于10mL丙酮,然后稀释至4.6×10-6mol/L,备用。往3.6mL 已配好的一系列浓度的AGPR水溶液(取实施例二步骤2制得的 ACBT-GGFLG-PEG8-R8GD粉末溶于去离子水中配置而成)加入45μL 4.6×10-6 mol/L芘的丙酮溶液,配置成样品。将配置好的样品放入37℃摇床(150r/min)震荡24h,确保丙酮完全挥发。最后,利用荧光分光光度计测定样品溶液在360-400 nm内的发射光谱(激发波长为342nm,发射光和激发光的狭缝宽度均设为5nm)。以AGPR溶液在393nm与374nm处的荧光强度比值(I/I0)作为纵坐标,以AGPR 溶液浓度的对数(lgC)为横坐标作图。找出两条直线的交点对应的横坐标值,从而计算得到该两亲性功能嵌合肽的CMC值。AGPR的CMC值测得为8.5mg/L (见图6),远远小于传统的两亲性多肽的CMC值,表明该两亲性功能嵌合肽在极低的浓度下能自组装形成胶束,且结构稳定,非常适合于药物载体的设计和制备。
实施例四、AIE性能测试实验
(1)ACBT的AIE性能测试
配制一系列ACBT(实施例一中制备的)浓度为30mg/L的溶液(以四氢呋喃-水作溶剂),其中水的体积占比(Water fraction,指水与THF的体积比)分别为90%、80%、70%、60%、50%、40%、30%、20%、10%和0%。设定激发波长为495nm,利用荧光分光光度计测定以上溶液在500-750nm范围内的发射光谱,并记录其在荧光强度的峰值,见图8A,并以各溶液在荧光强度的峰值与初始荧光强度(在纯四氢呋喃溶剂中的荧光强度)之比为纵坐标,与水的体积占比为横坐标绘制曲线,见图8B。由图8A发射光谱显示,ACBT的发射光谱峰值在660nm,属于近红外一区(650-900nm),有助于生物成像;ACBT在良性溶剂(THF)中,在650nm处的荧光强度极弱,并且随着混合溶剂中水的体积占比增大,ACBT的荧光强度越来越强。由图8B显示,当水的体积占比为90%时, ACBT的荧光强度比初始强度增大了44倍,证明合成的AIEgen分子ACBT具有很好的AIE效应。
(2)AGPR的AIE性能测试
配制一系列AGPR(实施例二中制备的)浓度为53.5mg/L的溶液(以N,N- 二甲基甲酰胺-水作溶剂),其中N,N-二甲基甲酰胺与水的体积比分别为1:99、 1:9、2:8、3:7、4:6、5:5、6:4、7:3、8:2和9:1,即水的体积占比(Water fraction,指水与DMF的体积比)分别为99%、90%、80%、70%、60%、50%、40%、30%、 20%和10%。设定激发波长为480nm,利用荧光分光光度计测定以上溶液在 500-650nm范围内的荧光发射光谱(见图9A),并记录其峰值的荧光强度值,以水的体积占比为横坐标,以该水的体积占比的溶液的荧光强度为纵坐标,绘制曲线(见图9B)。AGPR在良性溶剂(N,N-二甲基甲酰胺)中,荧光强度极弱。随着N,N-二甲基甲酰胺/水混合溶液中水的体积比(Water fraction,指水与N,N- 二甲基甲酰胺的体积比)增加到80%时,AGPR在606nm处的荧光强度突然显著增强,激活了AIE过程。当水的体积比为90%时,AGPR的荧光强度是在水的体积比为10%时的N,N-二甲基甲酰胺中的荧光强度的4.3倍,证明了我们合成的多肽胶束型诊断试剂AGPR在人体内富含组织蛋白酶CathepsinB的肿瘤溶酶体/内涵体环境中具有良好的AIE效应。
实施例五、细胞实验
人宫颈癌细胞(HeLa)和非洲绿猴肾细胞(COS 7)均为本申请发明人实验室的冻存材料,细胞培养基则由1%的双抗(青霉素-链霉素)加入购买的含10%的胎牛血清蛋白的DMEM培养基中配制而成(以下简称DMEM)。所有细胞均在细胞培养基中孵育,在37℃条件下,含有5%CO2的细胞培养箱中培养。
