CN117105848A - 荧光分子、nir ii纳米荧光探针及其制备方法和应用 - Google Patents
荧光分子、nir ii纳米荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及荧光分子、NIR II纳米荧光探针及其制备方法和应用。所述的荧光分子是以花菁类FD 1080为母核,经脂肪侧链修饰制得稳定发光的FD1080‑(CH2)n(6a‑f),具备NIR II荧光成像和较好的生物透膜性能。本发明将靶向整合素αvβ3的多肽cRGD与高分子材料Mal‑PEG‑PLGA(‑PCL、‑PLA、‑聚碳酸酯)偶联,与所述荧光分子制得兼具良好NIR II荧光活体成像和主动靶向肿瘤转移无创识别功能的纳米胶束FD1080‑(CH2)n NPs cRGD,构建了一款成像质量高和生物安全性好并可实现荧光影像指导下的手术导航切除肿瘤转移灶肿瘤的荧光探针,有助于肿瘤诊疗一体化的临床应用的商业价值。
Description
技术领域
本发明属于生物技术领域,尤其是涉及荧光分子、NIR II纳米荧光探针及其制备方法和应用。
背景技术
荧光成像(Fluorescence imaging,FI)作为一种非侵入性的成像手段,具备成本低、分辨率和灵敏度高及安全性好等优点,被广泛用于生物医学成像。与可见光(390-780nm)相比,近红外光(780-1700nm)生物成像具有穿透深度高、可避免生物自体荧光干扰等优势,因此在医学生物成像领域被广泛应用,例如近红外光(NIR I)的吲哚菁绿(ICG)是目前唯一被FDA批准上市用于临床的显影剂(荧光探针)。但由于它在结直肠癌的临床切除手术中,仍存在荧光穿透深度低,缺乏肿瘤组织的靶向性,尤其是导航切除肿瘤组织的边界不清晰,无法精准定位等缺点,使得患者的肿瘤组织难以根除,极大地增加了肿瘤的复发率和转移率。因此,研发组织穿透率高且具靶向和安全性良好的荧光探针是突破手术导航临床应用瓶颈的关键技术难题。
目前的近红外荧光探针分为NIR I(780-950nm)荧光探针和NIR II(1000-1700nm)荧光探针。与NIR I荧光探针成像相比,NIR II荧光探针成像质量更高,原因在于NIR II探针组织穿透深度深,光散射效应低,可以达到更高的信噪比从而获得高分辨率的荧光成像。根据材料可将目前的NIR II荧光探针大致分为三类:基于有机小分子的NIR II探针、基于有机共轭聚合物的NIR II探针和无机纳米探针(Chem Soc Rev:Optical nano-agents inthe second near-infrared window for biomedical applications.2019,48(1):22-37.)。其中,无机纳米荧光探针存在潜在生物毒性不易代谢等缺点,基于有机共轭聚合物的NIR II荧光探针因分子结构不确定可能存在潜在生物毒性,抗光漂白能力弱等缺点(Chinese Chem Lett:Recent advances on small-molecule fluorophores withemission beyond 1000nm for better molecular imaging in vivo.2019,30:1731-1737.),都限制了它们进一步的生物应用。
发明内容
本发明的目的是补充现有技术的不足,提供荧光分子、NIR II纳米荧光探针及其制备方法和应用。
本发明的目的可以通过以下技术方案来实现:
本发明首先提供一种荧光分子FD1080-(CH2)n,其结构如通式I所示:
其中n是偶数,选自6、8、10、12、14或16等。
本发明所提供的荧光分子FD1080-(CH2)n,兼具NIR II成像能力和高透膜性。
本发明进一步提供所述荧光分子FD1080-(CH2)n的制备方法,包括以下步骤:
按合成路线1制备:
以1,8-环内酰亚胺为原料,经取代、格氏反应和羟醛缩合反应得到目标产物6a-f,目标产物6a-f即通式I所示结构:
合成路线1
其中,n是偶数,选自6、8、10、12、14或16等。
在本发明的一个实施方式中,所述荧光分子FD1080-(CH2)n的制备方法中采用试剂如下:
(i)CH3-CH2-(CH2)n-Cl(其中n是6、8、10、12、14、16等偶数),二甲基甲酰胺(DMF),氢化钠(NaH),室温;
(ii)甲基氯化镁(CH3MgCl),N2,60℃;
(iii)二甲基甲酰胺(DMF),二氯甲烷(DCM),200℃;
(iv)乙酸酐,乙醇,N2,70℃。
本发明进一步提供一种可靶向肿瘤的高分子材料,其为通过硫醇化法将cRGD多肽与高分子材料聚乙二醇类嵌段共聚物连接得到的可靶向肿瘤的高分子材料。
在本发明的一个实施方式中,所述聚乙二醇类嵌段共聚物选自聚乙二醇-聚乳酸羟基乙酸嵌段共聚物(PEG-PLGA)、聚乙二醇-聚己内酯嵌段共聚物(PEG-PCL)、聚乙二醇-聚乳酸嵌段共聚物(PEG-PLA)或聚乙二醇-聚碳酸酯嵌段共聚物的一种或者几种。当所述聚乙二醇类嵌段共聚物分别选择以上材料时,所述可靶向肿瘤的高分子材料分别表示为cRGD-PEG-PLGA、cRGD-PEG-PCL、cRGD-PEG-PLA、cRGD-PEG-聚碳酸酯。
在本发明的一个实施方式中,所述聚乙二醇-聚乳酸羟基乙酸嵌段共聚物(PEG-PLGA)选自聚乳酸和羟基乙酸共聚物-聚乙二醇2000、聚乳酸和羟基乙酸共聚物-聚乙二醇5000、聚乳酸和羟基乙酸共聚物-聚乙二醇-氨基、聚乳酸和羟基乙酸共聚物-聚乙二醇-马来酰亚胺、聚乳酸和羟基乙酸共聚物-聚乙二醇-巯基中的一种或几种的混合物。
本发明进一步提供所述可靶向肿瘤的高分子材料的制备方法,以cRGD多肽和Mal-PEG-PLGA、Mal-PEG-PCL、Mal-PEG-PLA或Mal-PEG-聚碳酸酯为原料,通过迈克尔加成反应,得到目标产物cRGD-PEG-PLGA、cRGD-PEG-PCL、cRGD-PEG-PLA、cRGD-PEG-聚碳酸酯。
在本发明的一个实施方式中,给出了具体可靶向肿瘤的高分子材料cRGD-PEG-PLGA的制备方法,按合成路线2制备:
合成路线2
在本发明的一个实施方式中,可靶向肿瘤的高分子材料cRGD-PEG-PLGA的制备方法中采用试剂如下:
(i)二甲基甲酰胺(DMF),三乙胺(Et3N),室温。
