CN110898233A - 三模态前列腺癌靶向纳米粒子显像剂及其制备方法 - Google Patents
三模态前列腺癌靶向纳米粒子显像剂及其制备方法 Download PDFInfo
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Abstract
本发明提供一种新型的三模态前列腺癌靶向纳米粒子显像剂及其制备方法。以具有内源性、高生物学相容的超微粒径有机黑色素纳米粒子(UMNPs)作为载体,将具有PSMA靶向功能的小分子基团与纳米粒子偶联,获得具有前列腺癌靶向的纳米分子影像探针PSMA‑PEG‑UMNPs,并利用UMNPs表面活性基团直接标记核素89Zr及T1加权磁共振造影剂Mn2+,得到具备PAI、MRI和PET三模态成像功能的纳米探针(89Zr,Mn)‑PSMA‑PEG‑UMNPs。该探针能与前列腺癌细胞表面的PSMA抗原特异性结合,并分别通过光学、核磁以及核医学手段准确定位PSMA高表达的组织,实现肿瘤的靶向多模态分子影像诊断目的。
Description
技术领域
本发明涉及放射医学、核医学以及纳米医学领域,具体地说,涉及三模态前列腺癌靶向纳米粒子显像剂及其制备方法。
背景技术
分子影像的研究多集中在光学成像、核素成像以及磁共振成像。光学成像是一种新型无创的成像技术,但其依然缺乏组织渗透能力;核素成像具有较高的敏感性,在全身成像中具有重要的作用,但是缺乏组织分辨率;MRI具有超高的组织分辨率,能够清晰地显示病灶的解剖结构和分界,但敏感性较差,且缺乏分子成像能力。在多模态成像领域以及前列腺癌诊疗方面,以前列腺特异性膜抗原(PSMA)为靶点的特异性小分子探针在前列腺癌的诊断、分期、预后及复发监测方面取得重大突破。通过构建基于黑色素纳米粒子的肿瘤靶向分子探针,进行前列腺癌特异性PAI、PET和MRI成像,从而整合多模态成像技术的优势。常用的PET和MRI造影剂分子量小,体内清除较快,肿瘤摄取相对较低,难以长期连续显像。肿瘤靶向药物与纳米技术的结合将是今后发展新型诊断和治疗药物的研究热点。
发明内容
本发明的目的是提供三模态前列腺癌靶向纳米粒子显像剂及其制备方法。
为了实现本发明目的,第一方面,本发明提供一种前列腺癌靶向纳米粒子,所述前列腺癌靶向纳米粒子的制备方法包括:先用双氨基聚乙二醇对UMNPs表面进行修饰,得到表面带有氨基的PEG-UMNPs,然后用Sulfo-SMCC对PEG-UMNPs表面上的氨基进行修饰,得到SMCC-PEG-UMNPs,最后将SMCC-PEG-UMNPs与经过巯基修饰的PSMA靶向基团偶联,即得前列腺癌靶向纳米粒子,记为PSMA-PEG-UMNPs。
本发明中,UMNPs为黑色素纳米粒子,纳米粒子的平均粒径大小为5-10nm,分子量约为30KDa。制备方法在文献(Fan Q,Cheng K,Hu X,et al.Transferring Biomarker intoMolecular Probe:Melanin Nanoparticle as a Naturally Active Platform forMultimodality Imaging[J].Journal of the American Chemical Society,2014,136(43):15185-15194)的基础上,进行改进,获得结构更规则的纳米颗粒。UMNPs的制备方法简述如下:
采用从植物细胞中提取的黑色素作为原材料,超声破碎法制备超微粒径UMNPs(5-10nm)。具体地,黑色素纳米粒子的制备方法如下:取10mg黑色素,在剧烈搅拌下加入3mL的NaOH溶液中(0.1M)。在超声细胞粉碎机(工作强度15%,功率20W)作用下,于1分钟内加入约2.3-2.5mL的0.1M HCl溶液,调体系pH至7.5,得到黑亮的UMNPs分散液;使用截留分子量为30kDa的超滤离心管去除溶液中的游离Na+、Cl-,并用去离子水清洗两次,得到纯净的黑色素纳米粒子UMNPs(平均粒径5-10nm)。
所述双氨基聚乙二醇为NH2-PEG5000-NH2。
所述经过巯基修饰的PSMA靶向基团记为PSMA-SH,PSMA-SH的合成过程如图1所示,具体方法如下:
(1)中间体A的合成
①将1g的H2N-Lys(Bzl)-Otbu和2eq的二异丙基乙胺用50ml DCM溶解于250ml单口瓶中,室温搅拌10min后使其活化;
②向①的体系中加入0.33eq三光气,氮气保护下,保持0℃,搅拌3h;
③向②的体系中加入H2N-Glu(Otbu)-Otbu,反应缓慢升至室温后,搅拌过夜,至反应完全;
④通过减压蒸馏除去溶剂,用EA和饱和食盐水水萃取2-3次,有机相减压蒸馏,得到粗品;
⑤用EA溶解上述粗品于100ml单口瓶内,加入10%钯碳催化剂(Pd/C),氢气保护下室温过夜;
⑥HPLC检测反应,除去钯碳催化剂,减压蒸馏,得到中间体A;
(2)中间体B的合成
①树脂溶涨:将2-氯三苯甲基氯树脂放入反应管中,每克树脂加入15ml DCM,振荡30min;
