CN114699540A - 一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针 - Google Patents
一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针 Download PDFInfo
- Publication number
- CN114699540A CN114699540A CN202210234058.7A CN202210234058A CN114699540A CN 114699540 A CN114699540 A CN 114699540A CN 202210234058 A CN202210234058 A CN 202210234058A CN 114699540 A CN114699540 A CN 114699540A
- Authority
- CN
- China
- Prior art keywords
- magnetic resonance
- fluorescence
- probe
- hsa
- dtpa
- 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.)
- Pending
Links
- 239000000523 sample Substances 0.000 title claims abstract description 82
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 13
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 13
- 238000002619 cancer immunotherapy Methods 0.000 title claims abstract description 8
- 229960004657 indocyanine green Drugs 0.000 claims abstract description 26
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 claims abstract description 20
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 17
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 17
- 230000008859 change Effects 0.000 claims abstract description 16
- 230000002902 bimodal effect Effects 0.000 claims abstract description 13
- 239000002105 nanoparticle Substances 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 239000002253 acid Substances 0.000 claims abstract description 6
- 102000008096 B7-H1 Antigen Human genes 0.000 claims abstract 5
- 108010074708 B7-H1 Antigen Proteins 0.000 claims abstract 5
- 206010028980 Neoplasm Diseases 0.000 claims description 25
- 230000008685 targeting Effects 0.000 claims description 15
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 12
- 238000002512 chemotherapy Methods 0.000 claims description 12
- 238000000799 fluorescence microscopy Methods 0.000 claims description 11
- 238000002595 magnetic resonance imaging Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 238000012544 monitoring process Methods 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- 238000002560 therapeutic procedure Methods 0.000 claims description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- MEANOSLIBWSCIT-UHFFFAOYSA-K gadolinium trichloride Chemical compound Cl[Gd](Cl)Cl MEANOSLIBWSCIT-UHFFFAOYSA-K 0.000 claims description 6
- 238000001959 radiotherapy Methods 0.000 claims description 5
- 230000005540 biological transmission Effects 0.000 claims description 4
- 230000035876 healing Effects 0.000 claims description 4
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims description 3