本发明还提供一种NIR II纳米荧光探针,其组成包括所述荧光分子FD1080-(CH2)n、所述可靶向肿瘤的高分子材料、泊洛沙姆或其衍生物。
本发明提供的NIR II纳米荧光探针可靶向肿瘤成像。
在本发明的一个实施方式中,所述荧光分子FD1080-(CH2)n、所述可靶向肿瘤的高分子材料、泊洛沙姆或其衍生物的质量比为1:10-100:10-100。
在本发明的一个实施方式中,所述泊洛沙姆或其衍生物选自泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338、泊洛沙姆407中的一种或几种的混合物。
在本发明的一个实施方式中,所述可靶向肿瘤的高分子材料选自cRGD-PEG-PLGA,具体为聚乳酸和羟基乙酸共聚物-聚乙二醇5000,所述泊洛沙姆或其衍生物选自泊洛沙姆188。
在本发明的一个实施方式中,所述荧光分子FD1080-(CH2)n、可靶向肿瘤的高分子材料cRGD-PEG-PLGA、泊洛沙姆188的质量比为1:25:25。
在本发明的一个实施方式中,所述NIR II纳米荧光探针的胶束的粒径为50-1000nm。
本发明还提供所述NIR II纳米荧光探针的制备方法,所述NIR II纳米荧光探针记为FD1080-(CH2)n NPs cRGD,其步骤包括:
(1)将泊洛沙姆或其衍生物、所述可靶向肿瘤的高分子材料和所述荧光分子FD1080-(CH2)n溶解于有机溶剂中,得到混合溶液;
(2)将步骤(1)所述的混合溶液混合均匀后旋转蒸发除去有机溶剂形成薄膜,置于油泵上将残留有机溶剂全部除去;
(3)向步骤(2)中形成的薄膜加入一定量的缓冲溶液,充分震荡使薄膜水化脱落;
(4)将步骤(3)得到的水溶液通过聚碳酸酯膜得到粒径均一的胶束,即所述NIR II纳米荧光探针。
在本发明的一个实施方式中,步骤(1)中所述有机溶剂选择氯仿、二氯甲烷、四氢呋喃或它们的混合溶剂中的一种或几种的组合。优选的,步骤(1)中的有机溶剂为氯仿。
在本发明的一个实施方式中,步骤(2)中,旋蒸温度在30-60℃之间。
在本发明的一个实施方式中,步骤(3)中,缓冲溶剂pH在5-8之间,体积在20-30mL之间。
本发明还进一步提供所述NIR II纳米荧光探针的应用。
在本发明的一个实施方式中,所述NIR II纳米荧光探针在制备近红外二区成像试剂中的应用。
在本发明的一个实施方式中,所述NIR II纳米荧光探针在制备近红外二区活体荧光成像试剂中的应用。
在本发明的一个实施方式中,所述NIR II纳米荧光探针在制备近红外二区靶向肿瘤荧光成像试剂中的应用。
在本发明的一个实施方式中,所述肿瘤为包括结直肠癌、黑色素瘤、头颈部鳞状细胞癌或乳腺癌等在内的实体瘤。
本发明基于有机小分子NIR II荧光探针具备低毒、易修饰、光学性能好等特性,优选花菁类NIR II荧光分子FD 1080作为先导化合物,为提高其生物透膜性能,在保留其共轭杂环和环己烯结构的前提下,将脂肪烃引入其侧链,合成得到一系列稳定发光的NIR II荧光分子,进而改善其脂溶性,不仅使其NIR II的发光性能更加稳定,而且能够提高探针分子的生物相容性,促进锚定细胞膜的能力,更易穿透细胞膜并扩散进入细胞。
本发明通过筛选和比对靶向和非靶向功能的胶束配方,获得具有主动靶向功能的荧光探针胶束。经文献检索发现(The American Journal of Surgery:Targetedtherapies for the treatment of cancer.2003,186(3):264-268.),整合素αvβ3是一种跨膜异二聚体糖蛋白,由多肽链α和多肽链β以非共价键结合形成,其肿瘤新生血管内皮细胞中高表达,是肿瘤的新生血管生成、侵袭和转移过程中的重要角色。整合素αvβ3可以介导细胞内外的信号转导,当其与其配体相特异性结合,生理功能会受到严重影响。根据文献(JMed CH&Em,:Strategies to inhibit tumor associated integrin receptors:rationale for dual and multi-antagonists.2014,57(15):6301-6315.),含RGD序列的多肽能够被整合素αvβ3中α链的胞膜外区特异性识别,从而影响整合素的生理功能。其中,RGD(精氨酸-甘氨酸-天冬氨酸)环肽是靶向(包括肺癌、黑色素瘤、结肠癌、头颈部鳞状细胞癌,乳腺癌等)肿瘤制剂中应用最为广泛和成熟的一种多肽,在肿瘤靶向药物递送系统的制备中,将其与整合素αvβ3结合是一项重要策略。由此,本发明优选多肽Cyclo(RGDfK)作为靶向肿瘤细胞表面整合素αvβ3特异性结合的配体,通过硫醇化法修饰包载材料PEG-PLGA,得到具有靶向肿瘤功能的包载材料;通过摸索最优处方配比并对一系列稳定发光的NIR II荧光分子FD1080-(CH2)n进行包载,制备得到靶向荧光探针FD1080-(CH2)n NPs cRGD,其粒径、PDI、电位和包封率等测定值均符合最优处方制备的表征值范围。
通过以小鼠结直肠癌肿瘤模型为例,在确定注射剂量为200μM(2mg/kg)的前提下,再分别设置阳性对照组(ICG)、非靶向胶束对照组(FD1080-(CH2)18NPs)和靶向胶束组(FD1080-(CH2)18NPs cRGD)共三组开展小鼠异位结直肠癌肿瘤模型的成像研究,观察它们在小鼠体内的成像结果。实验表明,FD1080-(CH2)18NPs cRGD能够较好地靶向小鼠结直肠癌生物成像,使得肿瘤和周围正常组织发光界限清晰,明显优于ICG组和FD1080-(CH2)18NPs组,信噪比可达到9,揭示FD1080-(CH2)18NPs cRGD是一个具备优良光学性质可主动靶向结直肠癌成像的NIR II荧光纳米探针。
此外,FD1080-(CH2)18NPs cRGD具备了良好的安全性。一方面,通过细胞毒性实验证明FD1080-(CH2)18NPs cRGD在200μM的浓度下,细胞依旧保持90%以上的生存率。另一方面,通过对小鼠的血常规指标、肝功能指标(谷丙氨酸转氨酶和天冬氨酸氨基转移酶)和肾功能指标(肌酐、尿酸和尿素氮)进行考察以及各器官组织HE染色结果病理分析,与空白组和ICG组相比,FD1080-(CH2)18NPs cRGD组各项指标并无显著性差异;病理分析各器官组织也未出现明显损伤。