②接Fmoc-2-Nal-OH:通过砂芯抽滤掉溶剂,加入3eq对应的Fmoc-2-Nal-OH,再加入10eq的DIEA,最后加入DMF溶解,振荡30min;用甲酯封住未反应完全的基团30min,防止后续参与反应;
③脱保护:去掉溶剂,按照每克树脂15ml的量加入由20%哌啶和80%DMF组成的混合液,孵育5min,抽滤去除溶液,再按照每克树脂15ml的量加入由20%哌啶和80%DMF组成的混合液,孵育15min;
④检测:吸走③中的混合液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,变深蓝色为阳性反应;
⑤清洗:按照每克树脂10ml的量先用DMF清洗两次,再按照每克树脂10ml的量用甲醇清洗两次,最后按照每克树脂10ml的量用DMF清洗两次;
⑥缩合:投入3eq Fmoc-Tranexamic Acid,3eq HBTU,用少量DMF溶解,加入反应管,立刻加入10eq DIEA反应30min;
⑦检测:吸走溶液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,无色为阴性反应,表明反应完全;
⑧清洗:按照每克树脂10ml的量先用DMF清洗两次,再按照每克树脂10ml的量用甲醇清洗两次,最后按照每克树脂10ml的量用DMF清洗两次;
⑨缩合:投入3eq Mpa(Trt),3eq HBTU,用少量DMF溶解,加入反应管,立刻加入10eq DIEA反应30min;
⑩检测:吸走溶液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,无色为阴性反应,表明反应完全;抽滤得到的滤液用旋转蒸发仪蒸干,得到中间体B;
(3)中间体A与中间体B的连接
①将步骤(2)得到的中间体B溶解于50ml DCM溶液中,加入1.5eq DCC和1.1eqNHS,室温搅拌过夜;
②反应液有固体析出,抽滤,取滤液,减压蒸馏;
③将②减压蒸馏得到的产物用20ml DMF溶解于100ml单口瓶内,加入1eq中间体A和0.5eq TEA,室温反应过夜;
④向③室温反应过夜所得产物中加入10%柠檬酸水溶液,析出晶体,抽滤,得到固体粗产品;
⑤裂解产物:按照每克固体粗产品10ml的量加入裂解液,裂解120min;其中,所述裂解液为TFA、水、EDT和TIS的混合物,它们的体积比为95:1:2:2;
⑥裂解产物用氮气吹干,用乙醚洗六次,然后常温挥干,得到PSMA-SH粗品;
(4)利用高效液相色谱对PSMA-SH粗品进行纯化,冷冻干燥。
第二方面,本发明提供所述前列腺癌靶向纳米粒子的制备方法,包括以下步骤:
1)将5-10mg UMNPs分散于5-10mL超纯水中,用NaOH溶液调体系pH至9,按UMNPs与NH2-PEG5000-NH2摩尔比1:20-30的比例,将NH2-PEG5000-NH2加入到上述pH为9的体系中,室温下搅拌反应12-24h,超滤去除未反应的NH2-PEG5000-NH2,得到表面带有氨基的PEG-UMNPs,每个UMNPs表面结合20-30个PEG;
2)按PEG-UMNPs表面上的氨基与Sulfo-SMCC摩尔比1:20的比例,将PEG-UMNPs与Sulfo-SMCC混合,室温下搅拌反应2h,反应结束后用PD-10柱纯化,得到SMCC-PEG-UMNPs;
3)按SMCC-PEG-UMNPs与PSMA-SH摩尔比1:20的比例,将SMCC-PEG-UMNPs与PSMA-SH混合,室温下搅拌反应12-24h,反应结束后用PD-10柱纯化。
第三方面,本发明提供所述前列腺癌靶向纳米粒子或按照上述方法制备得到的前列腺癌靶向纳米粒子在制备三模态前列腺癌靶向纳米粒子显像剂中的应用,所述三模态是指光声成像(PAI)、正电子发射成像(PET)和磁共振成像(MRI)。
第四方面,本发明提供三模态前列腺癌靶向纳米粒子显像剂,所述显像剂是将所述前列腺癌靶向纳米粒子或按照上述方法制备得到的前列腺癌靶向纳米粒子与Mn2+偶联,再用89Zr进行核素标记得到的,记为(89Zr,Mn)-PSMA-PEG-UMNPs。
第五方面,本发明提供所述显像剂的制备方法,包括以下步骤:
A、将前列腺癌靶向纳米粒子PSMA-PEG-UMNPs分散于超纯水中,得到纳米粒子溶液,按纳米粒子与Mn2+摩尔比1:200-500的比例,将纳米粒子溶液与可溶性锰盐溶液(MnCl2)混合,反应结束后用PD-10柱纯化,PBS缓冲液作为淋洗液,得到Mn-PSMA-PEG-UMNPs;
B、将5mg Mn-PSMA-PEG-UMNPs分散于5-10mL 0.1M PBS缓冲液中,将溶液超滤浓缩至10mg/mL,然后依次加入0.1M HEPES溶液200-400μL、2M Na2CO3 40-80μL、185-370MBq89ZrCl4,调体系pH至7.0-7.4,于25℃-40℃反应20-30min,反应结束后用PD-10柱纯化。产物(89Zr,Mn)-PSMA-PEG-UMNPs的放射化学纯度大于95%。
