CN114835723B - Psma荧光分子探针、制备方法及试剂盒 - Google Patents
Psma荧光分子探针、制备方法及试剂盒 Download PDFInfo
- Publication number
- CN114835723B CN114835723B CN202210763244.XA CN202210763244A CN114835723B CN 114835723 B CN114835723 B CN 114835723B CN 202210763244 A CN202210763244 A CN 202210763244A CN 114835723 B CN114835723 B CN 114835723B
- Authority
- CN
- China
- Prior art keywords
- psma
- solution
- molecular probe
- fluorescent molecular
- fitc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 title claims abstract description 47
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 title claims abstract description 47
- 239000003068 molecular probe Substances 0.000 title claims abstract description 26
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 239000000243 solution Substances 0.000 claims description 36
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 33
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 28
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 239000002994 raw material Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 1
- -1 p-methylbenzylbutyryl Chemical group 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- 239000011259 mixed solution Substances 0.000 description 18
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 16
- 206010060862 Prostate cancer Diseases 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 229940125904 compound 1 Drugs 0.000 description 12
- 229940125782 compound 2 Drugs 0.000 description 12
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 12
- 239000003643 water by type Substances 0.000 description 12
- 239000000126 substance Substances 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 238000003384 imaging method Methods 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 229940126214 compound 3 Drugs 0.000 description 7
- 238000000799 fluorescence microscopy Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012737 fresh medium Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- ZEEBGORNQSEQBE-UHFFFAOYSA-N [2-(3-phenylphenoxy)-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound C1(=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)C1=CC=CC=C1 ZEEBGORNQSEQBE-UHFFFAOYSA-N 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 238000009206 nuclear medicine Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102000007066 Prostate-Specific Antigen Human genes 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000483399 Ipimorpha retusa Species 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 238000013188 needle biopsy Methods 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000010882 preoperative diagnosis Methods 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 238000011472 radical prostatectomy Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1011—Condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
