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Preparation method and application of alpha-GalCer nanoparticles

A nanoparticle and solution technology, applied in biochemical equipment and methods, microorganisms, blood/immune system cells, etc., can solve the problems of PET cytotoxicity and time-consuming, and achieve the effect of simple preparation process and short preparation time

Inactive Publication Date: 2020-06-30
诺莱生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the defects of long time-consuming preparation process of iNKT and cytotoxicity of PET in the prior art, the present invention provides a preparation method and application of α-galactosylceramide nanoparticles

Method used

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  • Preparation method and application of alpha-GalCer nanoparticles
  • Preparation method and application of alpha-GalCer nanoparticles
  • Preparation method and application of alpha-GalCer nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the preparation method of α-GalCer nanoparticle

[0034] 1. Solution preparation:

[0035] Preparation of PEI solution: dissolve PEI with a molecular weight of 1800 in the diluent so that the final concentration of PEI is 1 mg / ml; preparation of α-GalCer solution: dissolve α-GalCer in the diluent so that the final concentration of α-GalCer is 1 mg / ml.

[0036] 2. Preparation of α-GalCer nanoparticles: α-GalCer and PEI can form nanoparticles through electrostatic interaction. Put the prepared PEI solution in a round-bottomed centrifuge tube, then slowly drop into the same volume of α-GalCer solution to mix, while gently vortexing the test tube, after mixing well, let stand at room temperature for 10-25 hours to form α-GalCer nano particle mixture. Take 2 μl of the solution and apply it on the copper grid, and after natural drying, observe the morphology of the nanoparticles with an electron microscope, as shown in figure 1 As shown, the results show tha...

Embodiment 2

[0039] Embodiment 2: cytotoxicity test

[0040] Peripheral blood mononuclear cells were seeded on a 96-well plate at a cell density of 2×10 4 / well, cultured. PEI or nanoparticles are formulated into different mass concentration gradients in the culture medium. Aspirate the original culture medium in each well, add different concentrations of PEI culture medium, 0.2ml per well, and incubate with the cells for 72 hours, and detect the growth of the cells by the cck8 method. The cell survival rate at different time points after the test was used to infer the toxicity of PEI and α-GalCer nanoparticles to mononuclear cells, and the feasibility of their application to cell induction has been judged. Experimental results such as figure 2 It was shown that α-GalCer nanoparticles reduced the cytotoxicity of PEI, and the cell survival rate was over 90%.

Embodiment 3

[0041] Example 3: α-GalCer nanoparticles stimulate iNKT proliferation experiment

[0042] 1. Day 0

[0043] i. Collect peripheral blood, pour the collected peripheral blood directly into a sterile centrifuge tube for 30-40ml, balance the centrifuge tube, centrifuge at 3500rpm for 10min. At the end of the centrifugation, slowly collect the upper plasma with a pipette and put it into another new sterile centrifuge tube, and the remaining part is used in the subsequent step b. The collected plasma was inactivated in a water bath at 56°C for 30 minutes, and the supernatant was collected by centrifugation for later use.

[0044] j. Dilute the rest with the same volume of normal saline (room temperature).

[0045] k. Add 15ml Ficoll (room temperature) to a 50ml conical tube. The top of the Ficoll is slowly covered with 25ml of diluted blood mixture. Centrifuge at 400 x g for 30 minutes without brake at room temperature.

[0046] l. Carefully remove the interface buffy coat and ...

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Abstract

The invention discloses a preparation method and application of alpha-GalCer nanoparticles. The preparation method comprises the following steps: respectively preparing a polyethylenimine (PEI) solution and an alpha-galactosyl ceramide (alpha-GalCer) solution; preparing an alpha-GalCer nanoparticle mixed solution by using the PEI solution and the alpha-GalCer solution through electrostatic action;and performing drying to obtain the alpha-GalCer nanoparticles. The invention provides the method for preparing the alpha-GalCer nanoparticles by combining PEI and alpha-GalCer, wherein the preparedalpha-GalCer nanoparticles reduce cytotoxicity of the PEI. The prepared alpha-GalCer nanoparticles are used for amplification of iNKT to prepare Valpha24+iNKT cells. Compared with the prior art, the preparation method has a simple preparation process and shorter preparation time.

Description

technical field [0001] The invention belongs to the technical field of cellular immunotherapy, and in particular relates to a preparation method and application of alpha-galactosylceramide nanoparticles. Background technique [0002] α-Galactosylceramide (α-GalCer) was originally a glycolipid extracted from sponges off the coast of Okinawa, and then synthesized to produce analogues, such as the commercial product KRN7000. Studies have shown that α-GalCer can stimulate the proliferation of iNKT cells and has strong anti-tumor activity in vivo. In addition to α-GalCer, other glycosylceramides (such as α-glucosylceramide) with sphingosine glycosyl and 3,4-hydroxyl α-heteromeric conformation have been shown to stimulate the proliferation of mouse iNKT cells, But less efficient. Studies have demonstrated that in vivo administration of α-GalCer not only activates iNKT cells through a CD1d-restricted mechanism, induces NK activity and production of cytokines (such as IL-4, IL-12,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/30C12N2500/50
Inventor 杨继建吕彬
Owner 诺莱生物医学科技有限公司
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