第六方面,本发明提供所述前列腺癌靶向纳米粒子或按照上述方法制备得到的前列腺癌靶向纳米粒子在制备前列腺癌分子探针中的应用。
第七方面,本发明提供一种前列腺癌靶向分子探针,其有效成分为所述前列腺癌靶向纳米粒子或按照上述方法制备得到的前列腺癌靶向纳米粒子。
第八方面,本发明提供一种试剂盒,所述试剂盒包含所述前列腺癌靶向纳米粒子或按照上述方法制备得到的前列腺癌靶向纳米粒子,或所述三模态前列腺癌靶向纳米粒子显像剂,或所述前列腺癌分子探针。
本发明提供的(89Zr,Mn)-PSMA-PEG-UMNPs的PET显像结果表明,(89Zr,Mn)-PSMA-PEG-UMNPs能够准确定位PSMA受体阳性前列腺癌模型,并与对照组PSMA受体阴性前列腺癌模型形成对比。(89Zr,Mn)-PSMA-PEG-UMNPs的光声显像显示,(89Zr,Mn)-PSMA-PEG-UMNPs能够准确定位PSMA受体阳性肿瘤,并在人前列腺癌细胞(LNCaP)种植的荷瘤鼠肿瘤部位具有较高信号。(89Zr,Mn)-PSMA-PEG-UMNPs的PET-MR显像显示,(89Zr,Mn)-PSMA-PEG-UMNPs能够明显增强PSMA受体阳性肿瘤部位的T1加权信号强度以及相对应的PET信号,并将两部分信号完美融合
本发明以具有优良生物学性能的黑色素纳米粒子(UMNPs)作为载体,将高生物相容性物质PEG和具有肿瘤靶向性的基团PSMA小分子抑制剂与纳米粒子偶联,获得新型的前列腺癌靶向的纳米分子影像探针PSMA-PEG-UMNPs,并以磁共振造影剂Mn2+、放射性核素89Zr进行标记,得到的(89Zr,Mn)-PSMA-PEG-UMNPs能够与前列腺癌表面特异性膜抗原PSMA特异性结合,分别通过PET、PAI以及MRI手段准确定位PSMA高表达肿瘤组织及转移灶,实现前列腺癌的靶向分子影像诊断目的,在同一种分子上进行多种分子影像探针的研究,以达到肿瘤早发现、早诊断、早治疗的目标。
附图说明
图1为经过巯基修饰的PSMA靶向基团PSMA-SH的合成过程示意图。
图2为本发明三模态前列腺癌靶向纳米粒子显像剂(89Zr,Mn)-PSMA-PEG-UMNPs的合成流程示意图。
图3为本发明实施例1中UMNPs的高分辨率透射电镜扫描图。
图4为本发明实施例3中细胞摄取和竞争实验,左侧为本发明(89Zr,Mn)-PSMA-PEG-UMNPs在阳性细胞LNCaP和阴性细胞PC-3中的摄取实验对比,右侧图为(89Zr,Mn)-PSMA-PEG-UMNPs在阳性细胞LNCaP的竞争抑制实验。
图5为本发明实施例4中三模态前列腺癌靶向纳米粒子显像剂(89Zr,Mn)-PSMA-PEG-UMNPs的药物代谢-动力学实验。
图6为本发明实施例5中中(89Zr,Mn)-PSMA-PEG-UMNPs在PSMA阳性模型LNCaP及PSMA阴性模型PC-3种植的Nod-SCID鼠体内的PET显像图(箭头指示为肿瘤)。
图7为本发明实施例6中(89Zr,Mn)-PSMA-PEG-UMNPs在PSMA阳性模型LNCaP荷瘤鼠中的光声显像图。
图8为本发明实施例7中(89Zr,Mn)-PSMA-PEG-UMNPs在PSMA阳性模型LNCaP荷瘤鼠中的PET-MR双模态融合显像示意图。
具体实施方式
本发明首先提供一种前列腺癌靶向纳米粒子,其是将对前列腺癌特异性膜抗原具有靶向作用的PSMA小分子基团偶联到生物有机黑色素纳米粒子表面,得到PSMA-PEG-UMNPs。所述PSMA小分子基团为含有活性巯基的不对称脲类结构。
其中,UMNPs纳米粒子,即生物来源有机黑色素纳米粒子,其构建方法参考文献(Fan Q,Cheng K,Hu X,et al.Transferring Biomarker into Molecular Probe:MelaninNanoparticle as a Naturally Active Platform for Multimodality Imaging[J].Journal of the American Chemical Society,2014,136(43):15185-15194),并进行改进,以获得结构更规则的纳米颗粒。UMNPs的制备方法简述如下:
采用从植物细胞中提取的黑色素作为原材料,超声破碎法制备超微粒径UMNPs(5-10nm)。具体地,黑色素纳米粒子的制备方法如下:取10mg黑色素,在剧烈搅拌下加入3mL的NaOH溶液中(0.1M)。在超声细胞粉碎机(工作强度15%,功率20W)作用下,于1分钟内加入约2.5mL的0.1M HCl溶液,调体系pH至7.5,得到黑亮的UMNPs分散液;使用截留分子量为30kDa的超滤离心管去除溶液中的游离Na+、Cl-,并用去离子水清洗两次,得到纯净的黑色素纳米粒子UMNPs(平均粒径5-10nm)。
