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Construction method of living cell probe based on neutrophilic granulocyte

A neutrophil and construction method technology, applied in the field of neutrophil-based living cell probe construction, can solve the problem of affecting neutrophil activity and biological characteristics, affecting high T1 relaxation rate, and tumor delivery ability Damage and other problems, to achieve the effect of simple nano-construction method, little impact on cell physiological functions, and easy access

Inactive Publication Date: 2019-12-20
NANTONG TUMOR HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, delivery of contrast agents such as Gd by internalization may be limited because Gd3 + It is not easy to exchange with free water in the cell, thus affecting the Gd sequestered on the protein nanoparticles 3+ The high T1 relaxation rate
In addition, intracellular delivery of imaging agents may potentially affect neutrophil viability and biology, resulting in impaired tumor-specific delivery

Method used

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  • Construction method of living cell probe based on neutrophilic granulocyte
  • Construction method of living cell probe based on neutrophilic granulocyte
  • Construction method of living cell probe based on neutrophilic granulocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 NEs probe construction

[0043] 1. FITC-labeled BSA molecule

[0044] Dissolve in 10mg.mL -1 NaHCO 3 The BSA protein in the solution was mixed with FITC at a mass ratio of 1:40, stirred at a constant speed for at least 5 hours to fully react; then purified by ultrafiltration to obtain FITC-BSA.

[0045] 2. FITC-BSA reduction

[0046] The FITC-BSA protein solution was mixed with the reducing agent DTT at a molar ratio of 1:16, and was continuously stirred with 2% SDS at 90°C for 2 hours for the reduction reaction to fully expose the free sulfhydryl groups; then use a concentration of 0.1mg.mL -1 , MES solution with a pH of 4.4 was diluted to prepare a concentration of 1mg.mL -1 working solution.

[0047] 3. Synthesis of Gd@BSA-FITC nanoparticles (nanoparticles, NPs)

[0048] The above working solution was placed in a 24-well plate (0.5 mL / well) and incubated at 130 rpm at 37°C for 5 hours; subsequently, GdCl was added 3 solution (15 μL / well, 2 mM), after...

Embodiment 2

[0061] Example 2. Effect of NEs probes on the performance of neutrophils

[0062] To verify the effect of this cell surface engineering method on the viability, morphology, and membrane protein markers of neutrophils. Such as Figure 4 As shown in A, Gd@BSA NPs exhibited low cytotoxicity; subsequent apoptosis assays with NEs probes revealed negligible difference in the cellular viability of the modified neutrophils compared with pure neutrophils, indicating that this The cell surface engineering method in the study has better cell compatibility ( Figure 4 B); The morphology of neutrophils conjugated with Gd@BSA NPs did not change significantly ( Figure 4 C).

[0063] CD11b is a neutrophil-specific surface protein that regulates neutrophil adhesion and migration. Therefore, changes in CD11b expression in NPs-modified neutrophils were assessed with reference to naive neutrophils. Flow cytometry analysis results ( Figure 4 D) shows negligible changes in CD11b expression ...

Embodiment 3

[0064] Embodiment 3. Chemotactic ability of NEs probe in mice

[0065]In order to meet the requirements of practical applications, the present invention also investigates whether the migration ability of NEs probes is retained in vivo. Inspired by the process of collecting neutrophils from the peritoneal cavity, mice injected with NEs into the tail vein were given sterile thioglycollate medium intraperitoneally, and the peritoneal lavage fluid was collected 6 hours later to purify the neutrophils.

[0066] Flow cytometry analysis revealed that the obtained neutrophils were divided into two populations ( Figure 5 A), the lower left cell population does not show fluorescence, which is the autologous neutrophils of the mouse, while the upper right cell population shows a fluorescent signal, which is the NEs probe (labeled with bodipy) administered in the tail vein. The above results indicated that the NEs probe retained the physiological function of neutrophils and could migrat...

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Abstract

The invention relates to the technical field of biomedical science detection, and discloses a construction method of a living cell probe based on neutrophilic granulocyte. The construction method comprises the steps of: A, performing BSA reduction; B, synthesizing Gd loaded BSA nanoparticles; C, labeling the Gd loaded BSA nanoparticles with Bodipy; and D, constructing a neutrophilic granulocyte probe: firstly with PBS as a medium, adding DTNB to a product solution in the step C, performing interaction at room temperature for a certain time to finish activation of BSA nanoparticles, after ultrafiltration and purification are performed, diluting the activated BSA nanoparticles to 0.1mg mL<-1> with an RPMI 1640 culture medium without FBS, then performing incubation with 1.25*10<6>cells.mL<-1>of neutrophilic granulocyte at room temperature, and then performing washing with ice-cold PBS so as to obtain the neutrophilic granulocyte probe.

Description

technical field [0001] The invention relates to the technical field of biomedical detection, and relates to the construction of live cell probes, in particular to a neutrophil-based live cell probe construction method. Background technique [0002] Timely and accurate identification of tumor lesions is an important prerequisite for clinical cancer intervention and the goal of tumor imaging diagnosis. Advances in nanoparticle-based molecular imaging have greatly improved the sensitivity and specificity of tumor detection. For example, inspired by biomineralization, previous studies successfully constructed chelated gadolinium ions (Gd 3+ ) bovine serum albumin (BSA) nanoparticles, which have obvious advantages over commercial gadolinium contrast agent (DTPA) in magnetic resonance imaging (MRI) detection of tumors. Some reports have made further studies on Gd-loaded nanoparticles, especially modifying the ligands of overexpressed cancer cell receptors to endow them with the ...

Claims

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Application Information

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IPC IPC(8): A61K49/18A61K49/14A61K49/08
CPCA61K49/08A61K49/143A61K49/1869
Inventor 冯峰邱钱赛温亚
Owner NANTONG TUMOR HOSPITAL
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