技术领域
本发明是关于检测技术,特别是关于一种PSMA荧光分子探针、制备方法及试剂盒。
背景技术
前列腺癌早期的准确检测对患者的治疗效果和生存至关重要。前列腺癌由于进展相对缓慢往往较难及早发现,前列腺特异性抗原(PSA)联合磁共振成像以及穿刺活检能有效诊断PCa,但目前缺乏能界定其生物学行为的高灵敏度及特异性的功能性成像设备,近十年来PET/CT得到了长足的发展,PET/CT整合了PET兼具高灵敏度与特异度的核医学分子影像以及CT精细的解剖图像,已在原发性前列腺癌的术前诊断与分期以及治疗决策制定等多个方面发挥出巨大的潜力。在治疗上,根治性前列腺癌切除术是治疗局限性及局部进展前列腺癌最有效的方法之一,术者确定切缘范围主要依靠术前影像学检查、术中肉眼所见及探查和术者自身经验。手术切除范围过大会损伤正常组织,影响尿控等正常功能;而切除范围过小会导致阳性切缘,患者容易出现复发。因此,如何在手术中尽可能保留正常组织和功能,同时对前列腺癌侵犯区域进行彻底切除是临床医生经常需要面对和解决的问题。
靶向荧光术中导航技术正是解决这一问题的良方,在前列腺癌领域,存在高特异性的前列腺癌分子标志物—前列腺特异性膜抗原(PSMA)。PSMA在90%的前列腺癌中过度表达,而正常组织如泪腺唾液腺、肾脏近端小管中仅有少量表达。PSMA在前列腺癌中的表达水平与肿瘤的侵袭和恶性程度高度相关。因此,PSMA成为前列腺癌病灶精准定位显像和术中导航的理想生物标志物。靶向PSMA的近红外荧光手术导航技术主要利用近红外染料具有波长较长、组织穿透力较强、散射较小等优点,可在术中点亮病灶,让术者清晰地了解病灶的范围,从而对其进行更加完整有效的切除,因此,靶向PSMA的近红外荧光手术导航将是治疗前列腺癌的一种非常有前景的手术方式。进一步,光学-核医学双模态的分子影像探针可结合两种显像模态的优势,高灵敏度、高精确度在手术前通过核医学模态识别和定位病变组织、在手术中精准荧光标记病灶位置引导外科手术的切除。
目前临床实验中使用的靶向PSMA的核素诊疗和手术导航药物都是以谷氨酸-脲基为基本骨架。这些探针对PSMA具有良好的靶向性和亲和力,但是存在膀胱内核素滞留严重及肾脏内核素背景高的问题,膀胱内大量核素滞留严重影响前列腺癌早期诊断准确率,而双肾高核素背景对肾脏具有一定的毒副作用。因此亟需通过改变药物结构,开发高特异性、低肾脏排泄的PSMA分子影像探针,对于实现前列腺癌病灶的精准定位与分级具有重要意义。但是目前现有探针的溶解度、染料穿透性等方面的不足限定了应用推广的前景。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
本发明的目的在于提供一种PSMA荧光分子探针、制备方法及试剂盒,优化了探针的分子结构,具有溶解性好,荧光穿透性强的特点,具有良好的应用性,同时采用优化选择的合成方案具有更高的合成产率和效率,改善产品的成本性能和推广应用性能。
在本发明的一个或多个实施方式中,PSMA荧光分子探针的制备方法,包括如下步骤:准备含有原料的原料液、第一溶液以及FITC溶液(FITC,CAS:3326-32-7),第一溶液为pH8-9的磷酸氢盐溶液,原料适应性地选自:
在本发明的一个或多个实施方式中,原料液为水溶液,且其中原料的浓度为:0.01-0.1mg/µL。
在本发明的一个或多个实施方式中,第一溶液的溶质选自K2HPO4、Na2HPO4。优选的,第一溶液为水溶液或者DMSO溶液。
在本发明的一个或多个实施方式中,FITC溶液中溶剂选自:DMSO、DMF、DME。
在本发明的一个或多个实施方式中,FITC溶液中FITC的浓度为:0.010-0.05mg/µL。
在本发明的一个或多个实施方式中,反应的条件为15-35℃避光条件下搅拌。
在本发明的一个或多个实施方式中,反应的反应时间为1-4h。
在本发明的一个或多个实施方式中,搅拌的速度为100-1000rpm。
在本发明的一个或多个实施方式中,试剂盒,包括如前述的PSMA荧光分子探针。
与现有技术相比,根据本发明实施方式的PSMA荧光分子探针、制备方法及试剂盒,优化了探针的分子结构,具有溶解性好,荧光穿透性强的特点,具有良好的应用性,同时采用优化选择的合成方案具有更高的合成产率和效率,改善产品的成本性能和推广应用性能。
附图说明
图1是根据本发明一实施方式的PSMA荧光分子探针的质谱图;
图2是根据本发明又一实施方式的PSMA荧光分子探针的质谱图;
图3是根据本发明一实施方式的PSMA荧光分子探针(化合物2)的结合力对照图,其中PC3是PSMA阴性的细胞,22RV1是PSMA阳性的细胞;
图4是根据本发明一实施方式的PSMA荧光分子探针(化合物2)在22RV1和PC3细胞染色的荧光成像,其中PC3是PSMA阴性的细胞,22RV1是PSMA阳性的细胞;
图5是根据本发明一实施方式的PSMA荧光分子探针(化合物2)在肿瘤中的荧光成像,其中PC3是PSMA阴性的细胞,22RV1是PSMA阳性的细胞;
图6是根据本发明一实施方式的PSMA荧光分子探针(化合物2)在肿瘤中的荧光成像,其中PC3是PSMA阴性的细胞,22RV1是PSMA阳性的细胞;
图7是根据本发明一实施方式的PSMA荧光分子探针(化合物2)在肿瘤和不同脏器中的荧光成像。
具体实施方式
下面结合附图,对本发明的具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。
除非另有其它明确表示,否则在整个说明书和权利要求书中,术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分,而并未排除其它元件或其它组成部分。
