CN114149482B - 一种螯合金属离子的智能转换双重刺激响应型探针及其制备方法和应用 - Google Patents
一种螯合金属离子的智能转换双重刺激响应型探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种螯合金属离子的智能转换双重刺激响应型探针及其制备方法与应用,该探针能够在水缓冲液中自组装成大的纳米颗粒,在LAP和GSH的作用下转变成小的纳米颗粒,从而增强肿瘤积聚和深部组织穿透,从而改善体内肿瘤的近红外成像。此外,首次发现这种LAP/GSH驱动的拆卸和尺寸缩小方法可以显著激活治疗药物的光动力效应,实现有效的肝肿瘤成像引导PDT,同时减少对正常组织的副作用。螯合探针Ce6‑Leu@Mn2+可改善氧供应以克服缺氧,并增强X射线辐射下ROS的生成,从而在活体小鼠中对人类肝脏HepG2肿瘤进行有效的MRI成像引导放疗。因此,本发明的尺寸可转换纳米系统可能提供一种强大的技术来提高药物输送效率,从而增强肿瘤诊断和治疗。
Description
技术领域
本发明属于肿瘤微环境介导的重组装技术领域,涉及一种螯合金属离子的智能转换双重刺激响应型探针及其制备方法和应用。
背景技术
随着纳米生物技术和纳米医学的快速发展,基于动态纳米组装的药物递送系统引起了极大的研究兴趣,并被认为是提高局部药物浓度以实现有效癌症诊断和治疗的一种有希望的手段。这种纳米系统有望提高抗肿瘤药物的特异性、积累和保留时间。刺激诱导的自组装方法可使分子在感兴趣的疾病部位局部组装,已被证明是实现成像信号放大、增强治疗效果和改善生物安全性的有效方法。例如,Xu及其同事开发的酶诱导自组装超分子水凝胶已证明能够改善小分子肽的积累和保留,以增强癌症成像和治疗。另外,Rao和Liang小组创新性地提出了基于双正交CBT-Cys的酶/GSH介导的自组装方法的概念。但是现在大多数这类造影剂仍处于临床前研究阶段,缺乏生物毒性、药代动力学和体内分布的实验评估,距离临床应用仍有一定的距离。
发明内容
为了克服上述现有造影剂中存在的问题,本发明合理地设计和合成了一种新型智能双刺激响应治疗探针,该探针保持较大的初始尺寸以延长血液循环,然后在肿瘤过度表达亮氨酸氨基肽酶(LAP)和还原性谷胱甘肽(GSH)的情况下变小尺寸在肿瘤部位进行增强肿瘤成像和治疗。探针最初可以自组装成大的纳米颗粒(~80纳米)。一旦纳米颗粒通过增强通透性和保留(EPR)效应到达肿瘤微环境(TME),亮氨酸基序和二硫键分别被LAP和GSH自发切割,通过分子间CBT-Cys缩合反应生成环状二聚体,它可以触发最初的大纳米粒子原位转化为微小的纳米粒子(~23纳米)。这种LAP/GSH驱动的原位形态/大小转换方法具有以下优点:(1)实现形态转换以促进探针穿透肿瘤组织;(2)放大荧光和磁共振信号,并提高1O2生成,以增强NIR/MRI成像引导PDT;(3)提高Ce6-Leu@Mn2+的O2产量,提高放射治疗(RT)的疗效。因此,本发明LAP/GSH响应性纳米系统克服了传统纳米医学面临的尺寸困境,为精确的癌症诊断和治疗提供了一种强大而令人惊讶的工具。
本发明采用以下技术方案:
一种螯合金属离子的智能转换双重刺激响应型探针,具有如下化学结构式:
上述螯合金属离子的智能转换双重刺激响应型探针在制备肿瘤诊断和/或治疗试剂中的应用。
上述螯合金属离子的智能转换双重刺激响应型探针的制备方法,包括以下步骤:
(1)化合物1与NH2-CBT进行酰胺缩合反应得到化合物2;
(2)化合物2脱掉保护基得到化合物3;
(3)化合物3与N-芴甲氧羰基-S-叔丁硫基-L-半胱氨酸进行酰胺缩合反应,得到化合物4;
(4)化合物4脱去保护基团得到化合物5;
(5)化合物5与光敏剂反应,得到化合物6;
(6)化合物6脱掉保护基得到化合物7;
(7)化合物7与N-叔丁氧羰基-L-亮氨酸进行酰胺缩合反应得到化合物8;
(8)化合物8脱掉保护基得到Ce6-Leu;
(9)将Ce6-Leu、无机锰盐在溶剂中混合,加入有机添加剂,搅拌得到螯合金属离子的智能转换双重刺激响应型探针。