- 230000002209 hydrophobic effect Effects 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 3
- 238000002648 combination therapy Methods 0.000 claims description 2
- 238000001819 mass spectrum Methods 0.000 claims description 2
- 241000764238 Isis Species 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- 230000001024 immunotherapeutic effect Effects 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 21
- 238000011156 evaluation Methods 0.000 abstract description 13
- 108091007744 Programmed cell death receptors Proteins 0.000 abstract description 12
- 238000003384 imaging method Methods 0.000 abstract description 11
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 abstract description 10
- 238000005516 engineering process Methods 0.000 abstract description 10
- 208000022679 triple-negative breast carcinoma Diseases 0.000 abstract description 10
- 241000699670 Mus sp. Species 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 238000001727 in vivo Methods 0.000 description 9
- 238000010586 diagram Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 239000003068 molecular probe Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 102000008070 Interferon-gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- DZSYJVXGONVNKA-UHFFFAOYSA-L NIR-1 dye Chemical group [K+].[K+].C1=CC2=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C=C2C(C2(C)C)=C1[N+](CC)=C2C=CC=CC=CC=C1C(C)(C)C2=CC(C(O)=O)=CC=C2N1CCCCS([O-])(=O)=O DZSYJVXGONVNKA-UHFFFAOYSA-L 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000002872 contrast media Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 229960003130 interferon gamma Drugs 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- AUQMGYLQQPSCNH-UHFFFAOYSA-L NIR-2 dye Chemical group [K+].[K+].C1=CC2=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C=C2C(C2(C)C)=C1[N+](CC)=C2C=CC=CC=C1C(C)(C)C2=CC(C(O)=O)=CC=C2N1CCCCS([O-])(=O)=O AUQMGYLQQPSCNH-UHFFFAOYSA-L 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000000679 relaxometry Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
- A61K49/103—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA
- A61K49/105—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA the metal complex being Gd-DTPA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/143—Peptides, e.g. proteins the protein being an albumin, e.g. HSA, BSA, ovalbumin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/14—Peptides, e.g. proteins
- A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针，涉及三阴性乳腺癌程序性死亡受体(PD‑1)治疗疗效评估。所述荧光/磁共振探针GdDTPA‑HSA@ICG‑Atezoilzumab(NPs‑Ate),包含二乙基三胺五乙酸、人血清白蛋白、吲哚青绿、PD‑L1抗体阿特珠单抗。制备方法：1)制备GdDTPA‑HSA@ICG纳米颗粒；2)制备NPs‑Ate抗体荧光/磁共振双模态探针。所述荧光/磁共振探针可应用于动态评价乳腺癌免疫治疗效果。实时动态监测ICT治疗有效时PD‑L1的表达变化，尽早、准确预测疗效和评估治疗的有效性。为三阴性乳腺癌ICT的疗效评估提供分子影像新技术和新方。