为了提高UMNPs的生物相容性,使用双氨基聚乙二醇(NH2-PEG(5000)-NH2)对UMNPs表面进行修饰。将5-10mg冷冻干燥后的UMNPs充分分散于5-10mL超纯水中,用NaOH溶液调体系pH至9,按UMNPs与NH2-PEG5000-NH2摩尔比1:20-1:30的比例,将NH2-PEG5000-NH2加入到上述pH为9的体系中,室温下搅拌反应12-24h,超滤去除未反应的NH2-PEG5000-NH2,冷冻干燥获得表面带有氨基的NH2-PEG-UMNPs固体。
本发明还提供制备所述肿瘤靶向纳米粒子的方法,利用NH2-PEG-UMNPs表面氨基,经交联剂Sulfo-SMCC进行活化后,得到SMCC-PEG-UMNPs纳米粒子,将该纳米粒子与经过巯基修饰的PSMA小分子基团进行反应,所得PSMA-PEG-UMNPs纳米粒子便具有靶向前列腺癌细胞功能。
本发明还提供所述肿瘤靶向纳米粒子在制备肿瘤三模态显像剂中的应用。
本发明还提供所述肿瘤靶向纳米粒子在PSMA受体表达前列腺癌三模态显像中的应用。
本发明还提供一种三模态肿瘤靶向纳米粒子显像剂,其是以所述肿瘤靶向纳米粒子作为标记前体,使用时进行Mn2+偶联和89Zr核素标记。制备方法包括以下步骤:
1、纳米粒子的靶向修饰:对PSMA小分子抑制剂进行巯基修饰,得到PSMA-SH,然后将PSMA-SH偶联至SMCC-PEG-UMNPs纳米粒子表面,形成肿瘤靶向纳米粒子PSMA-PEG-UMNPs,用PD-10柱分离后作为标记前体。
2、Mn2+偶联:黑色素纳米粒子自身具备对金属离子的高亲和力,可无需偶联剂直接通过亲电反应进行Mn2+偶联,得到具备磁共振T1加权造影功能的Mn-PSMA-PEG-UMNPs纳米探针。
3、89Zr标记:同样利用黑色素纳米粒子自身对金属离子的高亲和力,直接进行89Zr核素标记,得到可用于PET、MRI、PAI三模态成像肿瘤靶向纳米粒子显像剂,即(89Zr,Mn)-PSMA-PEG-UMNPs。
其中,步骤1具体为:按照NH2-PEG-UMNPs表面上氨基与交联剂Sulfo-SMCC的摩尔比1:20-1:30混合,室温反应2h,PD-10柱纯化除去未反应的Sulfo-SMCC;然后按照SMCC-PEG-UMNPs纳米粒子与PSMA-SH摩尔比1:20混合,室温下搅拌反应12h。反应完成后用PD-10柱分离可得PSMA-PEG-UMNPs。
步骤2具体为:按照PSMA-PEG-UMNPs与MnCl2摩尔比1:500混合,反应完成后以PD-10柱分离,PBS缓冲液作为淋洗液,得到Mn-PSMA-PEG-UMNPs。
步骤3具体为:将5mg Mn-PSMA-PEG-UMNPs分散于5-10mL 0.1M PBS缓冲液中,将溶液超滤浓缩至10mg/mL,然后依次加入0.1M HEPES溶液200-400μL、2M Na2CO340-80μL、185-370MBq 89ZrCl4,调体系pH至7.0-7.4,于25℃-40℃反应20-30min,反应结束后用PD-10柱纯化。产物(89Zr,Mn)-PSMA-PEG-UMNPs的放射化学纯度大于95%。(89Zr,Mn)-PSMA-PEG-UMNPs的合成过程见图2。
本发明中涉及的术语:
Sulfo-SMCC:4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐。
H2N-Lys(Bzl)-Otbu:赖氨酸。
DCM:二氯甲烷。
H2N-Glu(Otbu)-Otbu:谷氨酸。
EA:乙酸乙酯。
Fmoc-2-Nal-OH:Fmoc-3-(2-萘基)-L-丙氨酸。
Fmoc-Tranexamic Acid:反式-4-(N-芴甲氧羰基氨基甲基)环己烷甲酸。DIEA:N,N-二异丙基乙胺。
DMF:二甲基甲酰胺。
HBTU:O-苯并三氮唑-四甲基脲六氟磷酸盐。
Mpa(Trt):巯基丙酸。
DCC:二环己基碳二亚胺。
NHS:N-羟基琥珀酰亚胺。
TEA:三乙胺。
TFA:三氟乙酸。
EDT:乙二胺四乙酸。
TIS:三异丙基硅烷。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1三模态前列腺癌靶向纳米粒子显像剂的制备方法
三模态前列腺癌靶向纳米粒子显像剂(89Zr,Mn)-PSMA-PEG-UMNPs的制备包括以下步骤:
1、PSMA-PEG-UMNPs的制备
1.1UMNPs的制备
采用从植物细胞中提取的黑色素作为原材料,超声破碎法制备超微粒径UMNPs。生物提取黑色素:取10mg黑色素,在剧烈的搅拌下溶于3mL的NaOH溶液中(0.1M)。在超声细胞粉碎机(工作强度15%,功率20W)作用下,于1分钟内加入约2.5mL的0.1M HCl溶液,调体系pH至7.5,得到黑亮的UMNPs分散液;使用截留分子量为30kDa的超滤离心管去除溶液中的游离Na+、Cl-,并用去离子水清洗两次,得到纯净的超微粒径黑色素纳米粒子UMNPs(平均粒径约为7nm)。UMNPs的高分辨率透射电镜扫描图见图3。