Ce6-Leu具有如下化学结构式:
上述技术方案中,步骤(1)中,化合物1与NH2-CBT的摩尔比为1∶1.2;酰胺缩合反应在N-甲基吗啡和氯甲酸异丁酯存在下进行;酰胺缩合反应为室温反应15~24小时。
上述技术方案中,步骤(2)中,化合物2脱去保护基团在N,N-二甲基甲酰胺/哌啶混合溶剂中进行;N,N-二甲基甲酰胺、哌啶的体积比为4∶1。
上述技术方案中,步骤(3)中,化合物3与N-芴甲氧羰基-S-叔丁硫基-L-半胱氨酸的摩尔比为1:1.2;酰胺缩合反应在1-羟基苯并三氮唑、O-苯并三氮唑-四甲基脲六氟磷酸盐和二异丙基乙胺存在下进行;酰胺缩合反应为室温反应2~4小时。
上述技术方案中,步骤(4)中,化合物4脱去保护基团在二氯甲烷/三氟乙酸混合溶剂中进行;二氯甲烷、三氟乙酸的体积比为4∶1。
上述技术方案中,步骤(5)中,化合物5与光敏剂的摩尔比为1.1∶1;所述光敏剂为NHS活化的二氢卟吩E6(Ce6-NHS)。
上述技术方案中,步骤(6)中,化合物6脱去保护基团在N,N-二甲基甲酰胺/哌啶混合溶剂中进行;N,N-二甲基甲酰胺、哌啶的体积比为4∶1。
上述技术方案中,步骤(7)中,化合物7与N-叔丁氧羰基-L-亮氨酸的摩尔比为1∶1.2;酰胺缩合反应在1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和二异丙基乙胺存在下进行;酰胺缩合反应为室温反应8~12小时
上述技术方案中,步骤(8)中,化合物8脱去保护基团在二氯甲烷/三氟乙酸混合溶剂中进行;二氯甲烷、三氟乙酸的体积比为4∶1。
上述技术方案中,步骤(9)中,无机锰盐为氯化锰,溶剂为甲醇,有机添加剂为吡啶;优选的,搅拌为35~40℃搅拌3~5小时。优选的,无机锰盐的摩尔量为Ce6-Leu摩尔量的4~6倍。
NHS活化的光敏剂二氢卟吩E6的化学结构式如下:
本发明中,步骤(9)搅拌结束后,使用高效液相色谱(HPLC)分离提纯,得到螯合金属离子的智能转换双重刺激响应型探针,为常规技术。优选的,所述的高效液相色谱分离方法为:C18柱,3.5μm,4.6×100 mm;流动相:A是水;B是乙腈;流速:3 mL/min;线性梯度洗脱程序:0 min,A∶B = 95∶5;13 min,A∶B = 0∶100。
本发明的探针通过肿瘤微环境中过表达的亮氨酸氨基肽酶和谷胱甘肽的双重刺激,使得纳米颗粒探针重新组装成纳米纤维,实现探针的荧光和产生ROS能力的恢复,从而达到肿瘤的特异性荧光成像和光动力治疗。与光学成像相比,磁共振成像(MRI)是一种无创成像方式,具有高空间分辨率和良好的穿透深度,是一种极具吸引力的临床诊断和肿瘤监测诊断技术,本发明Ce6-Leu螯合锰(II)离子(Mn2+),可作为MRI成像剂。
由于上述技术方案的运用,本发明与现有技术相比具有如下优点:
本发明中使用2-氰基苯并噻唑与1,2-氨基硫醇发生快速高效的点击缩合反应形成两亲性的二聚体,并通过分子间作用力的改变使得纳米颗粒重新组装成纳米纤维。当探针进入肿瘤细胞后,在肿瘤细胞内过表达的亮氨酸氨基肽酶和谷胱甘肽的刺激下,暴露半胱氨酸结构中原有的氨基和巯基,从而与2-氰基苯并噻唑(CBT)的氰基发生点击缩合反应,且不受外界环境影响;Mn2+螯合探针(Ce6-Leu@Mn2+)被证明具有催化内源性H2O2在缺氧肿瘤部位持续产生O2的能力,从而改善氧气供应以增强放射治疗效果。
本发明肿瘤微环境响应的智能型探针的诊断和治疗功能只有在特殊的肿瘤微环境触发下才能被激活,即使被正常组织截留,其诊断和治疗功能不会被激活,因此不会对癌症的诊断和治疗带来干扰。所以,肿瘤微环境响应的智能诊疗试剂能有效的提高癌症诊断的精准度和治疗的效果。
附图说明
图1为Ce6-Leu@Mn2+、Ce6-Ac@Mn2+的MALDI-TOF/MS。
图2为Ce6-Ac和Mn2+螯合Ce6-Ac的紫外-可见吸收光谱。