Description
技术领域
本发明涉及三阴性乳腺癌程序性死亡受体(PD-1)治疗疗效评估，尤其是涉及基于荧光、磁共振双模态成像对PD-1治疗三阴性乳腺癌过程中程序性死亡配体(PD-L1)表达的一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针。
背景技术
乳腺癌是女性发病率最高的恶性肿瘤，其中三阴性乳腺癌恶性程度高、复发率高、侵袭力强、患者的生存期短。随着免疫治疗的发展，免疫检查点治疗(ICT)，为三阴性乳腺癌的治疗带来新契机，但是有效率仅有20％～30％，对于不敏感患者，ICT不仅不能表现出应有的疗效反而造成时间和金钱的浪费，贻误治疗时机。如何预测疗效或者早期评估是发现敏感人群的关键,目前ICT疗效评价标准参照iRECIST，主要借助磁共振(MRI)等传统影像技术，依靠肿瘤大小与解剖学信息评估疗效，难以到达分子水平，存在时效性差、准确度低等缺点。因此，深入分子水平探索ICT疗效预测及早期评估的技术方法有望提高疗效评估的准确性，有助于个体化治疗。
PD-L1作为ICT的重要生物标志物之一，其表达水平对ICT疗效评估有重要价值。可视化PD-L1时空变化的分子影像技术及其医学应用的研究成为前沿领域关注焦点。但目前PD-L1检测的免疫组化方法存在无法动态监测、判断标准不一等缺点。而分子影像技术可深入分子水平，在体无创地对肿瘤组织整体内部的分子情况进行实时监测，不仅可用于疗效预测，还可用于早期疗效评估，特别是荧光、磁共振成像技术具有实时在体、动态显像、安全性高、灵敏度高、无电离辐射等诸多优势。
发明内容
本发明的目的在于为突破ICT在三阴性乳腺癌临床应用面临的瓶颈，解决如何提高可视化PD-L1动态变化的活体成像灵敏度，满足ICT疗效精准评估的需求这一科学问题，提供可推动ICT精准诊疗研究，满足乳腺癌防治的巨大需求的一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针。
本发明所述荧光/磁共振探针为靶向PD-L1的近红外二区荧光/磁共振双模态分子探针，包含二乙基三胺五乙酸(DTPA)、人血清白蛋白(HSA)、吲哚青绿(ICG)、PD-L1抗体阿特珠单抗(Atezoilzumab)，其分子式为：GdDTPA-HSA@ICG-Atezoilzumab(简称NPs-Ate)。
所述荧光/磁共振探针的制备方法，包括以下步骤：
1)制备GdDTPA-HSA@ICG纳米颗粒：将人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)在pH＝8.2的条件下进行连接，调节pH＝6.5后，加入氯化钆(GdCl3)得到GdDTPA-HSA纳米颗粒，用质谱验证钆(Gd)连接效率，利用人血清白蛋白(HSA)的疏水口袋装入吲哚青绿(ICG)，利用光谱验证吲哚青绿(ICG)包裹成功，利用透射电镜验证纳米颗粒的合成。
2)制备NPs-Ate抗体荧光/磁共振双模态探针：选择聚乙二醇(NHS-PEG2000-COOH)将步骤1)制备的GdDTPA-HSA@ICG纳米颗粒与靶向元件阿特珠单抗(Atezoilzumab)连接，利用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化，得到靶向肿瘤的所述荧光/磁共振探针GdDTPA-HSA@ICG-Atezoilzumab(NPs-Ate)。
所述荧光/磁共振探针可应用于动态评价乳腺癌免疫治疗效果。
所述应用的具体方法可为：对肿瘤进行ICT单用及联用治疗(PD-1治疗加化疗/放疗)，利用治疗前后探针的荧光成像及磁共振成像监测组织中PD-L1表达变化，评估PD-L1变化与ICT愈后的关系。
与现有技术相比，本发明的有益效果在于：
本发明采用高灵敏度的近红外荧光成像与高分辨率的MRI相结合的策略，将人血清白蛋白(HSA)、吲哚青绿(ICG)、PD-L1抗体阿特珠单抗(Atezoilzumab)等安全材料合成具有临床转化潜能的靶向PD-L1双模态分子影像探针，活体实时动态监测ICT治疗有效时PD-L1的表达变化，尽早、准确预测疗效和评估治疗的有效性。
本发明在ICT的疗效预测和早期评估方面的应用价值，为三阴性乳腺癌ICT的疗效评估提供分子影像新技术和新方法，推动ICT精准诊疗研究。
附图说明
图1为NPs-Ate的合成示意图。
图2为NPs-Ate的电镜图。
图3为NPs-Ate的光学表征图。
图4为NPs-Ate的磁学表征图。
图5为NPs-Ate磁共振靶向性验证图。
图6为使用流程参考图。
图7为第一次荧光检测图。
图8为第二次荧光检测图。
图9为PD-L1免疫组化图
图10为使用效果监测图。
图11为病理安全性验证图。
图12为体重安全性验证图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下实施例将结合附图对本发明进行作进一步的说明。应当理解，此处所描述的具体实施例仅仅用于解释本发明，并不用于限定本发明。相反，本发明涵盖任何由权利要求定义的在本发明的精髓和范围上做的替代、修改、等效方法以及方案。进一步，为了使公众对本发明有更好的了解，以下对本发明的细节描述中，详尽描述了一些特定的细节部分。对本领域技术人员来说没有这些细节部分的描述也可以完全理解本发明。
本发明所述靶向肿瘤的所述荧光/磁共振探针GdDTPA-HSA@ICG-Atezoilzumab(NPs-Ate)的合成示意图如图1所示。
1.设计并制备靶向PD-L1的GdDTPA-HSA@ICG-Atezoilzumab(NPs-Ate)近红外二区荧光/磁共振双模态分子探针