综上所述,本发明所获得的具有主动靶向功能的荧光探针胶束FD1080-(CH2)nNPscRGD,是一个分辨率和灵敏度高且兼具生物利用度、生物相容性和安全性好的靶向小鼠结直肠癌成像的NIR II荧光探针,不仅特异性靶向结直肠癌,而且成像的边界清晰度等方面均明显优于NIR I ICG。
本发明提供的荧光分子在侧链引入具有脂肪烃以改善脂溶性,增强荧光分子与细胞膜之间的亲和力;同时,为提高体内成像具有靶向性和生物利用度,选择cRGD多肽修饰高分子包载材料PEG-PLGA-泊洛沙姆,制备获得主动靶向肿瘤功能的荧光探针胶束FD 1080-(CH2)n cRGD NPs。
本发明所制备的NIR II纳米荧光探针FD1080-(CH2)n NPs cRGD与游离的荧光分子相比,兼具高灵敏度、高分辨率,且水溶性和安全性均显著提高,与同类NIR II荧光成像探针相比优势表现在:
1、本发明的NIR II的纳米荧光探针发光性能更加稳定,荧光分子具有较好的生物透膜性能,较好地锚定细胞膜的能力,更易穿透细胞膜并扩散进入细胞。
2、本发明的NIR II的纳米荧光探针具有主动靶向肿瘤的成像功能,较好地示踪小鼠结直肠癌生物成像,使得肿瘤和周围正常组织发光界限清晰,明显优于ICG组,信噪比可达到9,具有良好的临床应用前景,有望实现肿瘤的靶向示踪。
3、本发明的NIR II的纳米荧光探针具有的成药性质:1)光物理学性质良好:量子产率为2.3%,摩尔消光系数为1.22×105;2)光稳定性远高于ICG。通过激光照射30min后,其荧光强度依旧保持在原有基础的80%以上;3)化学稳定好。在FBS和PBS环境中,粒径和荧光强度在36h内均无明显变化。
4、本发明的NIR II的纳米荧光探针具有较好地安全性。
附图说明
图1:(a).探针FD1080-(CH2)18NPs cRGD的粒径和TEM图;
(b).探针FD1080-(CH2)18NPs cRGD紫外可见近红外吸收及发射光谱;
(c).探针FD1080-(CH2)18NPs cRGD的相对量子产率;
(d).探针FD1080-(CH2)18NPs cRGD的摩尔吸光系数。
图2:(a).ICG、FD1080-(CH2)18NPs及FD1080-(CH2)18NPs cRGD小鼠体内成像;
(b).各组探针小鼠体内成像信噪比(肿瘤组织/正常组织);
(c).ICG、FD1080-(CH2)18NPs及FD1080-(CH2)18NPs cRGD各组小鼠体内探针器官蓄积量;
(d).各组小鼠体内探针器官蓄积量情况比较。
图3FD1080-(CH2)18NPs cRGD的光稳定性;
图4FD1080-(CH2)18NPs cRGD的体外稳定性;
图5FD1080-(CH2)18NPs cRGD的体外释放实验结果;
图6FD1080-(CH2)18NPs cRGD的细胞毒性实验结果;
图7FD1080-(CH2)18NPs cRGD的肾功能指标评价结果;
图8FD1080-(CH2)18NPs cRGD的肝功能指标评价结果;
图9FD1080-(CH2)18NPs cRGD的H&E染色病理分析结果;
图10FD1080-(CH2)18的1HNMR谱图;
图11FD1080-(CH2)18的13CNMR谱图;
图12FD1080-(CH2)18的MALDI-MS谱图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。
以下实施例中荧光分子所涉及的合成路线如合成路线1:
合成路线1
其中n是6、8、10、12、14、16等偶数。
上述反应中采用试剂:
(i)CH3-CH2-(CH2)n-Cl(其中n是6、8、10、12、14、16等偶数),二甲基甲酰胺(DMF),氢化钠(NaH),室温;
(ii)甲基氯化镁(CH3MgCl),N2,60℃;
(iii)二甲基甲酰胺(DMF),二氯甲烷(DCM),200℃;
(iv)乙酸酐,乙醇,N2,70℃;
实施例1:6a-f的制备:
中间体2a:1-辛基苯并[c,d]吲哚-2(1H)-酮
取圆底烧瓶(50mL),将原料1,8-环内酰亚胺(1.7g,10mmol)和1-溴辛烷(2.304g,12mmol)以及NaH(0.72g,30mmol)溶于20mL DMF中,N2保护,于室温反应3h,TLC(PE:EA=5:1)跟踪反应终点,加入适量乙醇淬灭反应,有固体直接析出,过滤并柱层析纯化(PE:EA=5:1),得到黄色固体化合物2(3.8g,收率90%)。1H NMR(400MHz,CDCl3)δ8.01(d,J=6.7Hz,1H),7.99(d,J=8Hz,1H),7.65(t,J=7.5Hz,1H),7.48(dd,J=8.7,3.3Hz,1H),7.41(t,J=7.6Hz,1H),6.86(d,J=6.5Hz,1H),1.74(p,J=7.3Hz,2H),1.33–1.12(m,12H),0.83(dd,J=8.8,5.3Hz,3H)ppm.ESI-MS:m/z 282.2[M+H]+,C19H24NO.1H NMR和MS数据与文献报道一致(ACS Appl.Mater.Interfaces 2018,10,13,11063–11069.)。
中间体3a:2-甲基-1-辛基苯并[c,d]吲哚-1-鎓
取斜两口瓶(50mL),将中间体2a(1.7g,4mmol)溶于20mL无水THF,N2保护,抽真空三次,在0℃下缓慢加入甲基氯化镁(3.2mL,9.6mmol),60℃条件下反应1h,TLC(PE:EA=6:1)跟踪反应终点,加入适量乙醇淬灭反应,逐滴滴加3M盐酸水溶液,调节pH至酸性,中途有绿色粘稠固体析出可不管,浓缩反应液后加入饱和碘化钾溶液,有暗红色固体析出,直接过滤得到中间体3a(1g,收率60%)。1H NMR(400MHz,CDCl3)δ9.05(d,J=7.2,1H),8.50(d,J=8.0,1H),8.27(d,J=7.4,1H),8.16(d,J=8.2,1H),7.93(t,J=7.7,1H),7.83(t,J=7.8,1H),4.93(t,J=7.5,2H),3.40(s,3H),2.05(m,2H),1.43(m,2H),1.30(m,2H),1.25-1.13(m,6H),0.86(t,J=6.9,3H)ppm.ESI-MS:m/z 280.2,C20H26N+.1H NMR和MS数据与文献报道一致(ACS Appl.Mater.Interfaces.2018,10,13,11063–11069.)。