图3为Ce6-Leu@Mn2+探针的合成及MRI表征,(a)Ce6-Leu和Mn2+螯合Ce6-Leu(Ce6-Leu@Mn2+)的紫外-可见吸收光谱,(b)TEM图像和(c)Ce6-Leu@Mn2+和用LAP和GSH处理的Ce6-Leu@Mn2+粒度分布,(d)Ce6-Leu@Mn2+T1加权MR图像与纵向弛豫(r1),(e)Ce6-Leu@Mn2+和Ce6-Ac@Mn2+的T1磁共振信号变化,使用或不使用LAP和GSH处理,(f)Mn2+标记的Ce6-Leu或Ce6-Ac(500μM,200μL)T1加权MR图像与肿瘤聚集的时间依赖性和(g)肿瘤中定量MR信号强度变化(I/I0),I是特定时间点的MR信号,I0是前时间点小鼠的MR信号。Pre表示Mn2+标记探针处理前的小鼠***P<0.001,**P<0.01,*P<0.05。
图4为类过氧化氢酶探针Ce6-Leu@Mn2+的表征,(a)Ce6-Leu@Mn2+的纳米粒示意图,LAP/GSH引发了纳米粒的重新组装,具有更小的尺寸和更好的过氧化氢酶样性能,以缓解肿瘤缺氧和增强放疗疗效。(b) 在不同溶液中从H2O2(1 mM)生成O2。(插图:Ce6-Leu@Mn2+溶液中H2O2用LAP和GSH处理)。(c)40μM Ce6-Leu@Mn2+用LAP和GSH处理,不同浓度的H2O2的O2浓度。(d)H2O2(1mM)下,与不同浓度的Ce6-Leu@Mn2+用LAP和GSH处理的O2浓度。
图5为Ce6-Leu@Mn2+、Ce6-Ac@Mn2+对3T3细胞的毒性。
图6为Ce6-Leu@Mn2+探针体外增强放射治疗效率,(a)HepG2细胞经Hoechst 33342(蓝色,细胞核)和缺氧探针(红色,缺氧细胞)不同处理后的共聚焦荧光图像。标尺:20μm,(b)Ce6-Leu, Ce6-Ac@Mn2+或Ce6-Leu@Mn2+ (30 μM)孵育4h,HepG2细胞中HIF-1α的蛋白表达,(c)通过γ-H2AX评估不同处理对HepG2细胞DNA的损伤。比例尺:20μm(d)每个细胞数量的相应定量分析***P<0.001。(e) X射线照射(0和6Gy)下不同探针处理的HepG2细胞的细胞迁移测定。(f)通过活/死染色评估不同处理的HepG2细胞的放射增敏效应。比例尺:200μm。
图7为细胞缺氧的检测。
图8为细胞迁移定量分析。
图9为体内增强放射治疗研究。(a)静脉注Ce6-Leu, Ce6-Ac@Mn2+或Ce6-Leu@Mn2+(200 μM, 200 μL)后HepG2肿瘤中氧合血红蛋白(HbO2,850 nm)浓度的时间依赖性变化和(b)肿瘤部位HbO2 PA强度的相应定量分析。(c)不同组肿瘤切片的免疫荧光图像,用抗HIF-1α抗体染色。细胞核用DAPI(蓝色)染色。标尺:60μm,(d)接受不同治疗的小鼠的肿瘤生长曲线。(e)不同组14天时解剖肿瘤的照片。(f)放射治疗后48小时处死的不同组小鼠肿瘤组织的隧道染色和H&E染色。标尺:60μm***P<0.001,**P<0.01,*P<0.05。
图10为静脉注射Ce6-Leu、Ce6-Ac@Mn2+或Ce6-Leu@Mn2+后不同时间点肿瘤的活体PA图像和PA信号。
图11为免疫荧光染色用于肿瘤缺氧评估。
图12为不同处理后第0、2、6、10、14天的小鼠照片。
图13为各组小鼠的平均体重和平均体重。
图14为Ce6-Leu、Ce6-Ac的制备示意图。
具体实施方式
本发明开发了一种集核磁成像及光动力治疗于一体的亮氨酸氨基肽酶和谷胱甘肽双响应型智能分子探针,具有重大的研究及应用价值,该造影剂可以在肿瘤微环境的刺激下从球形纳米颗粒重新组装为纳米纤维,并且荧光以及产生ROS的能力恢复,从而实现体肿瘤的特异性荧光成像和光动力治疗,该尺寸可转换纳米系统将提供一种新的先进技术,以提高药物输送效率,实现精确的肿瘤诊断和治疗。
具体而言,本发明提供的方法,其步骤如下:
(1)构建、合成双重刺激响应型探针:
按照设计的合成步骤:首先化合物1与NH2-CBT发生酰胺缩合反应,随后用20%的哌啶(N,N-二甲基甲酰胺∶哌啶 = 4∶1,v/v)脱去保护基团Fmoc;接着与N-芴甲氧羰基-S-叔丁硫基-L-半胱氨酸发生酰胺缩合反应,随后用20%的三氟乙酸(二氯甲烷∶三氟乙酸 = 4∶1,v/v)将中间体化合物的Boc保护基团脱掉;接着与已经用NHS活化好的光敏剂二氢卟吩E6反应,所得的中间体化合物再用20%的哌啶(N,N-二甲基甲酰胺∶哌啶 = 4∶1,v/v)脱去保护基团Fmoc所得的中间体与N-叔丁氧羰基-L-亮氨酸反应得到产物再20%的三氟乙酸(二氯甲烷∶三氟乙酸= 4∶1,v/v)将Boc保护基团脱掉得到最终的探针Ce6-Leu;
(2)螯合金属离子的智能转换双重刺激响应型探针Ce6-Leu@Mn2+
将Ce6-Leu和MnCl2溶解在甲醇中,然后添加吡啶,搅拌,最后经HPLC纯化,得到化合物Ce6-Leu@Mn2+。
活体磁共振成像。HepG2荷瘤小鼠静脉注射Ce6-Leu@Mn2+或Ce6-Ac@Mn2+(500μM),分别加入200μL PBS中。然后用3%异氟烷混合氧气(0.5 L/min)麻醉小鼠,并使用配备小动物成像线圈的3.0 T临床MR扫描仪(MR solutions,UK)成像。
细胞缺氧的检测。HepG2细胞以每孔8×103细胞的密度接种在玻璃底培养皿上过夜。然后,在不同试剂(30μM)培养4h后,将缺氧探针(缺氧红检测试剂)加入HepG2细胞中,在37℃下5%CO2下培养30min。用PBS洗涤三次,去除多余试剂。然后,用Hoechst 33342对细胞核进行15分钟的染色,然后用共焦激光扫描显微镜对其进行表征。(CLSM;λex=596 nm,λem=670 nm)。
细胞内HIF-1α水平的测定。通过westernblot分析测定细胞HIF-1α水平。在不同试剂(30μM)孵育4h后,用冰冷PBS洗涤HepG2细胞3次,并使用含有完全蛋白酶抑制剂的RIPA裂解缓冲液裂解。通过SDS-PAGE分离每个样品中的60μg蛋白质,并转移到PVDF膜上。在用5%脱脂乳封闭2小时后,PVDF膜在4℃下与HIF-1α抗体孵育过夜,然后在室温下与相应的二级抗体结合辣根过氧化物酶孵育2小时。用ECL plus检测系统观察PVDF膜。
DNA双链断裂和细胞迁移试验。对于DNA双链断裂分析,将HpeG2细胞以5×104的密度接种到35mm培养皿中并培养过夜。将细胞分为RT、Ce6-Leu+RT、Ce6-Ac@Mn2++RT 、Ce6-Leu@Mn2++RT四组,将含有不同试剂的培养基(浓度为30μM)添加到HpeG2细胞中。培养4小时后,用PBS洗涤三次,去除多余的试剂。然后用6Gy剂量的X射线处理HpeG2细胞。处理后,用4%多聚甲醛固定细胞0.5小时,用1%Triton X-100渗透细胞10分钟,使细胞膜破裂,然后在37℃下用5%BSA封闭细胞1小时。之后,将固定细胞与400μLγ-H2AX抗体在4℃下孵育过夜,然后在洗涤后与二级抗体Cy3标记的山羊抗Rabbit IgG(H+L)在37℃下孵育1h。最后,用Hoechst 33342对细胞核进行染色,然后使用Olympus共焦显微镜(日本东京奥林巴斯)进行检查,以分析红色磷酸化H2AX信号。
对于细胞迁移试验,将HpeG2细胞以每孔4×105个细胞接种在6孔板中过夜。在达到约95%的汇合后,将含有不同试剂的培养基(浓度为30μM)添加到HpeG2细胞中。培养4小时后,用PBS洗涤三次,去除多余的试剂。然后用0或6Gy剂量的X射线处理HpeG2细胞,并使用1mL无菌移液管尖端刮取细胞层以形成间隙。通过孵育前和孵育后120小时的细胞照片手动量化细胞迁移。
肿瘤的活体PA成像。使用实时多光谱光声断层成像系统(MOST,德国伊瑟拉医疗公司)进行活体PA成像。荷HepG2皮下肿瘤的小鼠静脉注射Ce6-Leu、Ce6-Leu@Mn2+或者Ce6-Ac@Mn2+ (200 μM, 200 μL)。然后用异氟醚麻醉小鼠,并将其置于水浴中,以将其体温保持在34℃,以便通过MSOT进行PA成像。图像重建后,用MSOT软件将肿瘤部位HbO2的PA信号与PA图像分离。