实施例1-1
其一种合成路线为:
本实施例的合成方案为:向化合物1的水溶液(浓度:0.01mg/µL)50µL中加入0.01MNa2HPO4(150µL,pH 8.4)得到混合溶液。FITC(0.25mg)溶于10µL DMSO中,再加入到前述混合溶液中,在20℃避光条件下,200rpm搅拌2小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为90%,收率为85%)其化学结构由Waters的LC-MS进行表征,图谱如图1所示(M/2+1)。
流式结合力测试:
细胞:22Rv1、PC3细胞
实验设备:流式细胞仪
方法:
1)将细胞接种到T75瓶中。形成单层后使用胰酶消化,将细胞转入离心管(1×106个/管),离心。
2)用添加化合物1不同浓度的新鲜培养液代替培养液,在37℃下孵育30min后使用PBS洗涤3次。用1%SDS(十二烷基硫酸钠)在ddH2O (1.0 mL)中裂解细胞,用流式细胞仪分析细胞结合荧光。使用GraphPad Prism 6绘制细胞结合荧光百分比与浓度的关系图,化合物1选择性结合PSMA高表达细胞22RV1,计算结合亲和力Kd=61.31nM,化合物1不与PC3结合,体现了探针具有良好的靶向性能。
细胞荧光结合检测:
细胞:22Rv1、PC3细胞
实验设备:荧光显微镜
方法:
1)将细胞接种到T75瓶中。形成单层后使用胰酶消化,将细胞转入离心管(1×106个/管),离心。
2)用添加化合物1不同浓度的新鲜培养液代替培养液,在37℃下孵育30min后使用PBS洗涤3次。用1%SDS(十二烷基硫酸钠)在ddH2O (1.0 mL)中裂解细胞,用流式细胞仪分析细胞结合荧光。结果表明化合物1与PSMA高表达细胞22RV1有良好的结合性,并且具有良好的荧光穿透性,响应清晰。
以化合物1探针为例的前列腺癌PSMA靶向荧光成像实验
实验对象:22Rv1细胞或HEK293T-PSMA(武汉普诺赛)皮下荷瘤小鼠
实验设备:IVIS活体成像仪 【IVIS Lumina LT】
1)动物模型构建:22Rv1或HEK293T-PSMA细胞培养于T75培养瓶中,收集细胞5×107 重悬于1ml的PBS中(含有50%高浓度的基质胶BD 356234。
2)于裸鼠【动物来源:军事医学科学院实验动物中心(北京),饲养设施(南开大学实验动物中心)】腋下进行种植,每只鼠皮下100μL缓慢注射一个皮丘
3)将裸鼠放回笼中继续饲养,待肿瘤体积达到300-500mm3时开始进行实验
4)尾静脉注射探针,探针使用含有5%DMSO的PBS溶液溶解(先用DMSO助溶),配置浓度为500μM的溶液,尾静脉注射,每只鼠250μL。
5)于不同的时间点(1,1.5,3,6和9h)进行IVIS活体荧光成像,成像参数为激发750nm,发射为800nm。结果表明化合物1对PSMA高表达,灵敏度高。
实施例1-2
本实施例的合成方案为:向化合物1的水溶液(浓度:0.05mg/µL)50µL中加入pH 8的 Na2HPO4(150µL)得到混合溶液。FITC(0.1mg)溶于10µL DMSO中,再加入到前述混合溶液中,在15℃避光条件下,1000rpm搅拌3小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为93%,收率为84%)其化学结构由Waters的LC-MS进行表征,图谱如图1所示(M/2+1)。
实施例1-3
本实施例的合成方案为:向化合物1的水溶液(浓度:0.1mg/µL)50µL中加入 pH 9的K2HPO4(150µL)得到混合溶液。FITC(0.5mg)溶于10µL DME中,再加入到前述混合溶液中,在35℃避光条件下,800rpm搅拌1小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为91%,收率为86%)其化学结构由Waters的LC-MS进行表征,图谱如图1所示(M/2+1)。
实施例1-4
本实施例的合成方案为:向化合物1的水溶液(浓度:0.08mg/µL)50µL中加入pH8.6的K2HPO4(150µL)得到混合溶液。FITC(0.25mg)溶于10µL DMF中,再加入到前述混合溶液中,在30℃避光条件下,100rpm搅拌4小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为87%,收率为80%)其化学结构由Waters的LC-MS进行表征,图谱如图1所示(M/2+1)。
对比例1
本对比例的合成方案为:向化合物1的水溶液(浓度:0.08mg/µL)50µL中加入FITC溶液(0.25mgFITC溶于10µL DMF中)得到混合溶液。再将pH8.6的K2HPO4(150µL)加入到前述混合溶液中,在30℃避光条件下,100rpm搅拌4小时,通过高效液相色谱仪(Agilent588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为80%,收率为62%)其化学结构由Waters的LC-MS进行表征,图谱如图1所示(M/2+1)。
对比例2
本对比例的合成方案为:将化合物1的水溶液(浓度:0.08mg/µL)50µL、pH8.6的K2HPO4(150µL)以及FITC溶液(0.25mgFITC溶于10µL DMF中)共混后,在30℃避光条件下,100rpm搅拌4小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为73%,收率为55%)其化学结构由Waters的LC-MS进行表征,图谱如图1所示(M/2+1)。
实施例2-1
本实施例中PSMA荧光分子探针(化合物2)为:
其一种合成路线为:
其一种具体的合成方案为:
