Targeting CpG nucleic acid drug delivery system and preparation method thereof

A technology for delivering systems and compounds, applied in the field of biomedicine, can solve problems such as low efficiency and low stability, and achieve the effects of simple preparation process, good biocompatibility, and high-efficiency biocompatibility

Pending Publication Date: 2021-08-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since CpG ODN is essentially a deoxynucleotide with a phosphodiester bond skeleton structure, it is easily degraded by nucleases in bloo...

Method used

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  • Targeting CpG nucleic acid drug delivery system and preparation method thereof
  • Targeting CpG nucleic acid drug delivery system and preparation method thereof
  • Targeting CpG nucleic acid drug delivery system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of targeted CpG nucleic acid drug delivery carrier:

[0038] Add DMSO to CaH 2 Stir and dry overnight, weigh yeast β-glucan, N,N-carbonyldiimidazole (CDI), and diethylenetriamine respectively according to a certain mass ratio, and dissolve them completely in dried anhydrous dimethyl sulfoxide, Prepare 12mL of β-glucan solution with a concentration of 13.5mg / mL and 10mL of CDI solution with a concentration of 50mg / mL for later use;

[0039] Stir 12mL of β-glucan solution at room temperature under nitrogen, and add NaH solid with a purity of 60% under nitrogen protection, according to the mass ratio of NaH:β-glucan as 1:5, 1:10, 1:20, Add NaH at 1:30 respectively, and react for 0.5~1h. Then add 10mL of the prepared CDI solution, under the protection of nitrogen, stir at room temperature for 1-4h, then add dropwise into 120ml of absolute ethanol, precipitate out, centrifuge, and then wash with absolute ethanol for 6-8 times, the washing is complete ...

Embodiment 2

[0051] Example 2 Preparation of carrier-loaded CpG nucleic acid drug:

[0052] Take 7mg of NH 2 -Glucan was dissolved in 10mL deionized water, stirred at 1000-3000rmp at room temperature to fully dissolve, it can be assisted by ultrasonic cleaning and dissolution, the power is 90W, ultrasonic cleaning 0.5-1h, and the concentration of 0.7mg / mL NH was obtained 2 -Glucan solution. Immediately, the resulting NH 2 -Glucan solution and CpG ODN with a concentration of 50-100ng / μL were mixed according to the mass ratio (1:2, 1:5, 1:10, 1:20, 1:30), and placed on a high-speed shaker at 100r / Shake for 1-2 hours in min, and load CpG ODN through electrostatic interaction to obtain the CpG ODN delivery system, which is stored at 4°C.

[0053] Figure 5 The retention experiment of the agarose gel electrophoresis of the described carrier loaded CpG ODN prepared for embodiment 2

[0054] The carrier and CpG ODN were mixed according to the above-mentioned different mass ratios, and the c...

Embodiment 3

[0057] In this example, RAW264.7 was used as the host cell to evaluate the cell uptake efficiency of the carrier in Example 1. According to the method provided in Example 2, NH was obtained at a mass ratio of 1:10. 2 -Glu / CpG ODNs complex, which was subjected to cell uptake experiments. Determination of cellular response to NH by flow cytometry 2 - Uptake of Glu / CpG ODNs complexes and PEI / CpG ODNs complexes.

[0058] Prepare NH respectively according to the method in Example 2 2 -Glucan, PEI and the complex of fluorescent FAM-CpG ODN with a mass ratio of 1:10, and the resulting sample was allowed to stand at room temperature for 1 h. Dilute with DMEM complete medium to a carrier concentration of 30 μg / mL, and set FAM-CpG ODN dissolved in DMEM complete medium as a control group. Add 500 μL RAW264.7 cell suspension (1×10 5 cell / well), the culture plate was pre-cultured overnight in an incubator to allow the cells to adhere to the wall (37°C, 5% CO 2 ); after 24 hours, add 3...

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Abstract

The invention discloses a targeting CpG nucleic acid drug delivery system and a preparation method thereof, and belongs to the field of biological medicine. The CpG nucleic acid drug delivery system comprises a carrier and a CpG nucleic acid drug, a carrier is made into water-soluble ammonification yeast beta-1,3-D-glucan through a specific mode, the surface of the water-soluble ammonification yeast beta-1,3-D-glucan has high positive charges, and the water-soluble ammonification yeast beta-1,3-D-glucan can be tightly combined with CpG through electrostatic interaction. The carrier is low in toxicity and good in biocompatibility, and can be combined with a specific recognition receptor Dectin-1 to target macrophages; and flow cytometry and laser confocal scanning microscope analysis show that the transfection efficiency is high. The low-toxicity CpG nucleic acid drug delivery system with good biocompatibility can realize targeted delivery of CpG nucleic acid drugs and improve the cell uptake efficiency.

Description

technical field [0001] The invention relates to a targeted CpG nucleic acid drug delivery system and a preparation method thereof, belonging to the field of biomedicine. Background technique [0002] CpG oligonucleotide (hereinafter referred to as CpG ODN) is a kind of DNA rich in unmethylated cytosine-guanine dinucleotide. Unmethylated CpG motifs widely exist in the genomic DNA of bacteria or viruses, and the frequency of occurrence is much higher than that of vertebrates, and 80% of the carbon atom at the 5th position of cytosine in vertebrates is methylated. This significant difference allows CpG ODN to be regarded as a "danger signal" by the mammalian immune system to initiate an immune response. CpG ODN can be recognized by Toll-like receptor 9 (hereinafter referred to as TLR9) of immune cells to promote the secretion of various cytokines and induce innate or adaptive immune responses. It can be used as an effective immunotherapeutic tool in anti-infection immunity, ca...

Claims

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Application Information

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IPC IPC(8): A61K47/61A61K47/69A61K31/711A61P37/02C08B37/02
CPCA61K47/61A61K47/6939A61K31/711A61P37/02C08B37/0024
Inventor 陈敬华张慧杰王志清胡成龙赖莉柳恒青
Owner JIANGNAN UNIV
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