体外细胞内吞实验:将HeLa细胞和COS 7细胞以105个/孔的密度分别接种到共聚焦培养皿中培养24h,然后各加入1mL含有多肽胶束型诊断试剂 AGPR(53.5mg/L)的DMEM溶液,并将共聚焦培养皿放入细胞培养箱中继续培养4h。随后用pH为7.4的PBS溶液(10mmol/L)洗涤细胞三次,再加入1mL 含Hoechst 33342(10μg/mL)的DMEM(不含胎牛血清)对细胞核进行染色15min。完成细胞核染色后吸出培养基,用pH为7.4的PBS溶液(10mmol/L)洗涤细胞三次,加入2mL DMEM培养基,并通过超分辨显微镜观测(激发波长:488nm,荧光接收通道:590nm±30nm)。
图10为实施例五中多肽胶束型诊断试剂AGPR的体外细胞内吞实验结果图;图10从左至右依次为:细胞核的Hoechst 33342蓝色荧光场(Hoechst 33342), AGPR的红色荧光场(AGPR),明场(Bright)和叠加场(Merge)。从上至下依次为:与多肽胶束型诊断试剂AGPR共培养的HeLa细胞、与多肽胶束型诊断试剂AGPR共培养的COS7细胞。从图10所知,HeLa细胞中出现了大量ACBT 的红色荧光,这是由于AGPR表面修饰的PEG8能有效提高AIEgen分子(ACBT) 的水溶性,使材料在靶向肽RGD的作用下,特异性识别并与HeLa细胞表面过度表达的整合素αvβ3结合,并且在穿膜肽R8的辅助下快速穿膜进入HeLa细胞,从而将AIEgen分子(ACBT)有效运输到细胞质内。接着,HeLa细胞质内过表达的Cathepsin B能有效裂解GGFLG序列,从而引发多肽胶束结构瓦解,实现 ACBT的定点释放。与此同时,疏水性AIEgen分子(ACBT)在细胞内会迅速团聚,激活其AIE效应,进而发挥细胞内成像功能。相比而言,AGPR在正常细胞(COS7)中的荧光强度很弱,有效证明了RGD肽的靶向性能。
肿瘤细胞内AIE成像性能实验:将HeLa细胞以105个/孔的密度接种于共聚焦培养皿中培养24h,随后加入一系列1mL含有多肽胶束型诊断试剂AGPR(25 mg/L、53.5mg/L和100mg/L)的DMEM溶液,并继续共培养4h后。随后用 pH为7.4的PBS溶液(10mmol/L)洗涤细胞三次,然后再加入1mL含Hoechst 33342(10μg/mL)的DMEM(不含胎牛血清)对细胞核进行染色15min。完成细胞核染色后吸出培养基,用pH为7.4的PBS溶液(10mmol/L)洗涤细胞三次,加入2mL DMEM培养基,并通过超分辨显微镜观测(激发波长:488nm,荧光接收通道:590nm±30nm)。
图11从左至右依次为:细胞核的Hoechst 33342蓝色荧光场(Hoechst 33342),AGPR的红色荧光场(AGPR),明场(Bright),和叠加场(Merge)。从上至下依次为:与25mg/L、53.5mg/L和100mg/L的多肽胶束型诊断试剂AGPR共培养的HeLa细胞。如图11所示,在AGPR浓度为25mg/L时,HeLa细胞内的红色荧光出现的较少,随着浓度的提高,HeLa细胞内出现了大量红色荧光。这是由于AGPR胶束被HeLa细胞内吞后,HeLa细胞内的组织蛋白酶B将胶束瓦解,浓度较低时,瓦解后的荧光片段不易聚集,表现出荧光强度较弱的状态;随着AGPR胶束浓度的增加,瓦解后的荧光片段能更好的发生聚集,同时彰显出 AIE的性能,使得荧光更强。表明可以在一定浓度内,通过增加红色AIE荧光探针AGPR的浓度,使其在近红外区成像更明显。进一步证明了AGPR胶束在细胞内具有很好的肿瘤靶向性荧光探针作用。
细胞毒性实验:采用MTT法评估多肽胶束型诊断试剂AGPR分别对正常细胞和肿瘤细胞的毒性大小。将HeLa细胞、COS 7细胞以5000个/孔的密度分别接种到96孔板中,每个孔加入100μL含有10%FBS的DMEM培养基,于37℃下在5%CO2培养箱中培养。48h后,加入含有固定浓度的AGPR的DMEM溶液,每孔加入100μL。将细胞放入培养箱培养24h。然后各个孔加入20μL的 MTT溶液(5mg/mL),继续放入培养箱中培养4h。然后除去孔中的培养基,并在每孔中加入150μL的DMSO。使用酶标仪测量96孔板在570nm处的吸光度(OD),并计算出细胞的存活率。