向化合物3的水溶液(浓度:0.01mg/µL)50µL中加入0.01M Na2HPO4(150µL,pH8.4)。FITC(0.25mg)溶于10µL DMSO中,加入到向化合物3的水溶液中,在25℃避光条件下,600rpm搅拌2小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6 x 150mm,5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1 毫升)纯化(产物纯度为92%,收率为78%)。其化学结构由Waters的LC-MS进行表征,质谱如图2所示。
流式结合力测试:
细胞:22Rv1、PC3细胞
实验设备:流式细胞仪
方法:
1)将细胞接种到T75瓶中。形成单层后使用胰酶消化,将细胞转入离心管(1×106个/管),离心。
2)用添加化合物2不同浓度的新鲜培养液代替培养液,在37℃下孵育30min后使用PBS洗涤3次。用1%SDS(十二烷基硫酸钠)在ddH2O (1.0 mL)中裂解细胞,用流式细胞仪分析细胞结合荧光。使用GraphPad Prism 6绘制细胞结合荧光百分比与浓度的关系图,化合物2选择性结合PSMA高表达细胞22RV1,计算结合亲和力Kd=62.21nM,化合物2不与PC3结合,如图3所示。
细胞荧光结合检测:
细胞:22Rv1、PC3细胞
实验设备:荧光显微镜
方法:
1)将细胞接种到T75瓶中。形成单层后使用胰酶消化,将细胞转入离心管(1×106个/管),离心。
2)用添加化合物2不同浓度的新鲜培养液代替培养液,在37℃下孵育30min后使用PBS洗涤3次。用1%SDS(十二烷基硫酸钠)在ddH2O (1.0 mL)中裂解细胞,用流式细胞仪分析细胞结合荧光,荧光显微镜拍照结果如图4所示。
以化合物2探针为例的前列腺癌PSMA靶向荧光成像实验
实验对象:22Rv1细胞或HEK293T-PSMA(武汉普诺赛)皮下荷瘤小鼠
实验设备:IVIS活体成像仪 【IVIS Lumina LT】
1)动物模型构建:22Rv1或HEK293T-PSMA细胞培养于T75培养瓶中,收集细胞5×107 重悬于1ml的PBS中(含有50%高浓度的基质胶BD 356234。
2)于裸鼠【动物来源:军事医学科学院实验动物中心(北京),饲养设施(南开大学实验动物中心)】腋下进行种植,每只鼠皮下100μL缓慢注射一个皮丘
3)将裸鼠放回笼中继续饲养,待肿瘤体积达到300-500mm3时开始进行实验
4)尾静脉注射探针,探针使用含有5%DMSO的PBS溶液溶解(先用DMSO助溶),配置浓度为500μM的溶液,尾静脉注射,每只鼠250μL。
5)于不同的时间点(1,1.5,3,6和9h)进行IVIS活体荧光成像,成像参数为激发750nm,发射为800nm。成像结果如图5、6所示,解剖的脏器荧光显示如图7所示。
实施例2-2
本实施例的合成方案为:向化合物3的水溶液(浓度:0.05mg/µL)50µL中加入pH 8的 Na2HPO4(150µL)得到混合溶液。FITC(0.1mg)溶于10µL DMSO中,再加入到前述混合溶液中,在15℃避光条件下,1000rpm搅拌3小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为91%,收率为83%)其化学结构由Waters的LC-MS进行表征,图谱如图2所示(M/2+1)。
实施例2-3
本实施例的合成方案为:向化合物3的水溶液(浓度:0.1mg/µL)50µL中加入 pH 9的K2HPO4(150µL)得到混合溶液。FITC(0.5mg)溶于10µL DME中,再加入到前述混合溶液中,在35℃避光条件下,800rpm搅拌1小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为90%,收率为84%)其化学结构由Waters的LC-MS进行表征,图谱如图2所示(M/2+1)。
实施例2-4
本实施例的合成方案为:向化合物3的水溶液(浓度:0.08mg/µL)50µL中加入pH8.6的K2HPO4(150µL)得到混合溶液。FITC(0.25mg)溶于10µL DMF中,再加入到前述混合溶液中,在30℃避光条件下,100rpm搅拌4小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为93%,收率为82%)其化学结构由Waters的LC-MS进行表征,图谱如图2所示(M/2+1)。
对比例3
本对比例的合成方案为:向化合物3的水溶液(浓度:0.08mg/µL)50µL中加入FITC溶液(0.25mgFITC溶于10µL DMF中)得到混合溶液。再将pH8.6的K2HPO4(150µL)加入到前述混合溶液中,在30℃避光条件下,100rpm搅拌4小时,通过高效液相色谱仪(Agilent588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为88%,收率为65%)其化学结构由Waters的LC-MS进行表征,图谱如图2所示(M/2+1)。
对比例4
本对比例的合成方案为:将化合物3的水溶液(浓度:0.08mg/µL)50µL、pH8.6的K2HPO4(150µL)以及FITC溶液(0.25mgFITC溶于10µL DMF中)共混后,在30℃避光条件下,100rpm搅拌4小时,通过高效液相色谱仪(Agilent 588915-902,HC-C18(2) 4.6×150mm, 5µm,0-5分钟:5%乙腈,5-45分钟:5-95%甲醇,流速:每分钟1毫升)纯化(产物纯度为91%,收率为53%)其化学结构由Waters的LC-MS进行表征,图谱如图2所示(M/2+1)。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
Claims (10)
3.如权利要求2所述的PSMA荧光分子探针的制备方法,其特征在于,所述原料液为水溶液,且其中原料的浓度为:0.01-0.1mg/µL。
4.如权利要求2所述的PSMA荧光分子探针的制备方法,其特征在于,所述第一溶液的溶质选自K2HPO4、Na2HPO4。
5.如权利要求2所述的PSMA荧光分子探针的制备方法,其特征在于,所述FITC溶液中溶剂选自:DMSO、DMF、DME。