1.2表面带有氨基的PEG-UMNPs的制备
将5mg UMNPs分散于5mL超纯水中,用0.1M NaOH溶液调体系pH至9,按UMNPs与NH2-PEG5000-NH2摩尔比1:20的比例,将NH2-PEG5000-NH2加入到上述pH为9的体系中,室温下搅拌反应12h,使用截留分子量为30kDa的超滤离心管超滤去除未反应的NH2-PEG5000-NH2,得到表面带有氨基的PEG-UMNPs(NH2-PEG-UMNPs)。
1.3PSMA-SH的制备
1.3.1中间体A的合成
①将1g的H2N-Lys(Bzl)-Otbu和2eq的二异丙基乙胺用50ml DCM溶解于250ml单口瓶中,室温搅拌10min后使其活化;
②向①的体系中加入0.33eq三光气,氮气保护下,保持0℃,搅拌3h;
③向②的体系中加入H2N-Glu(Otbu)-Otbu,反应缓慢升至室温后,搅拌过夜,至反应完全;
④通过减压蒸馏除去溶剂,用EA和饱和食盐水水萃取2-3次,有机相减压蒸馏,得到粗品;
⑤用EA溶解上述粗品于100ml单口瓶内,加入10%钯碳催化剂(Pd/C),氢气保护下室温过夜;
⑥HPLC检测反应,除去钯碳催化剂,减压蒸馏,得到中间体A;
1.3.2中间体B的合成
①树脂溶涨:将2-氯三苯甲基氯树脂放入反应管中,每克树脂加入15ml DCM,振荡30min;
②接Fmoc-2-Nal-OH:通过砂芯抽滤掉溶剂,加入3eq对应的Fmoc-2-Nal-OH,再加入10eq的DIEA,最后加入DMF溶解,振荡30min;用甲酯封住未反应完全的基团30min;
③脱保护:去掉溶剂,按照每克树脂15ml的量加入由20%哌啶和80%DMF组成的混合液,孵育5min,抽滤去除溶液,再按照每克树脂15ml的量加入由20%哌啶和80%DMF组成的混合液,孵育15min;
④检测:吸走③中的混合液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,变深蓝色为阳性反应;
⑤清洗:按照每克树脂10ml的量先用DMF清洗两次,再按照每克树脂10ml的量用甲醇清洗两次,最后按照每克树脂10ml的量用DMF清洗两次;
⑥缩合:投入3eq Fmoc-Tranexamic Acid,3eq HBTU,用少量DMF溶解,加入反应管,立刻加入10eq DIEA反应30min;
⑦检测:吸走溶液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,无色为阴性反应,表明反应完全;
⑧清洗:按照每克树脂10ml的量先用DMF清洗两次,再按照每克树脂10ml的量用甲醇清洗两次,最后按照每克树脂10ml的量用DMF清洗两次;
⑨缩合:投入3eq Mpa(Trt),3eq HBTU,用少量DMF溶解,加入反应管,立刻加入10eq DIEA反应30min;
⑩检测:吸走溶液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,无色为阴性反应,表明反应完全;抽滤得到的滤液用旋转蒸发仪蒸干,得到中间体B;
1.3.3中间体A与中间体B的连接
①将步骤(2)得到的中间体B溶解于50ml DCM溶液中,加入1.5eq DCC和1.1eqNHS,室温搅拌过夜;
②反应液有固体析出,抽滤,取滤液,减压蒸馏;
③将②减压蒸馏得到的产物用20ml DMF溶解于100ml单口瓶内,加入1eq中间体A和0.5eq TEA,室温反应过夜;
④向③室温反应过夜所得产物中加入10%柠檬酸水溶液,析出晶体,抽滤,得到固体粗产品;
⑤裂解产物:按照每克固体粗产品10mL的量加入裂解液,裂解120min;其中,所述裂解液为TFA、水、EDT和TIS的混合物,它们的体积比为95:1:2:2;
⑥裂解产物用氮气吹干,用乙醚洗六次,然后常温挥干,得到PSMA-SH粗品;
1.3.4利用高效液相色谱对PSMA-SH粗品进行纯化,冷冻干燥。
1.4PSMA-PEG-UMNPs的制备
按照NH2-PEG-UMNPs表面上氨基与交联剂Sulfo-SMCC摩尔比1:20混合,室温反应2h,PD-10柱纯化去除未反应的Sulfo-SMCC;然后按照SMCC-PEG-UMNPs纳米粒子与PSMA-SH的摩尔比1:20混合,室温下搅拌反应12h,反应完成后用PD-10柱分离可得SMCC-PEG-UMNPs。PD-10柱使用前需用去金属离子化的、0.01M PBS缓冲液(pH=7.4)淋洗。
2、Mn-PSMA-PEG-UMNPs的制备
取5mg PSMA-PEG-UMNPs充分分散于超纯水中,按照PSMA-PEG-UMNPs与MnCl2的摩尔比1:500混合,室温下搅拌反应12h后使用PD-10柱分离,PBS缓冲液(0.1M,pH=7.4)作为淋洗液,得到Mn-PSMA-PEG-UMNPs。