免疫荧光染色用于肿瘤缺氧评估。荷HpeG2肿瘤的小鼠随机分为4组。静脉注射PBS(200μL)、Ce6-Leu(200μL、200μM)、Ce6-Ac@Mn2+(200μL,200μM)或Ce6-Leu@Mn2+(200μL,200μM),从小鼠上解剖肿瘤并切片以进行HIF-1α免疫荧光染色。用激光共聚焦显微镜观察切片。
肿瘤的HE和TUNEL染色接受。HepG2荷瘤小鼠(n=3)静脉注射PBS(200μL)、Ce6-Leu(200μL、200μM)、Ce6-Ac@Mn2+(200μL,200μM)或Ce6-Leu@Mn2+(200μL,200μM)。注射后6h对肿瘤进行8Gy的X线照射。在不同处理后48小时,从小鼠身上解剖肿瘤,并将其固定在中性缓冲福尔马林(10%)中。然后,将肿瘤切成4μm厚的切片,进行苏木精-伊红(H&E)和TUNEL染色。然后用激光共聚焦显微镜观察切片。
下文将结合附图和具体实施例来进一步阐述本发明。应当理解的是,这些实施例仅用于解释和说明本发明中的技术方案,而并非旨在限制本发明的范围。此外,除非另有说明,下列实施例中所使用的材料、试剂、仪器等均可通过商业手段获得。
实施例1:螯合金属离子的智能转换双重刺激响应型探针Ce6-Leu@Mn2+和对照组探针Ce6-Ac@Mn2+的合成与表征
(1)将化合物1(400 mg,0.85 mmol)溶于10 mL四氢呋喃中,再滴加入N-甲基吗啡(130 mg,1.28 mmol),然后将圆底烧瓶置于冰盐浴中,冷却至0oC,随后再滴加入氯甲酸异丁酯(175 mg,1.28 mmol),活化半小时后,再加入用干燥的四氢呋喃溶解的2-氨基-6-氰基苯并噻唑(NH2-CBT,179 mg,1.00 mmol),保持0oC反应1小时,然后室温搅拌过夜。反应结束后,通过旋转蒸发仪旋干溶剂,然后将残留固体复溶在乙酸乙酯(50 mL)中,并用碳酸氢钠的水溶液萃取三次,有机相用Na2SO4干燥后进行抽滤,旋干溶剂。以石油醚(PE)和乙酸乙酯(EA)= 2:1的体积比为洗脱液,用硅胶色谱柱纯化粗产物,得到中间体1;
(2)将中间体1(500 mg,0.80 mmol)溶于8 mL DMF中,然后将反应瓶放置在冰水浴中,随后滴加入2 mL哌啶,保持0oC反应5分钟,反应结束后,旋蒸除去溶剂和哌啶。以二氯甲烷(DCM)和甲醇(MeOH)= 80:1的体积比为洗脱液,用硅胶色谱柱纯化粗产物,得到中间体2;
(3)在20 mL的圆底烧瓶中加入中间体2(250 mg,0.62 mmol),然后用干燥的DMF溶清,随后再加入HBTU(282.15 mg,0.74 mmol),HOBT(100.44 mg,0.74 mmol)和DIPEA(213.68μL),搅拌15分钟后再加入化合物N-芴甲氧羰基-S-叔丁硫基-L-半胱氨酸(321.04mg,0.74 mmol)。继续搅拌,室温反应2小时,反应结束后通过旋蒸除去溶剂,然后再加入25mL乙酸乙酯复溶粗产物,随后有机相用25 mL的超纯水,饱和碳酸氢钠,氯化钠水溶液各洗一次。有机相用无水硫酸钠干燥后旋蒸除去溶剂,以石油醚(PE)和乙酸乙酯(EA)= 2:1的体积比为洗脱液,用硅胶色谱柱纯化粗产物,得到中间体3;
(4)在20 mL含20%(体积比)三氟乙酸的DMF溶液中加入中间体3,室温反应1小时后,通过旋蒸除去溶剂和三氟乙酸,得到中间体4(其结构如图1中的化合物5所示)。中间体4不做进一步纯化。准确称取40毫克中间体4(0.0558 mmol),加入20 mL的无水DMF溶液溶清,再加入45.71毫克Ce6-NHS(0.05 mmol)和7.76毫克DIPEA(0.06 mmol),室温搅拌2小时后,用HPLC进行分离提纯,收集吸收光谱在400 nm处的组分得到中间体5;
(5)将中间体5(45 mg,0.035 mmol)溶于8 mL DMF中,然后将反应瓶放置在冰水浴中,随后逐滴加入2 mL哌啶,保持0oC反应5分钟,反应结束后,用HPLC进行分离提纯,收集吸收光谱在400 nm处的组分得到中间体6;