中间体5:(E)-2-氯-3-(羟基亚甲基)环己基-1-烯-1-甲醛
取圆底烧瓶(250mL),加入DMF(34mL,435mmol)溶于40mL二氯甲烷中,在冰浴0℃条件下搅拌30min后,先将POCl3(32mL,348mmol)溶于30mL二氯甲烷,通过恒压漏斗缓慢加入上述反应液中;再将环己烷(8.53g,87mmol)缓慢加入上述反应液,在50℃条件下反应2h,反应结束后将油状物侧入大量冰水中,过夜,有黄色固体析出(10g,收率为60%)。1H NMR(400MHz,DMSO-d6)δ10.38(d,J=359.7Hz,1H),9.00–8.41(m,1H),2.15(t,J=6.1Hz,4H),1.45–1.26(m,2H)ppm.ESI-MS:m/z 173.2[M+H]+,C8H10Cl35O2.1H NMR和MS数据与文献报道一致(Angew.Chem.Int.Ed.2018,57,7483–7487.)。
荧光分子6a:2-((E)-2-((E)-2-氯-3-((E)-2-(1-辛基苯并[c,d])吲哚-2(1H)-亚甲基)亚乙基)环己基-1-烯-1-基)乙烯基)-1-辛基苯并[c,d]吲哚-1-鎓
取斜两口瓶(50mL),将中间体3(840mg,2mmol)和中间体5(172mg,1mmol)溶于20mL乙酸酐和乙醇中(7:3),N2保护,70℃反应4h,TLC检测反应终点(DCM:MeOH=20:1),直接旋干柱层析,得到黑绿色固体(200mg,收率25%)。1H NMR(400MHz,CDCl3)δ8.39(d,J=13.8Hz,2H),8.05(d,J=6.0Hz,2H),7.81(d,J=8.0Hz,2H),7.72(d,J=4.8Hz,2H),7.33(d,J=7.9Hz,2H),7.26(t,J=7.3Hz,2H),6.90(d,J=6.3Hz,2H),6.33(d,J=14.1Hz,2H),3.89(d,J=4.4Hz,4H),2.68(s,4H),1.89(s,2H),1.64–1.53(m,4H),1.20(s,4H),1.07(d,J=11.3Hz,16H),0.67(t,J=5.1Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ153.21,142.17,140.71,132.12,131.25,130.52,129.80,129.38,128.05,124.89,123.25,109.77,107.48,77.25,44.81,31.80,29.70,29.37,29.16,27.16,22.64,21.11,14.14ppm;MS(Maldi-Tof)calcdfor C48H56Cl35N2 +695.5619,found 695.5612.
中间体2b:1-癸基苯并[c,d]吲哚-2(1H)-酮
合成方法同中间体2a,所用取代试剂为1-溴癸烷(2.2g,10mmol),1H NMR(400MHz,CDCl3)δ8.17(d,J=6.7Hz,1H),7.96(m,1H),7.67(t,J=7.5Hz,1H),7.50(dd,J=8.7,3.3Hz,1H),7.39(t,J=7.6Hz,1H),6.90(d,J=6.5Hz,1H),1.74(p,J=7.3Hz,2H),1.38–1.18(m,16H),0.83(dd,J=8.8,5.3Hz,3H)ppm.ESI-MS:m/z 310.1[M+H]+,C21H28NO.1H NMR和MS数据与文献报道一致(Journal of Photochemistry and Photobiology A:Chemistry200(2008)106–113.)。
中间体3b:2-甲基-1-癸基苯并[c,d]吲哚-1-鎓
合成方法同中间体3a,1H NMR(400MHz,CDCl3)δ8.97(d,J=7.2,1H),8.48(d,J=8.0,1H),8.25(d,J=7.4,1H),8.18(d,J=8.2,1H),7.93(t,J=7.7,1H),7.83(t,J=7.8,1H),4.93(t,J=7.5,2H),3.40(s,3H),2.05(m,2H),1.43(m,2H),1.30(m,2H),1.25-1.13(m,10H),0.88(t,J=6.9,3H)ppm.ESI-MS:m/z 308.1,C22H30N+.1H NMR和MS数据与文献报道一致(Journal of Photochemistry and Photobiology A:Chemistry 200(2008)106–113.)。
荧光分子6b:2-((E)-2-((E)-2-氯-3-((E)-2-(1-癸基苯并[c,d])吲哚-2(1H)-亚甲基)亚乙基)环己基-1-烯-1-基)乙烯基)-1-癸基苯并[c,d]吲哚-1-鎓
荧光分子6b合成操作同6a,反应得蓝黑色固体6b(1.72g,收率23%).1H NMR(600MHz,CDCl3)δ8.66(d,J=13.7Hz,2H),8.29(d,J=6.5Hz,2H),8.00(d,J=7.7Hz,2H),7.92–7.82(m,2H),7.55(d,J=8.1Hz,2H),7.49(t,J=7.5Hz,2H),7.12(d,J=7.0Hz,2H),6.51(d,J=13.9Hz,2H),4.09(d,J=6.7Hz,4H),2.86(s,4H),2.09(d,J=7.5Hz,2H),1.87–1.78(m,4H),1.36–1.32(m,4H),1.29–1.20(m,24H),0.86(t,J=7.0Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ153.21,142.17,140.71,132.12,131.25,130.52,129.80,129.38,128.05,124.89,123.25,109.77,107.48,77.25,44.81,31.80,29.70,29.37,29.16,27.16,22.64,21.11,14.14ppm;MS(Maldi-Tof)calcd for C52H64Cl35N2 +751.6281,found751.6283.