3、将Mn-PSMA-PEG-UMNPs溶液超滤至10mg/mL,向其中依次加入0.1M HEPES溶液(无金属离子,200μL)、185MBq 89ZrCl4草酸溶液、2M Na2CO3(无金属离子,40μL),调体系pH至7.0,室温反应20-30min,即得(89Zr,Mn)-PSMA-PEG-UMNPs。测定标记率及放射化学纯度,当标记率小于90%,需用PD-10柱纯化,所得(89Zr,Mn)-PSMA-PEG-UMNPs的放射化学纯度大于95%。
标记率的测定采用快速薄层层析法。采用的体系如下:ITLC-SG(快速硅胶薄层层析-硅胶纸);展开剂:4mM EDTA,0.01M PBS溶液,pH7.4;标记物在原点,游离的89Zr在前沿。结果显示,标记率和放射化学纯度均大于95%。
三模态前列腺癌靶向纳米粒子显像剂(89Zr,Mn)-PSMA-PEG-UMNPs的合成流程示意图见图2。
实施例2三模态前列腺癌靶向纳米粒子显像剂的制备方法
制备方法同实施例1,仅将步骤1中NH2-PEG-UMNPs表面上氨基与交联剂Sulfo-SMCC的摩尔比改为1:30,SMCC-PEG-UMNPs纳米粒子与PSMA-SH摩尔比改为1:30,步骤2中PSMA-PEG-UMNPs纳米粒子与MnCl2摩尔比改为1:1000,其余反应条件相同。
结果显示,单个纳米粒子偶联PSMA-SH数由20提高到23个,Mn2+偶联数未发生明显变化。所得(89Zr,Mn)-PSMA-PEG-UMNPs标记率和放射化学纯度均大于95%。
实施例3三模态前列腺癌靶向纳米粒子显像剂细胞摄取及竞争抑制实验
细胞摄取实验:将生长至对数期的LNCaP细胞和PC-3细胞分别以2×105个/孔均匀铺到24孔板中,向每孔加入500μL不含胎牛血清的PRIM 1640培养基,培养箱孵育24h。将一定量(89Zr,Mn)-PSMA-PEG-UMNPs使用生理盐水溶液稀释(37kBq/μL),每孔均匀加入10μL(89Zr,Mn)-PSMA-PEG-UMNPs溶液,放入培养箱孵育一段时间,分别于1h、2h、4h、24h将孔板取出,使用1M NaOH裂解细胞(n=6)并收集,γ-counter检测放射性活度。实验结果如图4左图显示,探针在LNCaP细胞摄取量各个时间点均高于PC-3细胞。
细胞竞争抑制实验:使用LNCaP细胞,实验步骤与细胞摄取实验大致相同,仅在加入放射性药物之前30min向部分孔内(n=6)加入1μg/孔PSMA-SH生理盐水溶液,然后每孔加入10μL(89Zr,Mn)-PSMA-PEG-UMNPs(37kBq/μL)溶液,分别于2h、4h收集裂解后的细胞溶液,使用γ-counter检测放射性活度。实验结果如图4右图所示,探针在LNCaP细胞的摄取可被PSMA-SH抑制,证明其对PSMA受体特异靶向性。
实施例4三模态前列腺癌靶向纳米粒子显像剂药代动力学检测
取5只正常BALB/c小鼠(均为雄性,4周龄,16-18g),将一定量(89Zr,Mn)-PSMA-PEG-UMNPs经生理盐水稀释,89Zr活度检测为11.1MBq/mL,每只小鼠经尾静脉注射200μL探针溶液,并严格记录注射时间。小鼠经尾静脉注射药物后,分别于1min、3min、5min、10min、15min、30min、60min、2h、8h、24h、48h使用毛细取血针经眼底静脉取血,并分别置于γ计数管中。称量计数管中血液质量,使用γ-counter计数仪检测样品放射性活度,同时取每只小鼠注射药物量的1%即22.2kBq作为测定参考标记品。结果使用单位组织的放射活度占注射总活度的百分比(%ID/g)显示,使用分析软件经衰减校正后计算药物生物半衰期。结果显示(图5):(89Zr,Mn)-PSMA-PEG-UMNPs的药物代谢-时间曲线符合二室模型,其分布相和清除相的半衰期分别为0.705h和10.67h。
实施例5三模态前列腺癌靶向纳米粒子显像剂在荷瘤鼠体内的PET显像
将人前列腺癌细胞LNCaP与PC-3用含10%的FBS的PRIM 1640培养基培养至对数期后,选择NOD-SCID鼠(雄性,4-6周龄,18-20g),每只于左侧腋下分别接种LNCaP与PC-3细胞(2×106个),将接种肿瘤的荷瘤鼠饲养于SPF级动物实验室,待瘤径达到0.8-1cm时开始用于体内成像实验。取300μL(11.1MBq)(89Zr,Mn)-PSMA-PEG-UMNPs(实施例1制备)生理盐水稀释溶液,经0.22μm有机滤膜过滤后,使用1mL注射器分别进行LNCaP荷瘤鼠和PC-3荷瘤鼠尾静脉注射(n=3),并分别于2h、24h、48h进行PET成像采集,显像时间为15min。小鼠显像前使用异氟烷麻醉,显像期间维持麻醉(体积系数1%)。结果见图6,该结果表明LNCaP模型肿瘤部位有明显的放射性摄取,与对照组PC-3模型肿瘤部位摄取形成鲜明对比,证明本探针的PSMA靶向PET造影能力。
实施例6三模态前列腺癌靶向纳米粒子显像剂在荷瘤鼠体内的光声显像