细胞毒性实验中检测得到的OD值均基于4个独立平行样的平均值,结果表示为平均值±标准偏差(SD)。细胞的相对存活率根据以下公式可计算得到:
Cell Viability(%)=(ODtreated/ODcontrol)×100
其中,ODcontrol为MTT试剂加入96孔板前检测到的吸光度值,ODtreated为MTT试剂加入96孔板后检测到的吸光度值。
图12为实施例五中多肽胶束型诊断试剂AGPR的细胞毒性实验结果图,其中,*:p<0.05,**:p<0.01。如图12所示,在100mg/L的浓度内,COS 7细胞和HeLa细胞的存活率都在90%以上,表明多肽胶束型诊断试剂AGPR在100 mg/L浓度内对细胞的毒性小,有着较低的生物毒性,具有在低毒性状态下,追踪癌细胞的潜能。
以上细胞实验足以证明多肽胶束型诊断试剂AGPR表面修饰的RGD肽对肿瘤细胞的靶向性和近红外区AIE成像性能。
Claims (6)
2.权利要求1所述的聚集诱导发光多肽胶束型诊断试剂AGPR在制备肿瘤细胞示踪药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述肿瘤细胞为整合素蛋白过表达的肿瘤细胞。
4.根据权利要求3所述的应用,其特征在于,所述肿瘤细胞为人宫颈癌细胞、人神经胶质瘤细胞、乳腺癌细胞或人肝癌细胞。
5.根据权利要求1所述的聚集诱导发光多肽胶束型诊断试剂AGPR,其特征在于,所述两亲性小分子功能肽ACBT-GGFLG-PEGn-R8GD,n=8。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN110312708A (zh) * | 2016-12-15 | 2019-10-08 | 香港科技大学 | 用于生物应用的发光材料 |
CN111471043A (zh) * | 2020-04-21 | 2020-07-31 | 浙江工业大学 | 一种含有苯并[c][1,2,5]噻二唑衍生物受体结构单元的有机发光材料及应用 |
CN112824403A (zh) * | 2019-11-21 | 2021-05-21 | 香港科技大学深圳研究院 | 一种靶向脂滴的AIEgen、制备方法及应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107849441A (zh) * | 2015-06-24 | 2018-03-27 | 香港科技大学 | 用于癌症的可视化和治疗的aie发光体 |
CN110312708A (zh) * | 2016-12-15 | 2019-10-08 | 香港科技大学 | 用于生物应用的发光材料 |
CN109602703A (zh) * | 2019-01-15 | 2019-04-12 | 天津大学 | 一种复合双亲肽纳米胶束及其制备方法和应用 |
CN112824403A (zh) * | 2019-11-21 | 2021-05-21 | 香港科技大学深圳研究院 | 一种靶向脂滴的AIEgen、制备方法及应用 |
CN111471043A (zh) * | 2020-04-21 | 2020-07-31 | 浙江工业大学 | 一种含有苯并[c][1,2,5]噻二唑衍生物受体结构单元的有机发光材料及应用 |
Non-Patent Citations (1)
Title |
---|
构建酶敏感型嵌合肽用于肿瘤光动力学治疗;田翔等;《功能材料》;20200427;第4卷(第51期);4084-4095 * |
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