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Design, synthesis and application of tumor-targeted near infrared fluorescence imaging agent

A fluorescent imaging agent and tumor-targeting technology, applied in the field of precise resection surgery, can solve the problem of low tumor specificity and achieve good specificity and broad application prospects

Active Publication Date: 2018-11-02
WUHAN KDWS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the shortcomings of low tumor specificity in traditional ICG imaging, the present invention provides a tumor-targeting near-infrared fluorescent imaging agent ICG-OSu-(PEG4)n-TMTP1, which is based on the original tumor-targeting polypeptide TMTP1 (NVVRQ ) on the basis of design and modification, and obtained a fluorescent imaging agent with the structural formula ICG-OSu-(PEG)n-G(CGNVVRQGC), which is not only used for tumor diagnosis, but also for guiding lesions in fluorescent laparoscopic surgery Precise resection of cervical fluorescein colposcopy to guide cervical biopsy sampling

Method used

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  • Design, synthesis and application of tumor-targeted near infrared fluorescence imaging agent
  • Design, synthesis and application of tumor-targeted near infrared fluorescence imaging agent
  • Design, synthesis and application of tumor-targeted near infrared fluorescence imaging agent

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Preparation of ICG-OSu-PEG4-TMTP1

[0031] see figure 1 , the preparation process of ICG-OSu-PEG4-TMTP1 is as follows: 1) use the solid phase carrier to synthesize the polypeptide GCGNVVRQGC according to the designed amino acid sequence, and covalently link it to PEG4; 2) remove the polypeptide from the resin, and use MEOH / I 2 Oxidation, the polypeptide sequence is bridged by a disulfide bond to form a ring, that is, PEG4-G (CGNVVRQGC), and then separated, purified, and freeze-dried; 3) The product obtained in step 2) is mixed with ICG-Osu at a ratio of 1:1 , dissolved in DMF, then added 2 times the volume of DIEA, and reacted for 10 minutes. After the reaction was identified by LCMS, it was separated, purified, and freeze-dried to obtain the imaging agent ICG-OSu-PEG4-TMTP1 of the present invention. The synthesis process was entrusted to Shanghai WuXi AppTec New Drug Development Co., Ltd.

[0032] see figure 2 , figure 2 a and 2b are detected by liquid ...

Embodiment 2

[0033] Embodiment 2 Cytotoxicity test:

[0034] 1. Cell Culture

[0035] The mouse breast cancer cell line 4T1 (purchased from ATCC cell bank in the United States) was cultured with RPMI-1640 medium (Gibco, ThermoFish Company, USA) containing 10% fetal bovine serum by volume fraction, and the cervical cancer cell lines HeLa and SiHa (purchased from the U.S. ATCC cell bank) and normal skin keratinized epithelial cells (purchased from China CCTCC cell bank) were cultured in DMEM medium (Gibco, ThermoFish Company, USA) containing 10% fetal bovine serum by volume fraction, and cervical intraepithelial neoplasia cell line S12 (Gifted by Professor Kenneth Raj, UK) with a volume fraction of 5% fetal bovine serum, 5 μg / ml insulin (Sigma-Aldrich, USA), 8.4ng / ml cholera toxin (Sigma-Aldrich, USA), 24.3 μg / ml glandular Purine (Sigma-Aldrich, USA), 0.5 μg / ml hydrocortisone (Sigma-Aldrich, USA) and 10 ng / ml endothelial factor (EGF, Peprotech) in F12 and DF12 medium (Gibco, ThermoFish Comp...

Embodiment 3

[0039] Example 3 Experiment of tumor cell binding efficiency under different PEG numbers

[0040] The imaging agents with different numbers of PEG were synthesized according to the method shown in Example 1, taking n=2, 4, 6, 8, 10, 20 respectively. Cultivate 4T1 cells and HeLa cells as shown in Example 2, and 4T1 and HeLa cells were prepared according to the ratio of 1*10 5 Plant a 24-well plate at a density of one per well. After overnight incubation, add 500ul of 1μM ICG and ICG-OSu-(PEG)n-TMTP1 to each well, and add PBS to the well as a blank control. After incubation on ice for 20 minutes, use Wash with cold PBS 3 times, and place under a small animal in vivo imager to collect fluorescence pictures of the well plate. Then, the fluorescence value of each well was quantitatively analyzed by the ROI tool of the IVIS imaging software.

[0041] For experimental results, see Figure 4 , Figure 4 a is the fluorescence image of 4T1 cells incubated with different PEG numbers ...

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Abstract

The invention relates to design, synthesis and application of a tumor-targeted near infrared fluorescent imaging agent. The structural formula of the tumor-targeted near infrared fluorescent imaging agent is ICG-OSu-(PEG)n-G(CGNVVRQGC), wherein ICG is a near infrared fluorescent imaging agent, namely indocyanine green, ICG-OSu is sulfoindocyanine green activating lipid, and is an amino reactive derivative of ICG, G(CGNVVRQGC) is tumor-targeted cyclic polypeptide TMTP1 and is carboxyl reactive, and the two are bridged by polyethylene glycol (PEG), wherein n is an integer of 2 to 20. The novel tumor-targeted molecular imaging agent provided by the invention greatly improves the specificity of ICG in imaging cervical cancer, breast cancer focuses and lymph node metastasis, and provides a goodindication for clinical diagnosis of cervical cancer and breast cancer, and removal of tumor lymph nodes by fluorescent endoscopy.

Description

technical field [0001] The invention relates to the technical field of medical imaging imaging, in particular to the precise resection of malignant tumors under fluorescent laparoscopy. Background technique [0002] Malignant tumors have become the primary disease that threatens human health. In 2010, one out of every five deaths of Chinese residents died of cancer. It is estimated that there will be 20 million new cancer cases every year by 2020, and the death toll of cancer patients will Break through 10 million people. Surgical resection is still the main treatment method for patients with solid tumors, supplemented by chemotherapy, radiotherapy and molecular targeted therapy. The key to the treatment of cancer patients is early detection and early intervention. Imaging examinations CT / MR / PET / B-ultrasound and laboratory examinations have become the basis for clinicians to diagnose early tumors, but the specificity and sensitivity of existing diagnostic methods are still...

Claims

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Application Information

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IPC IPC(8): A61K49/00
CPCA61K49/0034A61K49/0054A61K49/0056
Inventor 马丁奚玲李飞周颖
Owner WUHAN KDWS BIOLOGICAL TECH CO LTD
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