(6)在10 mL圆底烧瓶中加入中间体6(26 mg,0.025 mmol),用5 mL无水DMF溶清,然后再加入NHS活化的N-叔丁氧羰基-L-亮氨酸(9.85 mg,0.03 mmol),室温反应2小时后旋蒸除去溶剂,使用半制备型高效液相色谱分离提纯得到中间体7;
(7)在5 mL含20%三氟乙酸的二氯甲烷溶液中加入中间体7(19 mg,0.015 mmol),室温反应1小时后,通过旋蒸除去溶剂和三氟乙酸,使用半制备型高效液相色谱分离提纯得到Ce6-Leu;
(8)在10 mL圆底烧瓶中加入中间体6(26 mg,0.025 mmol),用5 mL无水DMF溶清,然后再加入乙酸酐(3.06 mg,0.03 mmol),室温搅拌2小时后旋蒸除去溶剂,用HPLC进行纯化分离得到Ce6-Ac;
以上步骤(1)至步骤(8)与已经提交的申请CN2021113897458(一种亮氨酸氨基肽酶和谷胱甘肽双重刺激响应型探针及其制备方法和应用)一致,化学结构以及表征可参见该申请,本发明对反应示意过程记载于图14。
(9)将0.0084 mmol Ce6-Leu和MnCl2(5.28 mg,0.042 mmol)溶解在1 mL甲醇中,然后添加100μL吡啶,在37℃下搅拌4小时,最后经HPLC纯化,得到化合物Ce6-Leu@Mn2+(8.85mg,85%)。MS (MALDI-TOF) Calcd for: C61H73MnN11O8S3([M+Na]+): 1262.440, found:1262.793;
将Ce6-Leu替换为Ce6-Ac,得到Ce6-Ac@Mn2+ (7.84 mg 80%). MS (MALDI-TOF)Calcd for: C57H64MnN10O8S3([M]+): 1167.350, found: 1167.833。
图1为上述Ce6-Leu@Mn2+、Ce6-Ac@Mn2+的结构式与质谱图。
实施例二 Ce6-Leu@Mn2+性能研究
图2为Ce6-Ac螯合锰离子前后的吸收光谱,图3为探针以及对比探针的相关测试,结果表明,Ce6-Leu和Ce6-Ac在410nm处都有一个高强度的初始峰,在与Mn2+螯合后,该峰的强度被显著抑制,同时出现轻微的蓝移(图2、图3a),这表明Mn2+已成功地并入Ce6-Leu或Ce6-Ac中,得到Ce6-Leu@Mn2+、Ce6-Ac@Mn2+。通过透射电镜(TEM)研究了Ce6-Leu@Mn2+的形态,在PBS缓冲液中能自组装成平均粒径为59.44±7.83nm的大颗粒,LAP和GSH进行处理后,将其分解成尺寸为24.13±2.73 nm的小纳米粒子(图3b和3c)。Ce6-Leu@Mn2+的T1磁共振信号以浓度依赖的方式逐渐增加,测量并计算其T1弛豫度(r1)为4.23 mM-1 S-1(图3d),Ce6-Ac@Mn2+的T1磁共振信号也以浓度依赖的方式逐渐增加,测量并计算其T1弛豫度(r1)为3.50mM-1S-1。此外,一旦Ce6-Leu@Mn2+溶液经LAP和GSH处理后,T1 MR信号比未经LAP和GSH处理的溶液高1.62倍(图3e)。然而,对照探针Ce6-Ac@Mn2+经或未经LAP和GSH处理,几乎没有观察到T1 MR信号变化;因此,Ce6-Leu@Mn2+是一种有希望的T1加权造影剂,用于肿瘤的活体MRI成像。
进一步评估了Ce6-Leu@Mn2+探针在体内的MRI性能。图3f显示了荷皮下HepG2肿瘤异种移植物的小鼠(n=3)在静脉注射探针Ce6-Leu@Mn2+、Ce6-Ac@Mn2+(200µL,500µM)后在选定时间点获得的的一系列代表性MR图像,注射Ce6-Leu@Mn2+后小鼠肿瘤的T1 MR信号强度随着时间的推移逐渐增强,在注射后4小时达到平台,明显高于Ce6-Ac@Mn2+(图5g)。此外,高分辨率图像显示,Ce6-Leu@Mn2+小鼠的增强MR信号几乎分布在整个肿瘤组织中,表明LAP/GSH驱动了Ce6-Leu@Mn2+的尺寸减小能显著提高肿瘤的穿透能力。总的来说,这些证据有力地证明Ce6-Leu@Mn2+在体内精确显示肿瘤方面有很大的潜力。