1.1制备GdDTPA-HSA@ICG纳米颗粒：拟将一定量的人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)在PH＝8.2的条件下进行连接，后调PH＝6.5加入氯化钆(GdCl3)得到GdDTPA-HSA纳米颗粒，用质谱验证钆(Gd)连接效率，利用HSA的疏水口袋装入ICG，利用光谱验证ICG包裹成功，利用透射电镜验证纳米颗粒的合成。
1.2制备NPs-Ate抗体荧光/磁共振双模态探针：选择聚乙二醇(NHS-PEG2000-COOH)将GdDTPA-HSA@ICG纳米颗粒与靶向元件阿特珠单抗连接，利用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化，得到靶向肿瘤的功能化PD-L1抗体荧光/磁共振双模态探针(NPs-Ate)。
另外，用免疫球蛋白G(IgG)构建对照探针GdDTPA-HSA@ICG-IgG(NPs-IgG)。
2.体外和在体水平评估分别评估荧光/磁共振双模态分子探针的安全性以及靶向性
2.1体外水平研究
1)亚细胞水平分布：利用荧光显微镜检测探针在亚细胞水平的分布；2)探针对细胞性能的影响：在细胞水平设置探针孵育时间、浓度梯度，通过细胞增殖实验(CCK-8)，对分子探针进行体外安全性及适用性评价；3)靶向性研究：探针与不同PD-L1表达水平的乳腺癌细胞共孵育后，用荧光成像、流式等方法进行多角度分析，验证探针的PD-L1靶向性，进一步采用“竞争阻断法”，验证探针的PD-L1特异性；
2.2在体水平研究：
1)评估PD-L1荧光/磁共振双模态分子探针的在体安全性：根据探针在小鼠体内各个脏器的探针影像信号强度判断其代谢途径和生物分布，最后根据探针对小鼠体重及各个脏器组织学水平的影响初步评价在体毒性；2)靶向性研究：在构建的不同PD-L1表达水平三阴性移植瘤模型中，尾静脉注射含有PD-L1抗体的探针进行近红外一区、二区荧光成像及磁共振成像，验证探针对PD-L1的靶向性。
3.荧光/磁共振双模态分子影像技术用于三阴性乳腺癌ICT疗效预测
3.1构建乳腺癌移植瘤小鼠模型：将鼠源性三阴性乳腺癌细胞4T1(PD-L1阴性)种植于大腿外侧，利用放疗、化疗、干扰素-γ(INF-γ)诱导PD-L1表达。观察探针能否监测小鼠肿瘤内PD-L1的表达升高。
3.2评估双模态分子影像技术对ICT预测的价值:对肿瘤进行ICT单用及联用治疗(PD-1治疗加化疗/放疗)，利用治疗前后探针的荧光成像及磁共振成像监测组织中PD-L1表达变化，评估PD-L1变化与ICT愈后的关系。
实施例1：探针形貌和尺寸检测
将少量样品稀释至浅绿色。吸取10μl溶液滴加在超薄碳膜上，室温晾干。探针稀释于PBS，取部分悬浮液滴于铜网，自然干燥，并采用透射电子显微镜，观察样品的形貌、分布和大小，选取清晰、无污染的视野拍照保存。
实施例1测试结果：如图2的电镜图所示，探针在电镜下粒径较为均一,分散性好，大小约为7～8nm。
实施例2：探针光谱检测
取适量探针溶于不同溶剂中，将样品装进比色皿，置于样品仓中。利用酶标仪检测400～900nm探针吸光度。利用荧光光谱仪检测其荧光发射光谱(近红外1区：激发：720nm发射：750～900nm；近红外2区:激发:808nm发射:1000-1400nm)
实施例2测试结果：如图3a的吸收光谱所示，探针在720nm左右有最大吸收峰，与ICG荧光相似，说明NPs-Ate的合成未改变ICG的荧光性能。如图3b的近红外1区荧光光谱所示，探针在800nm左右有最大的发射强度，与ICG荧光相似，说明NPs-Ate的合成未改变ICG的荧光性能。如图3c的近红外二区荧光光谱所示，该探针在808的激发光下，在近红外二区有较强的荧光拖尾信号，说明该探针可用于近红外二区荧光成像。
实施例3：探针磁共振性能的检测
取适量探针在PBS中稀释稀释至不同浓度，利用去离子水作为对照，利用台式磁共振弛豫测量系统测量纵向弛豫时间(T1)，并利用1/T1与Gd浓度的曲线计算样品的纵向弛豫率(γ1)。所有样本采用T1加权成像。
实施例3测试结果：如图4磁共振信号显示，不同的Gd3+离子浓度具有不同T1磁共振信号，利用1/T1与Gd浓度的曲线计算样品的纵向弛豫率(γ1)为9.72。说明该探针有较好的T1弛豫信号，可用于磁共振成像。
实施例4：探针磁共振靶向性检测
MDA-MB-231皮下移植瘤小鼠9只，随机分为3组。每组小鼠通过尾静脉分别注射目的探针，NPs-IgG对照探针和Gd-DTPA对照造影剂，分别于注射前及注射后不同时间点(1、6、12、24、36、48、72h)使用9.4T小动物磁共振成像设备采集肿瘤磁共振图像，通过软件分析各组在不同时间点的信号强度差异。
实施例4测试结果：如图5分别为第1、6、12h NPs-Ate、NPs-IgG、Gd-DTPA对照造影剂在小鼠肿瘤平面的磁共振图像(左)及在1、6、12、24、36、48、72h的信号背景比定量图像(右)。靶向PD-L1探针较对照组探针在第12h可检测到更高的T1磁共振信号和探针的摄取。说明探针具有特异性靶向高表达PD-L1组织的能力，并且信号差异可被磁共振仪器检测。
实施例5：小鼠免疫治疗中利用探针监测PD-L1表达变化及愈后
4T1移植瘤小鼠模型放疗联合免疫小鼠治疗监测，皮下移植瘤小鼠通过皮下注射4T1构建治疗模型，连续4天白蛋白紫杉醇化疗25mg/kg后，尾静脉进行第一次注射探针，检测光学手段能否探测出表达的差异。化疗后腹腔注射PD-1抗体(20mg/kg 2天一次连续4次)，在第14天再次注入探针，检测光学手段能否探测出表达的差异。观测小鼠肿瘤大小的变化，以及小鼠愈后，检测探针信号变化与治疗效果的关系。(如图6为治疗流程图)
实施例5测试结果1：如图7为第一次荧光成像在体小鼠肿瘤荧光及定量。化疗后小鼠肿瘤组织处可在体检测到更高的荧光信号，说明探针可以监测到化疗干预所致的荧光信号增加。
实施例5测试结果2：如图8为第二次荧光成像在体小鼠肿瘤荧光及定量。化疗及PD-1治疗组小鼠肿瘤组织处可在体检测到更高的荧光信号，说明探针可以监测到化疗联合PD-1治疗所致的荧光信号增加。
