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Albumin-binding near-infrared fluorescent dye-maleimide conjugate

A maleimide conjugate, albumin-binding technology, which is applied to the near-infrared fluorescent dye maleimide conjugate, the application field of sentinel lymph node tumor metastasis diagnosis, and achieves stable binding and improved resolution. and imaging efficiency

Active Publication Date: 2021-12-07
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem solved by the present invention is to overcome the defects of the imaging method used in the existing sentinel lymph node navigation surgery, and provide an imaging material with good safety, strong lymph node targeting and high imaging resolution

Method used

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  • Albumin-binding near-infrared fluorescent dye-maleimide conjugate
  • Albumin-binding near-infrared fluorescent dye-maleimide conjugate
  • Albumin-binding near-infrared fluorescent dye-maleimide conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: the synthesis of near-infrared fluorescent dye-maleimide conjugate (IR-Mal)

[0048] IR820 (100mg, 0.118mmol) and 6-aminocaproic acid (45mg, 0.353mmol) were dissolved in 3.3ml of anhydrous DMF at a ratio of 1:3, triethylamine (67μl, 0.473mmol) was added, and nitrogen protection was carried out. The reaction system was heated to 85° C., and stirred for 3 h in the dark, and the reactant was observed to change from green to blue. The solvent was removed by rotary evaporation under reduced pressure, and the intermediate product (IR820-COOH) was separated by column chromatography. Take 84mg intermediate product IR820-COOH, 40.6mg 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 35.3μl N,N -Diisopropylethylamine (DIPEA) was dissolved in 20ml of anhydrous DMF, and after stirring at room temperature for 30min, N-6-aminomaleimide hydrochloride dissolved in 5ml of anhydrous DMF in advance was added to the reaction solution 42 mg of s...

Embodiment 2

[0051] Embodiment 2: In vitro binding experiment of near-infrared fluorescent dye-maleimide conjugate (IR-Mal) and bovine serum albumin

[0052] An appropriate amount of IR-Mal was weighed and added to bovine serum albumin (BSA) pH 7.4 phosphate buffer so that the concentrations of the conjugate and BSA were 81 μM and 35 μM, respectively. Then the mixed solution was incubated in a constant temperature shaker at 37° C., and samples were taken after 6 hours, and the combination was examined by 10% SDS-polyacrylamide gel (SDS-PAGE). In order to verify that the combination of IR-Mal and albumin is due to the maleimide group and the free sulfhydryl group of albumin 34-cysteine, another set of controls was set up, adding excess 6- Maleimide caproic acid was incubated for 12 hours to block the free sulfhydryl groups of albumin in advance, and then an equal amount of IR-Mal was added to perform in vitro binding with the same steps as above. After electrophoresis, the peeled gel plate...

Embodiment 3

[0055] Example 3: Research on protein binding stability, protein complex lyophilization stability and photostability of near-infrared fluorescent dye-maleimide conjugate (IR-Mal)

[0056] After IR-Mal is covalently bound to albumin in vivo, albumin needs to maintain its biological activity to exert its lymphatic targeting function, so the protein stability after covalent binding was investigated. Firstly, 180mg of BSA was dissolved in 3ml of 300μM IR-Mal pH7.4 phosphate buffer solution, after mixing, the mixture was placed in a 37°C constant temperature shaker and incubated for 18h, and then the mixed solution was lyophilized to obtain the BSA-IR-Mal complex freeze-dried powder. Take an appropriate amount of BSA and BSA-IR-Mal complex freeze-dried powder, dissolve in pH 7.4 phosphate buffer, so that the protein concentration is 0.5mg / ml, compare the protein in the two solutions with a circular dichroism spectrometer (CD). secondary structure.

[0057] We compared the fluores...

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Abstract

The invention belongs to the technical field of medicine, and relates to an albumin-binding near-infrared fluorescent dye-maleimide conjugate. The albumin-binding near-infrared fluorescent dye-maleimide conjugate specifically uses a maleimide-containing group to replace the chlorine in the conjugated chain of the cyanine dye, through the intramolecular charge transfer effect Its optical properties have been modified so that it can be used as a blue biological dye. Secondly, the maleimide group in the conjugate can be covalently bonded to the free sulfhydryl group of cysteine ​​34 on plasma albumin through Michael addition reaction, thus having excellent lymph node targeting. This near-infrared fluorescent bioimaging agent overcomes the shortcomings of the imaging method used in clinical sentinel lymph node navigation surgery, and is safe, reliable, accurate in positioning, and high in imaging efficiency. As a whole, it can realize the dual imaging function of lymph node positioning in vivo.

Description

technical field [0001] The invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, including designing and synthesizing a near-infrared fluorescent dye maleimide conjugate that can covalently bind albumin in vivo and target lymph nodes by using endogenous plasma albumin as a carrier , and its application in the diagnosis of sentinel lymph node tumor metastasis. Background technique [0002] Cancer is a serious threat to the health of all human beings. About 90% of cancer-related deaths are caused by cancer metastasis, among which lymphatic metastasis is a main way of tumor metastasis. Therefore, the discovery and diagnosis of metastatic lymph nodes is very important for patients. . Sentinel lymph node is the first checkpoint for tumor metastasis, and sentinel lymph node navigation (SNNS) is a commonly used clinical staging method for early breast cancer lymph node metastasis. The traditional lymph node navigation methods are bl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00
CPCA61K49/0002A61K49/0034A61K49/0056A61K49/006
Inventor 孙进张文娟何仲贵宋司晨
Owner SHENYANG PHARMA UNIVERSITY
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