Accelerator capable of improving functionalization process of adoptive DCs (Dendritic Cells), as well as preparation method and application of accelerator

A dendritic cell and enhancer technology, which is applied in the field of enhancer and its preparation for improving the functionalization process of adoptive dendritic cells, can solve the problem of low response rate, inability to effectively promote adoptive DCs to secrete IL-12, etc. problems, to achieve the effect of good biological safety and increase the possibility of clinical promotion and application

Active Publication Date: 2015-11-25
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the biggest defect of this enhancer is that it cannot effectively promote the secretion of IL-12 by adoptive DCs, and a large number of literatures have proved that the activation of T cells by adoptive DCs largely depends on the high secretion of IL-12, which is also a recent The main reason for the low clinical response rate (10–15%) of several years of adoptive DCs therapy

Method used

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  • Accelerator capable of improving functionalization process of adoptive DCs (Dendritic Cells), as well as preparation method and application of accelerator
  • Accelerator capable of improving functionalization process of adoptive DCs (Dendritic Cells), as well as preparation method and application of accelerator
  • Accelerator capable of improving functionalization process of adoptive DCs (Dendritic Cells), as well as preparation method and application of accelerator

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, the preparation of spherical gold nanoparticles

[0064] Place a three-necked flask containing 100mL of 0.01wt% chloroauric acid solution in an oil bath with stirring and heating to 130°C, quickly add 0.4-3.0mL of 1wt% sodium citrate solution, and keep stirring at this temperature for half an hour, Obtain the colloidal solution, centrifuge and concentrate at 3000-12000rpm for 10-20min, discard the supernatant, and obtain spherical gold nanoparticles, such as figure 1 shown. from figure 1 It can be seen from the transmission electron microscope photos of spherical gold nanoparticles that the hydrodynamic radius of the spherical gold nanoparticles is in the range of 10nm-90nm (gold nanoparticles are abbreviated as "AuNP+radius", and A-E are the hydrodynamic radii of 15nm, 30nm, 40nm, 60nm and 80nm spherical gold nanoparticles), and the distribution is relatively uniform (the scale in the figure is 200nm). In industrial applications, the scale of preparati...

Embodiment 2

[0065] Embodiment 2, preparation of rod-shaped gold nanoparticles

[0066] Ⅰ. Add 0.25mL0.01mol / L chloroauric acid solution to 7.5mL0.1mol / L cetyltrimethylammonium bromide (CTAB) solution, mix well and then add 0.6mL0.01mol / L The freshly prepared sodium borohydride solution placed in an ice-water bath was inverted back and forth for 2 minutes to mix well to obtain a bright brown-yellow gold nanometer seed solution. The gold nanometer seed solution was aged in a water bath at 25° C. for 3 h.

[0067]Ⅱ. Add 4mL of 10mmol / L chloroauric acid solution, 0.6mL of 10mmol / L silver nitrate solution and 0.64mL of 0.01mol / L ascorbic acid solution to 95mL of 0.01mol / L CTAB solution in turn, and then add 0.1mL step Ⅰ Mix the aged gold nano seed solution, put it in a water bath at 25°C for 3 hours, and obtain a colloidal solution of rod-shaped gold nanoparticles (stock solution); centrifuge the stock solution at least twice at 8000 rpm, each time 10min, to remove excess CTAB, centrifuge an...

Embodiment 3

[0068] Embodiment three, the preparation of CpG-OVAp spherical nano-gold cocktail

[0069] 1. Functional modification of spherical gold nanoparticles by Toll-like receptor 9 (TLR9) pathway stimulator oligonucleotide single strand (CpG):

[0070] (1) The reducing agent dithiothreitol (DTT) performs dedisulfide bond pretreatment on the thiolated CpG sequence (5'-SH-AAAAA-Spacer9-tccatgacgttcctgacgtt-3'):

[0071] Prepare 160 μL of DTT solution with a DTT concentration of 0.125 mol / L in phosphate buffered saline (PBS) with pH=8.0, 0.18 mol / L, add 40 μL of 100 μmol / L CpG into the DTT solution, react at room temperature for 1 hour, and pass through a nucleic acid purification column (GE Healthcare Life Science, NAP TM -5columes) for desalination and other impurity removal treatments.

[0072] (2), CpG functional modification of spherical gold nanoparticles:

[0073] Add the reaction solution after the impurity removal treatment in step (1) to the spherical gold nanoparticles obt...

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Abstract

The invention discloses an accelerator capable of improving a functionalization process of adoptive DCs (Dendritic Cells), as well as a preparation method and application of the accelerator. Main ingredients of the accelerator comprise an immunologic stimulant-nanometer gold compound and an antigen-nanometer gold compound, which are obtained by modifying gold nanoparticles with an immunologic stimulant and an antigen respectively; or the main ingredients comprise an immunologic stimulant-antigen-nanometer gold compound obtained by jointly modifying the gold nanoparticles with the immunologic stimulant and the antigen. According to the accelerator, nanometer gold with the characteristics of high biocompatibility, adjustable surface physicochemical properties and the like is used as a nanometer targeting vector of adoptive DCs vaccines, and the shapes or the particle sizes of the nanometer material are screened or effectively combined for properties of different junctional complexes, so that the homing efficiency of lymphoid organs under the local or intravenous infusion of the adoptive DCs vaccines is greatly improved, and a new idea is provided for clinical screening in the future.

Description

technical field [0001] The invention relates to the field of nano-immunity technology, in particular to an accelerator capable of improving the functionalization process of adoptive dendritic cells and its preparation method and application. Background technique [0002] Dendritic cells (Dendritic cells, DCs) are currently found to be the most powerful highly specialized antigen-presenting cells (Antigenpresenting cells, APCs) that control a series of immune responses. Dendritic cells can mobilize various immune resistance mechanisms, such as CD8 + T cells, cytotoxic T cells, CD4 + Helper T cells, natural killer (NK) cells, and natural killer T (NKT) cells. Each of these lymphocytes has the role of recognizing and killing diseased cells, as well as releasing protective cytokines such as IFN-γ and CD4 T cells. DCs can stimulate the proliferation of T cells in vivo and in vitro, induce the generation of antigen-specific cytotoxic T lymphocytes (Cytotoxic Tlymphocyte, CTL), ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/385A61K47/02A61P35/00C12N5/0784
Inventor 詹林盛王小慧周欠欠张玉龙
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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