实施例5测试结果3：如图9为第二次荧光成像后小鼠组织PD-L1免疫组化。化疗联合PD-1治疗组小鼠PD-L1表达最高。该免疫组化结果与荧光图像一致，说明该探针可监测治疗过程中PD-L1的表达。
实施例5测试结果4：如图10为予以治疗后小鼠肿瘤体积曲线以及小鼠愈后的生存曲线。在化疗联合PD-1治疗组观测到更小的肿瘤增加和更好的愈后。说明探针信号的增加可能和愈后有关。
实施例6：在正常小鼠中检测探针安全性。
在正常小鼠中注射诊断剂量的探针，将PBS对照，分别检测30天内的体重变化。在1、3、7、28天提取组织器官(心、肝、脾、肺、肾、胃、肠、脑)，观察是否有病理变化。
实施例6测试结果1：如图11为注射探针后在1、3、7、28天心、肝、脾、肺、肾、胃、肠、脑的病理图片。图片中各时间段及各器官病理未见明显改变，说明探针在小动物水平未见明显毒性。
实施例6测试结果2：如图12为注射探针30天内小鼠体重变化。注射探针组小鼠较PBS对照组无显著差异，说明探针在小动物水平未见明显毒性。
上述实施例仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。
Claims (4)
1.一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针，其特征在于由二乙基三胺五乙酸、人血清白蛋白、吲哚青绿、PD-L1抗体阿特珠单抗合成；荧光/磁共振探针的分子式为：GdDTPA-HSA@ICG-Atezoilzumab(NPs-Ate)。
2.如权利要求1所述荧光/磁共振探针的制备方法，其特征在于包括以下步骤：
1)制备GdDTPA-HSA@ICG纳米颗粒：将人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)在pH＝8.2的条件下进行连接，调节pH＝6.5后，加入氯化钆(GdCl3)得到GdDTPA-HSA纳米颗粒，用质谱验证钆(Gd)连接效率，利用人血清白蛋白(HSA)的疏水口袋装入吲哚青绿(ICG)，利用光谱验证吲哚青绿(ICG)包裹成功，利用透射电镜验证纳米颗粒的合成；
2)制备NPs-Ate抗体荧光/磁共振双模态探针：选择聚乙二醇(NHS-PEG2000-COOH)将步骤1)制备的GdDTPA-HSA@ICG纳米颗粒与靶向元件阿特珠单抗(Atezoilzumab)连接，利用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化，得到靶向肿瘤的所述荧光/磁共振探针GdDTPA-HSA@ICG-Atezoilzumab(NPs-Ate)。
3.如权利要求1所述荧光/磁共振探针在动态评价乳腺癌免疫治疗效果中的应用。
4.如权利要求3所述应用，其特征在于其具体方法为：对肿瘤进行ICT单用及联用治疗(PD-1治疗加化疗/放疗)，利用治疗前后探针的荧光成像及磁共振成像监测组织中PD-L1表达变化，评估PD-L1变化与ICT愈后的关系。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210234058.7A CN114699540A (zh) | 2022-03-10 | 2022-03-10 | 一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210234058.7A CN114699540A (zh) | 2022-03-10 | 2022-03-10 | 一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114699540A true CN114699540A (zh) | 2022-07-05 |
Family
ID=82169470
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210234058.7A Pending CN114699540A (zh) | 2022-03-10 | 2022-03-10 | 一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114699540A (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347625A (zh) * | 2007-07-20 | 2009-01-21 | 上海交通大学医学院附属瑞金医院 | 核磁共振血栓靶向对比剂及其制备方法 |
-
2022
- 2022-03-10 CN CN202210234058.7A patent/CN114699540A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347625A (zh) * | 2007-07-20 | 2009-01-21 | 上海交通大学医学院附属瑞金医院 | 核磁共振血栓靶向对比剂及其制备方法 |
Non-Patent Citations (1)
| Title |
|---|
| QIAN CHEN等: "An albumin-based theranostic nano-agent for dual-modal imaging guided photothermal therapy to inhibit lymphatic metastasis of cancer post surgery", 《BIOMATERIALS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Stone et al. | Oxygen in human tumors: Correlations between methods of measurement and response to therapy: Summary of a workshop held November 19-20, 1992, at the National Cancer Institute, Bethesda, Maryland | |