Mn2+螯合Ce6衍生物将H2O2转化为O2的催化能力与纳米颗粒的大小密切相关,严重的自聚集会削弱纳米颗粒的催化作用。接下来,评估探针Ce6-Leu@Mn2+表现出类似过氧化氢酶的活性,可将H2O2转化为O2,即Ce6-Leu@Mn2+溶液(40µM)在存在或不存在LAP和GSH的情况下用H2O2(1 mM)处理,然后使用溶解氧计测量O2生成量(图4a)。如图4b所示,Ce6-Leu@Mn2+经LAP和GSH处理(表示为Ce6-Leu@Mn2++LAP+GSH+H2O2)与对照Ce6-Ac@Mn2+相比,显示出显著的O2产生,表明Ce6-Leu@Mn2+对LAP和GSH的反应可以产生大量的O2,其产生量与使用的H2O2量呈正相关(图4c)。类似地,在H2O2(1mM)量不变的情况下,O2的产生随着探针Ce6-Leu@Mn2+浓度的增加而逐渐增强(图4d)。总之,这些结果高度证明探针Ce6-Leu@Mn2+在LAP和GSH的作用下提高了产生O2的能力,并用于克服肿瘤缺氧,以增强癌症的放射治疗。
进一步评估了Ce6-Leu@Mn2+用于在HepG2细胞中将H2O2转化为O2的能力。首先,MTT法检测Ce6-Leu@Mn2+和Ce6-Ac@Mn2+对3T3细胞的增殖情况,图5所示结果表明,两种探针在8至128μM浓度范围内对3T3细胞的细胞毒性可忽略不计。使用缺氧/氧化应激检测试剂盒检测细胞内缺氧,并检测缺氧探针的荧光信号(缺氧红检测试剂)用CLSM法检测不同处理的细胞中。如图6a所示,分别使用PBS和Ce6-Leu处理的两组细胞均检测到强烈的红色荧光,表明细胞内环境高度缺氧。相反,接受Ce6-Ac@Mn2+的HepG2细胞显示出相对较低的荧光,而含有Ce6-Leu@Mn2+的细胞呈现最微弱的荧光,甚至持续8小时(图7),表明O2的产生显著缓解了细胞缺氧。此外,通过westernblot(WB)分析也确定了HIF-1α的细胞内表达水平,该表达水平在常氧条件下可被某些酶降解,但在低氧条件下保持高表达。图6b清楚地表明,经Ce6-Leu@Mn2+处理后,HepG2细胞中HIF-1α的表达受到显著抑制,再次证明Ce6-Leu@Mn2+能在癌细胞内产生氧气,有效缓解细胞内缺氧。通过γ-H2AX免疫荧光染色分析细胞的DNA双链断裂。图6c和6d中给出的结果表明,在Ce6-Leu@Mn2+和X射线(6 Gy)处理下(表示为Ce6-Leu@Mn2++RT),HepG2细胞中检测到明显的DNA损伤和X射线辐射,而在Ce6-Ac@Mn2+和X射线照射(6 Gy)(表示为Ce6-Ac@Mn2++RT)细胞中仅观察到γ-H2AX的轻微增加,在X射线辐射(表示为RT)和PBS或Ce6-Leu(表示为Ce6-Leu+RT)处理的细胞中几乎没有检测到γ-H2AX。接下来,研究了不同处理的细胞的转移和细胞迁移能力。如图6e所示,如果不进行X射线照射,所有四组HepG2细胞在120小时内均显示出良好的增殖和迁移能力;然而,在X射线(6 Gy)照射后,含有Ce6-Leu@Mn2+的细胞仍保留约20%的细胞迁移能力,显著低于其他对照组(图8)。此外,在Ce6-Leu@Mn2+存在的情况下,X射线辐射的放射增敏效应通过活/死分析在活细胞中进行评估。如图6f所示,在Ce6-Leu@Mn2++RT细胞中明显检测到大量死亡细胞(红色荧光),而其他四个对照组的细胞仅观察到少量死亡细胞。总的来说,这些结果有力地证明了探针Ce6-Leu@Mn2+在X射线辐射下,对癌细胞具有良好的细胞毒性。