6.如权利要求5所述的PSMA荧光分子探针的制备方法,其特征在于,所述FITC溶液中FITC的浓度为:0.010-0.05mg/µL。
7.如权利要求2所述的PSMA荧光分子探针的制备方法,其特征在于,所述反应的条件为15-35℃避光条件下搅拌。
8.如权利要求7所述的PSMA荧光分子探针的制备方法,其特征在于,所述反应的反应时间为1-4h。
9.如权利要求7所述的PSMA荧光分子探针的制备方法,其特征在于,所述搅拌的速度为100-1000rpm。
10.试剂盒,包括如权利要求1所述的PSMA荧光分子探针。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210763244.XA CN114835723B (zh) | 2022-07-01 | 2022-07-01 | Psma荧光分子探针、制备方法及试剂盒 |
PCT/CN2022/105197 WO2024000648A1 (zh) | 2022-07-01 | 2022-07-12 | Psma荧光分子探针、制备方法及试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210763244.XA CN114835723B (zh) | 2022-07-01 | 2022-07-01 | Psma荧光分子探针、制备方法及试剂盒 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114835723A CN114835723A (zh) | 2022-08-02 |
CN114835723B true CN114835723B (zh) | 2022-09-16 |
Family
ID=82575028
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210763244.XA Active CN114835723B (zh) | 2022-07-01 | 2022-07-01 | Psma荧光分子探针、制备方法及试剂盒 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN114835723B (zh) |
WO (1) | WO2024000648A1 (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020184806A1 (ko) * | 2019-03-11 | 2020-09-17 | 서울대학교병원 | 망막맥락막 신생혈관성 질환을 진단하기 위한 인테그린 αVβ3 표적화 프로브 및 이의 제조 방법 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10406246B2 (en) * | 2013-10-17 | 2019-09-10 | Deutsches Kresbsforschungszentrum | Double-labeled probe for molecular imaging and use thereof |
HUE051161T2 (hu) * | 2014-12-19 | 2021-03-01 | Bracco Imaging Spa | Operáció közbeni képalkotás |
US9808538B2 (en) * | 2015-09-09 | 2017-11-07 | On Target Laboratories, LLC | PSMA-targeted NIR dyes and their uses |
EP3510399B1 (en) * | 2016-09-09 | 2023-03-01 | On Target Laboratories, LLC | Psma-targeted nir dyes and their uses |
EP3589295A4 (en) * | 2017-02-28 | 2020-11-04 | Endocyte, Inc. | COMPOSITIONS AND METHODS OF T CAR LYMPHOCYTE THERAPY |
US11448650B2 (en) * | 2017-05-08 | 2022-09-20 | Glyca Inc. | Methods for diagnosing high-risk cancer using polysialic acid and one or more tissue-specific biomarkers |
CN110564407A (zh) * | 2019-08-27 | 2019-12-13 | 浙江师范大学 | 一种基于双受体FRET的双发射比率型pH荧光探针及应用 |
CN112574280B (zh) * | 2020-12-21 | 2022-05-17 | 北京大学第一医院 | 一种双酶体系探针及其应用 |
-
2022
- 2022-07-01 CN CN202210763244.XA patent/CN114835723B/zh active Active
- 2022-07-12 WO PCT/CN2022/105197 patent/WO2024000648A1/zh unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020184806A1 (ko) * | 2019-03-11 | 2020-09-17 | 서울대학교병원 | 망막맥락막 신생혈관성 질환을 진단하기 위한 인테그린 αVβ3 표적화 프로브 및 이의 제조 방법 |
Non-Patent Citations (1)
Title |
---|
Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes;KatsuhikoMitachi 等;《Analytical biochemistry》;20160422;第505卷;第29-35页 * |