| Li et al. | Preoperative detection and intraoperative visualization of brain tumors for more precise surgery: A new dual‐modality MRI and NIR nanoprobe | |
| Detappe et al. | Advanced multimodal nanoparticles delay tumor progression with clinical radiation therapy | |
| Jiang et al. | Molecular MRI differentiation between primary central nervous system lymphomas and high-grade gliomas using endogenous protein-based amide proton transfer MR imaging at 3 Tesla | |
| Thorwarth et al. | A kinetic model for dynamic [18F]-Fmiso PET data to analyse tumour hypoxia | |
| Wang et al. | In vivo albumin labeling and lymphatic imaging | |
| Hoskin et al. | Hypoxia in prostate cancer: correlation of BOLD-MRI with pimonidazole immunohistochemistry—initial observations | |
| JP4607859B2 (ja) | 蛍光分析物と連動して作動するインビボ蛍光センサ、システム及び関連方法 | |
| RU2114430C1 (ru) | Способ обнаружения и лечения рака легких | |
| Kishimoto et al. | Pulsed electron paramagnetic resonance imaging: applications in the studies of tumor physiology | |
| US20060204443A1 (en) | Methods for tumor treatment using dendrimer conjugates | |
| US20140154185A1 (en) | Use of non-labeled sugars and detection by mri for assessing tissue perfusion and metabolism | |
| Rickard et al. | Clinical and pre-clinical methods for quantifying tumor hypoxia | |
| Wang et al. | Ultrashort echo time (UTE) imaging of receptor targeted magnetic iron oxide nanoparticles in mouse tumor models | |
| Daimiel | Insights into hypoxia: non-invasive assessment through imaging modalities and its application in breast cancer | |
| CN106075469A (zh) | Gd3+诱导金纳米团簇自组装成金纳米颗粒的方法及应用 | |
| Bianchi et al. | In vivo MRI for effective non‐invasive detection and follow‐up of an orthotopic mouse model of lung cancer | |
| Prasad et al. | Optical and magnetic resonance imaging approaches for investigating the tumour microenvironment: state-of-the-art review and future trends | |
| CN104288786B (zh) | 基于近红外量子点的肿瘤靶向诊疗系统及其制备方法 | |
| Van de Ven et al. | Optical imaging of the breast | |
| EP3037107B1 (en) | Polymer nanoparticle composite and composition for mri imaging including same | |
| He et al. | Molecular MRI differentiation of VEGF receptor-2 levels in C6 and RG2 glioma models | |
| Alonzi et al. | Functional imaging in clinical oncology: magnetic resonance imaging-and computerised tomography-based techniques | |
| Lemasson et al. | Multiparametric MRI as an early biomarker of individual therapy effects during concomitant treatment of brain tumours | |
| CN114699540A (zh) | 一种动态评价乳腺癌免疫治疗效果的荧光/磁共振探针 |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220705 |