取瘤径0.8-1cm的LNCaP荷瘤鼠,每只鼠经尾静脉注射一定量(89Zr,Mn)-PSMA-PEG-UMNPs(实施例1制备)生理盐水稀释液(0.06mM,300μL),分别于2h、24h、48h进行肿瘤部位光声成像采集,成像结果见图7,该结果表明LNCaP模型肿瘤部位光声信号随着时间变化逐渐增强,证明本探针的光声造影能力及PSMA靶向光声成像功能。
实施例7三模态前列腺癌靶向纳米粒子显像剂在荷瘤鼠体内的PET-MR双模态融合显像
取瘤径0.8-1cm的LNCaP荷瘤鼠,每只鼠经尾静脉注射一定量(89Zr,Mn)-PSMA-PEG-UMNPs(实施例1制备)生理盐水稀释液(0.06mM,11.1MBq/300μL),使用上海联影医疗科技有限公司生产的PET-MR成像仪搭配小动物线圈,分别于药物注射前、药物注射后4h进行PET-MR融合成像采集,MR序列信息为:TR(重复时间):531ms,TE(回波时间):9.1ms;翻转角(flipangle):30°;FOV(扫描视野):160×100mm2;扫描矩阵(matrix):256×256;扫描层厚(slicethickness):3mm,成像结果见图8,该结果表明探针经尾静脉注射后4h,PET-MR成像中肿瘤部位T1加权信号强度明显增高,且可与PET信号完美融合,证明探针可用于PSMA高表达前列腺癌特异性PET-MR融合成像。
本发明提供一种新型的三模态前列腺癌靶向纳米粒子显像剂及其制备方法,以具有内源性、高生物学相容的新型有机黑色素纳米粒子(UMNPs)作为载体,将具有前列腺癌特异性膜抗原(PSMA)靶向功能的小分子基团与纳米粒子偶联,获得新型的具有前列腺癌靶向的纳米分子影像探针PSMA-PEG-UMNPs,并利用UMNPs表面活性基团无需借助偶联剂,直接标记长半衰期正电子核素89Zr以及安全性较高的T1加权磁共振造影剂Mn2+,得到具备光声成像(PAI)、正电子发射成像(PET)和磁共振成像(MRI)三模态成像探针(89Zr,Mn)-PSMA-PEG-UMNPs。该探针能够与前列腺癌细胞表面的PSMA抗原特异性结合,分别通过光学、核磁以及核医学手段准确定位PSMA高表达的组织,实现肿瘤的靶向多模态分子影像诊断目的,在实现前列腺癌早发现、早诊断、早治疗的目标的同时,未来还可利用PET-MR新型设备对前列腺癌穿刺进行精确引导。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (9)
1.前列腺癌靶向纳米粒子,其特征在于,所述前列腺癌靶向纳米粒子的制备方法包括:先用双氨基聚乙二醇对UMNPs表面进行修饰,得到表面带有氨基的PEG-UMNPs,然后用Sulfo-SMCC对PEG-UMNPs表面上的氨基进行修饰,得到SMCC-PEG-UMNPs,最后将SMCC-PEG-UMNPs与经过巯基修饰的PSMA靶向基团偶联,即得前列腺癌靶向纳米粒子,记为PSMA-PEG-UMNPs;
其中,UMNPs为黑色素纳米粒子;纳米粒子的平均粒径大小为5-10nm;
所述双氨基聚乙二醇为NH2-PEG5000-NH2。
2.根据权利要求1所述的前列腺癌靶向纳米粒子,其特征在于,所述经过巯基修饰的PSMA靶向基团记为PSMA-SH,其制备方法包括以下步骤:
(1)中间体A的合成
①将1g的H2N-Lys(Bzl)-Otbu和2eq的二异丙基乙胺用50ml DCM溶解于250ml单口瓶中,室温搅拌10min后使其活化;
②向①的体系中加入0.33eq三光气,氮气保护下,保持0℃,搅拌3h;
③向②的体系中加入H2N-Glu(Otbu)-Otbu,反应缓慢升至室温后,搅拌过夜,至反应完全;
④通过减压蒸馏除去溶剂,用EA和饱和食盐水水萃取2-3次,有机相减压蒸馏,得到粗品;
⑤用EA溶解上述粗品于100ml单口瓶内,加入10%钯碳催化剂,氢气保护下室温过夜;
⑥HPLC检测反应,除去钯碳催化剂,减压蒸馏,得到中间体A;
(2)中间体B的合成
①树脂溶涨:将2-氯三苯甲基氯树脂放入反应管中,每克树脂加入15ml DCM,振荡30min;
②接Fmoc-2-Nal-OH:通过砂芯抽滤掉溶剂,加入3eq对应的Fmoc-2-Nal-OH,再加入10eq的DIEA,最后加入DMF溶解,振荡30min;用甲酯封住未反应完全的基团30min;
③脱保护:去掉溶剂,按照每克树脂15ml的量加入由20%哌啶和80%DMF组成的混合液,孵育5min,抽滤去除溶液,再按照每克树脂15ml的量加入由20%哌啶和80%DMF组成的混合液,孵育15min;
④检测:吸走③中的混合液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,变深蓝色为阳性反应;
⑤清洗:按照每克树脂10ml的量先用DMF清洗两次,再按照每克树脂10ml的量用甲醇清洗两次,最后按照每克树脂10ml的量用DMF清洗两次;
⑥缩合:投入3eq Fmoc-Tranexamic Acid,3eq HBTU,用少量DMF溶解,加入反应管,立刻加入10eq DIEA反应30min;
