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Preparation and application of nano-micelle imaging agent for cervical cancer sentinel node

A sentinel lymph node and nano-micelle technology, which is applied in the preparation of in vivo experiments, drug delivery, emulsion delivery, etc., can solve the problems of low specificity of lymph node imaging agents, inability to observe in real time, dye retention, etc., to achieve Maintain colloidal stability, reduce quenching, and improve stability

Active Publication Date: 2020-02-14
WUHAN KDWS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have some disadvantages: after the placenta blue dye is injected, the dye stays at the injection site for a long time
[0009] The purpose of the present invention is to provide a targeted nano-fluorescence imaging agent that can specifically identify and diagnose sentinel lymph nodes of cervical cancer metastasis in real time during surgery, thereby overcoming the low specificity of existing lymph node imaging agents, inconvenient operation, and inability to real-time Observation and other issues, in order to play an efficient and specific diagnostic role in clinical treatment

Method used

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  • Preparation and application of nano-micelle imaging agent for cervical cancer sentinel node
  • Preparation and application of nano-micelle imaging agent for cervical cancer sentinel node
  • Preparation and application of nano-micelle imaging agent for cervical cancer sentinel node

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of targeted peptide-modified fluorescent nanoimaging agent

[0056] 1. Synthetic peptide TMTP1

[0057] Use peptide solid-phase synthesis technology to synthesize cyclic polypeptide c (NVVRQC) head-to-tail amide bonds to form a ring. The synthesis process is completed by Wuhan Baiyixin Biotechnology Co., Ltd. The purity of the synthesized cyclic polypeptide c (NVVRQC) is 98%. RP-HPLC and MS technique detection, its molecular weight is 699.2, and quality is 20mg.

[0058] 2. Preparation of fluorescent nanoimaging agent

[0059] (1) Synthesis of MA-PEG-PLGA copolymer: first activate MA (maleimide butyric acid) in DCM (dichloromethane) with EDC and NHS for 5 hours, and then add polyethylene glycol diols of different molecular weights Amines (2KDa, 3KDa, 4KDa and 10KDa) were reacted with triethylamine for 48h to obtain a PEG solution with MA end groups; PLGA (50 / 50 20000) was activated in DCM with EDC and NHS for 5h to obtain the activated product of...

Embodiment 2

[0069] Example 2 adjusts the ratio of materials for micelles preparation and targeting peptide to improve the physicochemical properties and targeting of nanoparticles.

[0070] (1) According to the method for preparing nanoparticles in 2(2) of Example 1, "1mg cyclic TMTP1 polypeptide and the above 20mg MA-PEG-PLGA (mass ratio 1:20) are dissolved in 1ml DMF solution", we will 1mg The cyclic TMTP1 polypeptide and the above 20mg MA-PEG-PLGA (mass ratio 1:20) are adjusted to the following ratio:

[0071] A.10mgMA-PEG-PLGA+10mgPEG(5000)-PLGA(50 / 50 20000)+1mg TMTP1;

[0072] B.10mg MA-PEG-PLGA+10mgPLGA(50 / 50 20000)+1mg TMTP1;

[0073] C.15mg MA-PEG-PLGA+5mg PLGA (50 / 50 20000)+1mgTMTP1;

[0074] D.15mg MA-PEG-PLGA+5mg PEG (5000)-PLGA (50 / 50 20000)+1mg TMTP1. Wherein MA-PEG-PLGA is the copolymer synthesized by the 2 (1) method of embodiment 1, PEG (5000 )-PLGA (50 / 50 20000) is monomethoxypolyethylene glycol poly(lactic-co-glycolic acid) copolymer, purchased from Jinan Daigang Biol...

Embodiment 3

[0080] Example 3 Establishment of footpad lymph node metastasis model and near-infrared fluorescence imaging

[0081] 1 Prepare tumor cells

[0082] The cervical cancer cell line HeLa was cultured in DMEM medium containing 10% serum (37°C, 5% CO2 incubator), and the luciferase gene luc expression vector was constructed through the Lentivirus system, and Hela was stably transfected with puromycin drug After 3 days of screening, hela-luc cells stably transfected with luc were obtained. Amplify hela-luc cells in a culture flask, digest with 0.25% trypsin when the cells grow to 80%, stop the digestion with complete medium, collect the cells into a 15mL centrifuge tube, centrifuge at 800rpm for 5 minutes, discard the upper After clearing, the cells were resuspended in PBS, centrifuged again, the supernatant was discarded, resuspended in PBS, and the cell density was calculated by cell counting.

[0083] 2 Establishment of tumor model

[0084] Establishment of nude mouse footpad ...

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Abstract

The invention relates to preparation and application of a nano-micelle imaging agent for a cervical cancer sentinel node. A nanometer imaging agent high polymer PEG-PLGA is used as a carrier, micelleparticles are formed through polymerization, indocyanine green is loaded inside, TMTP1 targeting peptide is modified on the surface of a micelle, and maleic amide connected with a surface hydrophilicgroup PEG end of the micelle is connected with a sulfhydryl group of TMTP1 with ring formation by amido bonds at the head and the tail. The near infrared fluorescence nano-micelle diagnosis reagent provided by the invention can image the cervical cancer sentinel node in real time, can distinguish whether tumor cell metastasis occurs or not, and offers the assistance for the imaging diagnosis of intraoperative lymph nodes of cervical cancer in clinic in the future and the guidance of individualized treatment of tumor patients.

Description

technical field [0001] The invention relates to the preparation and application of a tumor-targeting nano-medicine, in particular to a nano-micelle imaging agent for sentinel lymph nodes of cervical cancer. Background technique [0002] Cervical cancer is the second most common gynecological cancer worldwide. The annual number of new cases in my country is close to 131,500, and the number of patients who die from cervical cancer is 33,000. Metastasis is the main cause of death in cervical cancer patients. The 2017 guidelines recommend that early-stage cervical cancer (stages IA and IB) mainly rely on surgical resection, and radical hysterectomy plus bilateral pelvic lymphadenectomy (with or without sentinel lymph node visualization) is a non-fertile requirement for patients with stages IA2, IB, and IIA. preferred treatment. Radical hysterectomy removes more parametrial tissues than simple hysterectomy, including part of the cardinal ligament, uterosacral ligament, upper v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K49/00C08G81/00
CPCA61K49/0082A61K49/0056A61K49/0034C08G81/00
Inventor 马丁奚玲罗丹枫魏军成陈刚魏睿蒋桂英
Owner WUHAN KDWS BIOLOGICAL TECH CO LTD
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