中间体2c:1-十二烷基苯并[c,d]吲哚-2(1H)-酮
合成方法同中间体2a,所用取代试剂为1-溴十二烷(2.26g,10mmol),1H NMR(400MHz,CDCl3):δ8.03(d,J=7Hz,1H),7.98(d,J=8Hz,1H),7.67(dd,J=7Hz,1H),7.50(d,J=9Hz,1H),7.42(dd,J=7Hz,1H),6.87(d,J=7Hz,1H),3.90(t,J=7Hz,2H),1.81-1.75(m,2H),1.43-1.37(m,2H),1.36-1.31(m,2H),1.30-1.24(m,14H),0.87(t,J=7Hz,3H)ppm.ESI-MS:m/z 338.4[M+H]+,C23H32NO.1H NMR和MS数据与文献报道一致(Chemistry ofMaterials,2018,30(19):6587-6588.)。
中间体3c:2-甲基-1-十二烷基苯并[c,d]吲哚-1-鎓
合成方法同中间体3a,1H NMR(400MHz,CDCl3)δ9.08(d,J=7.2,1H),8.61(d,J=8.0,1H),8.47(d,J=7.4,1H),8.26(d,J=8.2,1H),8.03(t,J=7.7,1H),7.91(t,J=7.8,1H),5.01(t,J=7.5,2H),3.43(s,3H),2.05(m,2H),1.43(m,2H),1.38(m,2H),1.30-1.19(m,14H),0.86(t,J=6.9,3H)ppm.ESI-MS:m/z 336.4,C24H34N+.1H NMR和MS数据与文献报道一致(Chemistry of Materials,2018,30(19):6587-6588.)。
荧光分子6c:2-((E)-2-((E)-2-氯-3-((E)-2-(1-十八烷基苯并[c,d])吲哚-2(1H)-亚甲基)亚乙基)环己基-1-烯-1-基)乙烯基)-1-十二烷基苯并[c,d]吲哚-1-鎓
荧光分子6c合成操作同6a,反应得蓝黑色固体6c(1.77g,收率22%).1H NMR(600MHz,CDCl3)δ8.66(d,J=13.7Hz,2H),8.29(d,J=6.5Hz,2H),8.00(d,J=7.7Hz,2H),7.92–7.82(m,2H),7.55(d,J=8.1Hz,2H),7.49(t,J=7.5Hz,2H),7.12(d,J=7.0Hz,2H),6.51(d,J=13.9Hz,2H),4.09(d,J=6.7Hz,4H),2.86(s,4H),2.09(d,J=7.5Hz,2H),1.87–1.78(m,4H),1.36–1.32(m,4H),1.29–1.20(m,32H),0.86(t,J=7.0Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ153.21,142.17,140.71,132.12,131.25,130.52,129.80,129.38,128.05,124.89,123.25,109.77,107.48,77.25,44.81,31.80,29.70,29.37,29.16,27.16,22.64,21.11,14.14ppm;MS(Maldi-Tof)calcd for C56H72Cl35N2 +807.5406,found807.5407.
中间体2d:1-十四烷基苯并[c,d]吲哚-2(1H)-酮
合成方法同中间体2a,所用取代试剂为1-溴十四烷(2.76g,10mmol),1H NMR(400MHz,CDCl3):δ8.03(d,J=7Hz,1H),7.98(d,J=8Hz,1H),7.67(dd,J=7Hz,1H),7.50(d,J=9Hz,1H),7.42(dd,J=7Hz,1H),6.87(d,J=7Hz,1H),3.90(t,J=7Hz,2H),1.81-1.75(m,2H),1.43-1.37(m,2H),1.36-1.31(m,2H),1.30-1.24(m,18H),0.88(t,J=7Hz,3H)ppm.13C NMR(150MHz,CDCl3)δ163.2,134.17,132.1,131.2,130.5,129.8,129.3,128.5,124.8,123.2,109.7,44.8,31.8,30.1,29.9,29.7,29.5,29.3,29.1,28.9,28.7,28.5,27.1,22.6,14.1ppm;ESI-MS:m/z 366.4[M+H]+,C25H36NO.
中间体3d:2-甲基-1-十四烷基苯并[c,d]吲哚-1-鎓
合成方法同中间体3a,1H NMR(400MHz,CDCl3)δ9.05(d,J=7.2,1H),8.50(d,J=8.0,1H),8.27(d,J=7.4,1H),8.16(d,J=8.2,1H),7.93(t,J=7.7,1H),7.83(t,J=7.8,1H),4.93(t,J=7.5,2H),3.40(s,3H),2.05(m,2H),1.43(m,2H),1.30(m,2H),1.25-1.13(m,18H),0.86(t,J=6.9,3H)ppm.13C NMR(150MHz,CDCl3)δ13C-NMR(CDCl3):170.2,139.2,138.4,135.9,131.5,131.1,129.8,128.9,128.7,122.6,121.4,48.7,31.5,30.7,30.4,30.1,29.9,29.7,29.5,29.3,29.0,28.9,26.9,22.4,16.3,13.9ppm;ESI-MS:m/z 364.4,C26H38N+.
荧光分子6d:2-((E)-2-((E)-2-氯-3-((E)-2-(1-十八烷基苯并[c,d])吲哚-2(1H)-亚甲基)亚乙基)环己基-1-烯-1-基)乙烯基)-1-十四烷基苯并[c,d]吲哚-1-鎓
荧光分子6d合成操作同6a,反应得蓝黑色固体6d(1.73g,收率20%).1H NMR(600MHz,CDCl3)δ8.66(d,J=13.7Hz,2H),8.29(d,J=6.5Hz,2H),8.00(d,J=7.7Hz,2H),7.92–7.82(m,2H),7.55(d,J=8.1Hz,2H),7.49(t,J=7.5Hz,2H),7.12(d,J=7.0Hz,2H),6.51(d,J=13.9Hz,2H),4.09(d,J=6.7Hz,4H),2.86(s,4H),2.09(d,J=7.5Hz,2H),1.87–1.78(m,4H),1.36–1.32(m,4H),1.29–1.20(m,40H),0.86(t,J=7.0Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ153.21,142.17,140.71,132.12,131.25,130.52,129.80,129.38,128.05,124.89,123.25,109.77,107.48,77.25,44.81,31.80,29.70,29.37,29.16,27.16,22.64,21.11,14.14ppm;MS(Maldi-Tof)calcd for C60H80Cl35N2 +863.8052,found863.8051.