众所周知,实体瘤通常对放疗不敏感。本发明探针的放射增敏效应提高了荷HepG2肿瘤的BALB/c小鼠的放疗效果。利用在生命系统中产生氧气的方法研究了Ce6-Leu@Mn2+探针的性能。光声成像(PA)作为光学和超声技术的联合体,是一种具有深层组织穿透性和高空间分辨率的优秀成像方式,利用PA成像分别检测氧合血红蛋白(HbO2)和探针在850 nm和680 nm处的PA信号,以监测肿瘤内的血氧饱和度变化。图9a和9b显示,注射Ce6-Leu或Ce6-Ac@Mn2+(200µL,200µM)的小鼠记录到肿瘤部位HbO2(红色,850 nm)的微弱PA信号,注射Ce6-Leu@Mn2+(200µL,200µM)的小鼠的PA信号随时间显著增加,并在6小时达到最大值(是Ce6-Leu的2.52倍)。此外,在680 nm处观察到探针PA信号增强的类似趋势(图10),表明探针可以有效地积聚在肿瘤区域。然后,解剖了不同治疗(PBS, Ce6-Leu, Ce6-Ac@Mn2+, Ce6-Leu@Mn2+)的小鼠肿瘤,切片进行抗HIF-1α抗体染色。发现Ce6-Leu@Mn2+小鼠肿瘤组织中HIF-1α水平升高与其他对照组相比显著降低(图9c和图11)。
进一步研究Ce6-Leu@Mn2+探针在体内的增强放疗性能,荷HepG2皮下肿瘤的BALB/c裸鼠被随机分为五组(n=5),分别接受不同的治疗:PBS、X射线照射(RT)、Ce6-Leu+X射线照射(Ce6-Leu+RT)、Ce6-Leu-Ac@Mn2++射线(Ce6-Ac@Mn2++RT)和Ce6-Leu@Mn2++X射线(Ce6-Leu@Mn2++RT)。通过尾静脉将探针注射到荷瘤小鼠体内6小时,随后对肿瘤进行X射线(8 Gy)照射。在14天内实时监测不同组小鼠的平均肿瘤大小。如图9d和12所示,RT和Ce6-Leu+RT组的肿瘤生长趋势相似,与PBS组相比,肿瘤大小分别减少了22.8%和18.6%。Ce6-Ac@Mn2++RT对小鼠肿瘤生长的影响较实验组的抑瘤率有下降,仅达到约42.9%的抑瘤率,与此形成鲜明对比的是,Ce6-Leu@Mn2++RT中肿瘤大小可以显著减小,肿瘤生长抑制率约为82%,与其他四个对照组比较(图9e和图13a)。同时,所有这些联合治疗对小鼠体重没有显著影响(图13b),表明探针Ce6-Leu@Mn2+在体内具有良好的生物相容性。为了进一步验证放射治疗效果,在X射线照射后48小时提取肿瘤组织,然后进行h&E和TUNEL染色。如图9f所示,在Ce6-Leu@Mn2++RT(V组)检测到严重的核固缩、凋亡和坏死,其他对照组未见明显坏死。总之,所有这些证据都高度证明,本发明LAP/GSH驱动的可转换治疗探针能够有效改善体内肿瘤的缺氧和放射治疗效果。
为了克服传统诊疗分子探针的缺点,构建一种肿瘤微环境响应型近红外分子探针,利用肿瘤细胞中过表达的亮氨酸氨基肽酶和谷胱甘肽触发缩合反应,进而进行重组装,使得探针在肿瘤部位荧光和产生ROS的能力的特异性恢复,进而有效的改善肿瘤的成像和治疗效果。它具有以下几个优点:首先,该缩合反应高效、温和、速度快、选择性高;第二,当探针进入肿瘤细胞后,在肿瘤细胞中过表达的亮氨酸氨基肽酶和谷胱甘肽的刺激下,暴露半胱氨酸结构中原有的氨基和巯基,从而发生点击缩合反应,不受外界环境影响。开发智能和形态可转换的纳米材料,可以时空地进行刺激响应性尺寸转换,这对于改善肿瘤渗透和有效的体内药物递送具有巨大的前景。Mn2+螯合探针(Ce6-Leu@Mn2+)被证明具有催化内源性H2O2在缺氧肿瘤部位持续产生O2的能力,从而改善氧气供应以增强放射治疗效果。因此,本发明公开的LAP/GSH驱动的尺寸可转换纳米系统将提供一种新的先进技术,以提高药物输送效率,实现精确的肿瘤诊断和治疗。
Claims (4)
1.一种螯合金属离子的智能转换双重刺激响应型探针,其特征在于,所述螯合金属离子的智能转换双重刺激响应型探针具有如下化学结构式:
。
2.权利要求1所述螯合金属离子的智能转换双重刺激响应型探针在制备肿瘤诊断和/或治疗试剂中的应用,其特征在于,所述肿瘤为肝癌。
3.根据权利要求2所述的应用,其特征在于,所述治疗为放射治疗。
4.权利要求1所述螯合金属离子的智能转换双重刺激响应型探针在制备肿瘤放疗增效试剂中的应用,其特征在于,所述肿瘤为肝癌。
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