Also Published As
Publication number | Publication date |
---|---|
CN114835723A (zh) | 2022-08-02 |
WO2024000648A1 (zh) | 2024-01-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20100215581A1 (en) | Contrast agents for detecting prostate cancer | |
CN108949147B (zh) | 一种分子影像探针及其应用 | |
JP2019531706A (ja) | 術前および術中の組織の局在化および視覚化のための組織特異的マーカー | |
Zhang et al. | Near-infrared dye-labeled anti-prostate stem cell antigen minibody enables real-time fluorescence imaging and targeted surgery in translational mouse models | |
JP5523282B2 (ja) | 蛍光コバラミンおよびその使用 | |
CN114380786B (zh) | 基于氨基肽酶激活型化学发光探针及其在恶性肿瘤的活体检测与手术导航方面的应用 | |
CN113209315A (zh) | 靶向肿瘤的多肽探针及应用 | |
US9801958B2 (en) | Polymer nanoparticle composite and composition for MRI imaging including same | |
CN114014843B (zh) | 一种psma靶向核素/荧光双模态配体和分子探针与应用 | |
CN114835723B (zh) | Psma荧光分子探针、制备方法及试剂盒 | |
Pouliot et al. | In vivo imaging of intraprostatic-specific gene transcription by PET | |
EP2584356B9 (en) | Method for detection of urothelial cancer | |
CN104560026A (zh) | 一种活体卵巢癌组织的靶向muc1的荧光探针及其制备方法 | |
JP2005226021A (ja) | マンノース受容体親和性化合物 | |
CN112592386B (zh) | 红光介导的核酸锚定型荧光探针及其制备方法和应用 | |
CN108743975B (zh) | 一种靶向肿瘤vegfr-3分子的近红外荧光显像剂的设计、合成及应用 | |
WO2012078647A2 (en) | Chemical composition to detect and treat amyloid in a patient's brain and retina | |
CN112933252A (zh) | 一种乳腺癌特异靶向分子探针及其制备方法和应用 | |
CN116407651B (zh) | 一种多肽mrwvyhpfq在肿瘤诊断药物中的应用 | |
CN115337413B (zh) | 一种针对非小细胞肺癌诊断的放射性分子探针的制备及应用 | |
Wei et al. | Advances in optical molecular imaging for neural visualization | |
RU2799760C2 (ru) | Наночастица, содержащее ее контрастное вещество для магнитно-резонансной томографии и цвиттер-ионное лигандное соединение | |
Chen et al. | Nanobody-loaded nanobubbles targeting the G250 antigen with ultrasound/photoacoustic/fluorescence multimodal imaging capabilities for specifically enhanced imaging of RCC | |
CN116510043A (zh) | 一种用于胶质瘤边界判定的分子影像纳米探针及其制备方法 | |
Fu et al. | Improving oral squamous cell carcinoma diagnosis and treatment with fluorescence molecular imaging |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CP01 | Change in the name or title of a patent holder |
Address after: Room 402, building 1, Northwest District, Suzhou nano City, No. 99 Jinjihu Avenue, Suzhou Industrial Park, Jiangsu 215000 Patentee after: Xindou Biotechnology (Suzhou) Co.,Ltd. Address before: Room 402, building 1, Northwest District, Suzhou nano City, No. 99 Jinjihu Avenue, Suzhou Industrial Park, Jiangsu 215000 Patentee before: Daigepurui Biotechnology (Suzhou) Co.,Ltd. |
|
CP01 | Change in the name or title of a patent holder |