中间体2e:1-十六烷基苯并[c,d]吲哚-2(1H)-酮
合成方法同中间体2a,所用取代试剂为1-溴十六烷(3.26g,10mmol),1H NMR(400MHz,CDCl3):δ8.19(d,J=7Hz,1H),8.08(d,J=8Hz,1H),7.87(dd,J=7Hz,J=7Hz,1H),7.47(d,J=9Hz,1H),7.40(dd,J=7Hz,J=7Hz,1H),6.83(d,J=7Hz,1H),3.95(t,J=7Hz,2H),1.75(m,2H),1.47(m,2H),1.35(m,2H),1.33-1.24(m,22H),0.88(t,J=7Hz,3H)ppm.ESI-MS:m/z 394.3[M+H]+,C27H40NO.1H NMR和MS数据与文献报道一致(Chemistry ofMaterials,2018,30(19):6587-6588.)。
中间体3e:2-甲基-1-十六烷基苯并[c,d]吲哚-1-鎓
合成方法同中间体3a,1H NMR(400MHz,CDCl3)δ9.05(d,J=7.2,1H),8.50(d,J=8.0,1H),8.27(d,J=7.4,1H),8.16(d,J=8.2,1H),7.93(t,J=7.7,1H),7.83(t,J=7.8,1H),4.93(t,J=7.5,2H),3.40(s,3H),2.05(m,2H),1.43(m,2H),1.30(m,2H),1.25-1.13(m,22H),0.86(t,J=6.9,3H)ppm.13C NMR(150MHz,CDCl3)δ168.2,140.2,139.4,136.9,133.1,132.1,129.8,128.9,128.7,122.6,121.4,48.7,32.5,31.4,30.9,30.7,30.1,29.9,29.7,29.5,29.3,29.1,29.0,28.9,26.9,22.4,16.3,14.1ppm;ESI-MS:m/z 392.3,C28H42N+.
荧光分子6e:2-((E)-2-((E)-2-氯-3-((E)-2-(1-十六烷基苯并[c,d])吲哚-2(1H)-亚甲基)亚乙基)环己基-1-烯-1-基)乙烯基)-1-十六烷基苯并[c,d]吲哚-1-鎓
荧光分子6e合成操作同6a,反应得蓝黑色固体6e(2.02g,收率22%).1H NMR(600MHz,CDCl3)δ8.66(d,J=13.7Hz,2H),8.29(d,J=6.5Hz,2H),8.00(d,J=7.7Hz,2H),7.92–7.82(m,2H),7.55(d,J=8.1Hz,2H),7.49(t,J=7.5Hz,2H),7.12(d,J=7.0Hz,2H),6.51(d,J=13.9Hz,2H),4.09(d,J=6.7Hz,4H),2.86(s,4H),2.09(d,J=7.5Hz,2H),1.87–1.78(m,4H),1.36–1.32(m,4H),1.29–1.20(m,48H),0.86(t,J=7.0Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ153.21,142.17,140.71,132.12,131.25,130.52,129.80,129.38,128.05,124.89,123.25,109.77,107.48,77.25,44.81,31.80,29.70,29.37,29.16,27.16,22.64,21.11,14.14ppm;MS(Maldi-Tof)calcd for C64H88Cl35N2 +919.8436,found919.8434.
中间体2f:1-十八烷基苯并[c,d]吲哚-2(1H)-酮
合成方法同中间体2a,所用取代试剂为1-溴十八烷,1H NMR(400MHz,CDCl3)δ8.04(d,J=7.0Hz,1H),7.99(d,J=8.1Hz,1H),7.69(t,J=7.6Hz,1H),7.51(d,J=8.4Hz,1H),7.45(t,J=7.7Hz,1H),6.90(d,J=6.9Hz,1H),3.90(t,J=7.3Hz,2H),1.22(d,J=5.8Hz,32H),0.85(t,J=6.7Hz,3H)ppm.ESI-MS:m/z 422.3[M+H]+,C29H44NO.1H NMR和MS数据与文献报道一致(Bioconjugate Chemistry,2019,30(10):2647-2663.)。
中间体3f:2-甲基-1-十八烷基苯并[c,d]吲哚-1-鎓
合成方法同中间体3a,1H NMR(400MHz,CDCl3)δ8.97(d,J=7.2,1H),8.60(d,J=8.0,1H),8.37(d,J=7.4,1H),8.20(d,J=8.2,1H),7.98(t,J=7.7,1H),7.87(t,J=7.8,1H),4.95(t,J=7.5,2H),3.37(s,3H),2.05(m,2H),1.43(m,2H),1.33(m,2H),1.28-1.15(m,26H),0.83(t,J=6.9,3H)ppm.ESI-MS:m/z 420.4,C30H46N+.1H NMR和MS数据与文献报道一致(Bioconjugate Chemistry,2019,30(10):2647-2663.)。
荧光分子6f:2-((E)-2-((E)-2-氯-3-((E)-2-(1-十八烷基苯并[c,d])吲哚-2(1H)-亚甲基)亚乙基)环己基-1-烯-1-基)乙烯基)-1-十八烷基苯并[c,d]吲哚-1-鎓
合成方法同6a,得到黑绿色固体(200mg,收率25%)。1H NMR(400MHz,CDCl3)δ8.63(d,J=13.6Hz,2H),8.27(d,J=6.6Hz,2H),8.01(d,J=7.6Hz,2H),7.87(d,J=10.3Hz,2H),7.54(d,J=8.2Hz,2H),7.48(t,J=7.6Hz,2H),7.12(d,J=7.0Hz,2H),6.56(d,J=13.7Hz,2H),4.12(s,4H),2.88(d,J=6.2Hz,4H),2.12(s,1=2H),1.80(s,6H),1.40(s,8H),1.22(d,J=3.3Hz,56H),0.86(t,J=6.6Hz,6H)ppm;13C NMR(150MHz,CDCl3)δ153.21,142.17,140.71,132.12,131.25,130.52,129.80,129.38,128.05,124.89,123.25,109.77,107.48,77.25,44.81,31.80,29.70,29.37,29.16,27.16,22.64,21.11,14.14ppm;MS(Maldi-Tof)calcd for C68H96Cl35N2 +975.2138,found 975.2134.