⑦检测:吸走溶液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,无色为阴性反应,表明反应完全;
⑧清洗:按照每克树脂10ml的量先用DMF清洗两次,再按照每克树脂10ml的量用甲醇清洗两次,最后按照每克树脂10ml的量用DMF清洗两次;
⑨缩合:投入3eq Mpa(Trt),3eq HBTU,用少量DMF溶解,加入反应管,立刻加入10eqDIEA反应30min;
⑩检测:吸走溶液,取十几粒树脂,用乙醇洗三次,加入Kaiser试剂,105℃-110℃加热5min,无色为阴性反应,表明反应完全;抽滤得到的滤液用旋转蒸发仪蒸干,得到中间体B;
(3)中间体A与中间体B的连接
①将步骤(2)得到的中间体B溶解于50mlDCM溶液中,加入1.5eq DCC和1.1eq NHS,室温搅拌过夜;
②反应液有固体析出,抽滤,取滤液,减压蒸馏;
③将②减压蒸馏得到的产物用20ml DMF溶解于100ml单口瓶内,加入1eq中间体A和0.5eq TEA,室温反应过夜;
④向③室温反应过夜所得产物中加入10%柠檬酸水溶液,析出晶体,抽滤,得到固体粗产品;
⑤裂解产物:按照每克固体粗产品10ml的量加入裂解液,裂解120min;其中,所述裂解液为TFA、水、EDT和TIS的混合物,它们的体积比为95:1:2:2;
⑥裂解产物用氮气吹干,用乙醚洗六次,然后常温挥干,得到PSMA-SH粗品;
(4)利用高效液相色谱对PSMA-SH粗品进行纯化,冷冻干燥。
3.权利要求2所述前列腺癌靶向纳米粒子的制备方法,其特征在于,包括以下步骤:
1)采用从植物细胞中提取的黑色素作为原材料,超声破碎法制备超微粒径UMNPs;具体地,取10mg黑色素,剧烈搅拌下溶于3mL的0.1M NaOH溶液中;在超声细胞粉碎机作用下,于1分钟内加入2.3-2.5mL的0.1M HCl溶液,调体系pH至7.5,得到黑亮的UMNPs分散液;使用截留分子量为30kDa的超滤离心管去除溶液中游离的Na+、Cl-,并用去离子水清洗两次,得到纯净的黑色素纳米粒子UMNPs;
2)将5-10mg UMNPs分散于5-10mL超纯水中,用NaOH溶液调体系pH至9,按UMNPs与NH2-PEG5000-NH2摩尔比1:20-30的比例,将NH2-PEG5000-NH2加入到上述pH为9的体系中,室温下搅拌反应12-24h,超滤去除未反应的NH2-PEG5000-NH2,得到表面带有氨基的PEG-UMNPs,每个UMNPs表面结合20-30个PEG;
3)按PEG-UMNPs表面上的氨基与Sulfo-SMCC摩尔比1:20的比例,将PEG-UMNPs与Sulfo-SMCC混合,室温下搅拌反应2h,反应结束后用PD-10柱纯化,得到SMCC-PEG-UMNPs;
4)按SMCC-PEG-UMNPs与PSMA-SH摩尔比1:20的比例,将SMCC-PEG-UMNPs与PSMA-SH混合,室温下搅拌反应12-24h,反应结束后用PD-10柱纯化。
4.权利要求1或2所述前列腺癌靶向纳米粒子或按照权利要求3所述方法制备得到的前列腺癌靶向纳米粒子在制备三模态前列腺癌靶向纳米粒子显像剂中的应用,所述三模态是指光声成像、正电子发射成像和磁共振成像。
5.三模态前列腺癌靶向纳米粒子显像剂,其特征在于,所述显像剂是将权利要求1或2所述前列腺癌靶向纳米粒子或按照权利要求3所述方法制备得到的前列腺癌靶向纳米粒子与Mn2+偶联,再用89Zr进行核素标记得到的。
6.权利要求5所述显像剂的制备方法,其特征在于,包括以下步骤:
A、将5mg前列腺癌靶向纳米粒子PSMA-PEG-UMNPs分散于超纯水中,得到纳米粒子溶液,按纳米粒子与Mn2+摩尔比1:200-500的比例,将纳米粒子溶液与可溶性锰盐溶液混合,反应结束后用PD-10柱纯化,PBS缓冲液作为淋洗液,得到Mn-PSMA-PEG-UMNPs;
B、将5mg Mn-PSMA-PEG-UMNPs分散于5-10mL 0.1M PBS缓冲液中,将溶液超滤浓缩至10mg/mL,然后依次加入0.1M HEPES溶液200-400μL、2M Na2CO3 40-80μL、185-370MBq89ZrCl4,调体系pH至7.0-7.4,于25℃-40℃反应20-30min,反应结束后用PD-10柱纯化。
7.权利要求1或2所述前列腺癌靶向纳米粒子或按照权利要求3所述方法制备得到的前列腺癌靶向纳米粒子在制备前列腺癌分子探针中的应用。
8.前列腺癌分子探针,其特征在于,有效成分为权利要求1或2所述前列腺癌靶向纳米粒子或按照权利要求3所述方法制备得到的前列腺癌靶向纳米粒子。
9.一种试剂盒,其特征在于,所述试剂盒包含权利要求1或2所述前列腺癌靶向纳米粒子或按照权利要求3所述方法制备得到的前列腺癌靶向纳米粒子,或权利要求5所述的显像剂,或权利要求8所述的分子探针。
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