实施例2:FD1080-(CH2)18NPs cRGD的制备及表征
采用薄膜分散法制备包载FD1080-(CH2)18的胶束,分别精密称取25mg的cRGD-PEG5000-PLGA5000和泊洛沙姆188以及1mg FD1080-(CH2)18,用少量氯仿溶解,短暂超声使溶解完全,移至烧瓶中,将烧瓶置于真空旋转蒸发仪旋干氯仿,随后用油泵除去可能存在的微量溶剂,使靶向高分子包载材料和FD1080-(CH2)18于烧瓶底部形成一层薄膜。于烧瓶内加入20mL的PBS溶液,超声2h后用0.22μm水系滤膜过滤溶液,得到胶束FD1080-(CH2)18NPs cRGD。所制备的纳米制剂的相关表征如表1所示,以及形态见附图1。
表1 FD1080-(CH2)18NPs cRGD的表征
FD1080-(CH2)18NPs cRGD经808nm激发,最大发射波长1040nm。相对量子效率为2.3%,摩尔吸光系数为1.22×105L·mol-1·cm-1。
实施例3:FD1080-(CH2)18NPs cRGD的体内成像研究
通过肿瘤细胞HCT 116建立异位肿瘤肿瘤模型小鼠,开展小鼠体内成像实验。通过尾静脉注射FD1080-(CH2)18NPs cRGD(2mg/kg),以ICG组、非靶向荧光探针FD1080-(CH2)18NPs组为对照,于0、2、4、8、12、24h采集图像,成像结果见附图2。由图2a可知,从成像时间看,ICG在24h后于小鼠体内已完全代谢,FD1080-(CH2)18NPs和FD1080-(CH2)18NPs cRGD组则依旧可成像。从靶向成像看,ICG和FD1080-(CH2)18NPs均不能靶向肿瘤成像,但是FD1080-(CH2)18NPs cRGD可靶向成像,于注射后2h开始在肿瘤处聚集,于12h达到最大值,此时肿瘤处荧光强度也最强,信噪比为9;图2b为几组探针在不同时间点肿瘤组织与正常组织的信噪比,结果表明FD1080-(CH2)18NPs cRGD的信噪比最高,12h时可达到9,可以明显辨别肿瘤组织和正常组织;图2c为几组探针在不同脏器及肿瘤中的蓄积程度,由图可知,ICG在24h已被代谢完全,各器官未见荧光。FD1080-(CH2)18NPs在肝脏中蓄积程度最高,其他器官鲜有发现。FD1080-(CH2)18NPs cRGD除了在肝脏中高度蓄积之外,在肿瘤处也有明显荧光,表明了FD1080-(CH2)18NPs cRGD的靶向性良好。
实施例4:FD 1080NPs cRGD的稳定性研究
(1)用808nm激光(激光功率密度分别为1、2、2.5、5W/cm2)照射FD1080-(CH2)18NPscRGD水溶液和ICG水溶液;在不同的时间点,测其荧光发射光谱和紫外-可见光吸收光谱,并进行对比。图3结果显示FD1080-(CH2)18NPs cRGD拥有更好的稳定性。
(2)将FD1080-(CH2)18NPs cRGD的PBS溶液以及被溶于10%FBS的PBS溶液两份溶液置于37℃水浴中,分别于0、12、24和36h,取部分样品,测其粒径和荧光发射波谱变化。图4结果显示FD1080-(CH2)18NPs cRGD的体外稳定性良好。
(3)于含有10%(w/v)FBS的95mL磷酸盐缓冲溶液(Phosphate buffer saline,PBS)中,37℃条件下开展FD1080-(CH2)18NPs cRGD的体外释放实验。图5结果显示,在含有10%FBS的释放介质中,36h内有约60%的探针释放并趋于稳定。
实施例5:FD1080-(CH2)18NPs cRGD的安全性研究
(1)体外安全性
选择对数生长期的NIH 3T3(小鼠胚胎成纤维贴壁细胞),通过CCK-8方法,测定FD1080-(CH2)18NPs cRGD对NIH 3T3的毒性。图6结果显示,在200μM范围内,FD1080-(CH2)18NPs cRGD对NIH 3T3的毒性较小,安全性高,即使浓度达到200μM,NIH 3T3细胞生存率依旧大于80%。
(2)体内安全性
通过肝肾功能指标评价、血液学及血清生物化学指标评价和H&E染色病理分析对FD1080-(CH2)18NPs cRGD进行体内安全性评价。
1)肾功能指标评价
体内成像实验完成后,随机挑选各组3只裸鼠通过眼球取血并收集分离血清对各组别小鼠血清中肾功能指标进行评价。肾功能指标即肌酐、尿酸和尿素氮,各项指标水平的升高在一定程度上反应肾功能出现异常和损伤,血清样本分析由武汉赛维尔生物科技有限公司完成,肾功能指标评价实验分析结果汇总于图7。
肾功能指标评价实验结果表明,FD1080-(CH2)18NPs cRGD对肾无明显毒性。图7a比较4组裸鼠血样中肌酐(CREA,μM)水平:肌酐是评价肾功能是否正常的重要指标,与空白组和ICG组比较,非靶向纳米荧光探针FD1080-(CH2)18NPs和靶向纳米荧光探针FD1080-(CH2)18NPs cRGD无明显差异;图7b比较4组裸鼠血样中尿素氮(UREA,mM):与空白组和ICG组比较,FD1080-(CH2)18NPs和FD1080-(CH2)18NPs cRGD均无明显差异;图7c比较4组裸鼠血样中尿酸(UA,μM):与空白组和ICG组比较,FD1080-(CH2)18NPs和FD1080-(CH2)18NPs cRGD均无明显差异。
2)肝功能指标评价
体内成像实验完成后,随机挑选各组3只裸鼠通过眼球取血并收集分离血清对各组别小鼠血清中肝功能指标进行评价。肝功能即谷丙氨酸转氨酶和天冬氨酸氨基转移酶,肝功能各项指标水平的升高在一定程度上反应肝功能出现异常和损伤,血清样本分析由武汉赛维尔生物科技有限公司完成,肝功能指标评价实验分析结果汇总于图8。肝功能指标评价实验结果表明,FD1080-(CH2)18NPs cRGD对肝无明显毒性。图8a比较4组裸鼠血样中谷丙氨酸转氨酶(ALT,U/L)水平:谷丙氨酸转氨酶是评价肝功能是否正常的重要指标,与空白组和ICG组比较,FD1080-(CH2)18NPs和FD1080-(CH2)18NPs cRGD均无明显差异;图8b比较4组裸鼠血样中天冬氨酸氨基转移酶(AST,U/L):与空白组和ICG组比较,FD1080-(CH2)18NPs和FD1080-(CH2)18NPs cRGD均无明显差异。
3)小鼠的血液学及血清生物化学研究
小鼠体内成像实验结束后,全部小鼠经眼球取血,配置抗凝剂混合血液一起放置或直接使用抗凝管采集,4℃保存所取血液,以未给药组小鼠和ICG组作为对照,对血液学各项指标进行评价,包括白细胞数目、淋巴细胞数目、单核细胞数目等。各项评价指标水平明显变化或超出参考范围,则表示探针注射入血后存在一定毒性。样本分析由武汉赛维尔生物科技有限公司完成,实验结果见表2。血液学指标检测结果表示,与ICG组和空白组相比,FD1080-(CH2)18NPs cRGD组各项指标均无明显差异,表明其体内安全性良好。
表2血液学各项指标评价结果
4)脏器H&E染色病理分析
体内成像实验完成后,通过随机挑选各组别裸鼠,取其心、肝、脾、肺和肾进行固定和H&E染色,并以正常裸鼠(Untreated group)作为对照,对其结果进行分析,考察其组织病理学变化。H&E染色切片制作由武汉赛维尔生物科技有限公司完成,H&E染色实验分析结果汇总于图10。结果显示,与ICG和空白组对比,FD1080-(CH2)18NPs cRGD对主要组织器官无明显病理毒性,具有较好的安全性。心脏(Heart)心肌横纹无明显增多;肝脏(Liver)各组肝细胞形态完整,细胞核居中且圆而大,可以清晰看到核仁结构,内部细胞核染为蓝色,无明显严重的糖原沉积、炎性细胞浸润和局灶性坏死等异常变化;脾脏(Spleen)各组的脾小体形态正常,滤泡无明显增多,无明显严重的糖原沉积等异常变化;肺(Lung)组织各组呈蜂窝网状结构,管腔扩张正常,无明显异常变化;肾(Kidney)无肾小球异样增生和肾小管坏死。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.一种荧光分子FD1080-(CH2)n,其特征在于,其结构如通式I所示:
其中n是偶数,包括6、8、10、12、14或16。
2.权利要求1所述荧光分子FD1080-(CH2)n的制备方法,其特征在于,包括以下步骤:
按合成路线1制备:
以1,8-环内酰亚胺为原料,经取代、格氏反应和羟醛缩合反应得到目标产物6a-f,目标产物6a-f即通式I所示结构:
其中n是偶数,包括6、8、10、12、14或16。
3.一种可靶向肿瘤的高分子材料,其特征在于,其为将cRGD多肽与高分子材料聚乙二醇类嵌段共聚物连接得到的可靶向肿瘤的高分子材料;
所述聚乙二醇类嵌段共聚物选自聚乙二醇-聚乳酸羟基乙酸嵌段共聚物、聚乙二醇-聚己内酯嵌段共聚物、聚乙二醇-聚乳酸嵌段共聚物或聚乙二醇-聚碳酸酯嵌段共聚物的一种或者几种;当所述聚乙二醇类嵌段共聚物分别选择以上材料时,所述可靶向肿瘤的高分子材料分别表示为cRGD-PEG-PLGA、cRGD-PEG-PCL、cRGD-PEG-PLA、cRGD-PEG-聚碳酸酯。
4.根据权利要求3所述的一种可靶向肿瘤的高分子材料,其特征在于,所述聚乙二醇-聚乳酸羟基乙酸嵌段共聚物选自聚乳酸和羟基乙酸共聚物-聚乙二醇2000、聚乳酸和羟基乙酸共聚物-聚乙二醇5000、聚乳酸和羟基乙酸共聚物-聚乙二醇-氨基、聚乳酸和羟基乙酸共聚物-聚乙二醇-马来酰亚胺、聚乳酸和羟基乙酸共聚物-聚乙二醇-巯基中的一种或几种的混合物。
5.权利要求3所述可靶向肿瘤的高分子材料的制备方法,其特征在于,以cRGD多肽和Mal-PEG-PLGA、Mal-PEG-PCL、Mal-PEG-PLA或Mal-PEG-聚碳酸酯为原料,通过迈克尔加成反应,得到目标产物cRGD-PEG-PLGA、cRGD-PEG-PCL、cRGD-PEG-PLA、cRGD-PEG-聚碳酸酯。
6.一种NIR II纳米荧光探针,其特征在于,其组成包括权利要求1所述荧光分子FD1080-(CH2)n、权利要求3所述可靶向肿瘤的高分子材料、泊洛沙姆或其衍生物;
所述荧光分子FD1080-(CH2)n、所述可靶向肿瘤的高分子材料、泊洛沙姆或其衍生物的质量比为1:10-100:10-100。
7.根据权利要求6所述NIR II纳米荧光探针,其特征在于,所述泊洛沙姆或其衍生物选自泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338、泊洛沙姆407中的一种或几种的混合物。
8.权利要求6所述NIR II纳米荧光探针的制备方法,其特征在于,所述NIR II纳米荧光探针记为FD1080-(CH2)n NPs cRGD,其步骤包括:
(1)将泊洛沙姆或其衍生物、所述可靶向肿瘤的高分子材料和所述荧光分子FD1080-(CH2)n溶解于有机溶剂中,得到混合溶液;
(2)将步骤(1)所述的混合溶液混合均匀后旋转蒸发除去有机溶剂形成薄膜,置于油泵上将残留有机溶剂全部除去;
(3)向步骤(2)中形成的薄膜加入缓冲溶液,充分震荡使薄膜水化脱落;
(4)将步骤(3)得到的水溶液通过聚碳酸酯膜得到粒径均一的胶束,即所述NIR II纳米荧光探针。
9.根据权利要求8所述NIR II纳米荧光探针的制备方法,其特征在于,
步骤(1)中所述有机溶剂选择氯仿、二氯甲烷、四氢呋喃或它们的混合溶剂中的一种或几种的组合;优选的,步骤(1)中的有机溶剂为氯仿;
步骤(2)中,旋蒸温度在30-60℃之间。
步骤(3)中,缓冲溶剂pH在5-8之间。
10.根据权利要求6所述NIR II纳米荧光探针的应用,其特征在于,选自以下应用中的一种或几种:
所述NIR II纳米荧光探针在制备近红外二区成像试剂中的应用;
所述NIR II纳米荧光探针在制备近红外二区活体荧光成像试剂中的应用;
所述NIR II纳米荧光探针在制备近红外二区靶向肿瘤荧光成像试剂中的应用;
所述肿瘤为包括结直肠癌、黑色素瘤、头颈部鳞状细胞癌或乳腺癌在内的实体瘤。
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