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Preparation method and application of manganese ion-CpG oligonucleotide nanoparticles

An oligonucleotide and nanoparticle technology, applied in the field of biomedicine, can solve the problems of poor treatment effect, adverse side effects, limited concentration, etc., and achieve the effect of good application prospect, growth inhibition and proliferation ability enhancement.

Inactive Publication Date: 2021-09-03
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complexity of the tumor microenvironment, most functional agents (such as small molecule chemotherapeutic drugs) are delivered to tumor cells at a limited concentration, and the therapeutic effect is not good, and even adverse side effects and toxicity problems occur.

Method used

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  • Preparation method and application of manganese ion-CpG oligonucleotide nanoparticles
  • Preparation method and application of manganese ion-CpG oligonucleotide nanoparticles
  • Preparation method and application of manganese ion-CpG oligonucleotide nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] This embodiment provides a method for preparing manganese ion-CpG oligonucleotide nanoparticles, comprising the following steps:

[0026] S1, dissolve anhydrous manganese chloride with deionized water to obtain 100mg / ml manganese chloride solution for use; the transmission electron microscope image of manganese chloride is as follows figure 1 Shown in A.

[0027] S2, dissolving CpG oligodeoxynucleotide (CpG ODN) with deionized water to obtain a 2.5 mg / ml CpG ODN solution for use; the transmission electron microscope image of CpG oligonucleotide is as follows figure 1 Shown in B.

[0028] In this example, the CpG oligodeoxynucleotide was synthesized by Suzhou Synbio Biotechnology Co., Ltd., and the sequence of the CpG oligodeoxynucleotide is shown in Table 1.

[0029] Table 1

[0030] Primer name Sequence(5'→3') CpG ODN 1826 T*C*C*A*T**G*A*C*G*T*T*C*C*T*G*A*C*G*T*T

[0031] S3. Mix the manganese chloride solution prepared in step S1 and the CpG...

Embodiment 2

[0034] In this example, the anti-tumor effect of the manganese ion-CpG oligonucleotide nanoparticles prepared in Example 1 is studied through experiments, and the specific operation process is as follows:

[0035] LLC (mouse lung cancer cells, 5*10 5 After that, the manganese ion-CpG oligonucleotide nanoparticles prepared in Example 1 were immunized by nasal drops to mice. The changes of tumor volume in mice were dynamically monitored, and the results showed that compared with mice immunized with Mncl2 and CpG ODN nasal drops, the tumor volume in mice receiving nasal drops of manganese ion-CpG oligonucleotide nanoparticles smaller, such as figure 2 , Figure 4 shown. After 25 days of inoculation of tumor cells, the tumor tissue was taken out and weighed. It was also found that the tumors in mice receiving intranasal immunization with manganese ion-CpG oligonucleotide nanoparticles were lighter, as shown in image 3 shown. This shows that the manganese ion-CpG oligonucleo...

Embodiment 3

[0037] In this embodiment, the influence of manganese ion-CpG oligonucleotide nanoparticles on immune cells in vivo is studied experimentally. The specific operation process is as follows:

[0038]The C57 / B6 mice inoculated with LLC were killed 25 days after inoculation, and the spleen was removed and placed in a 6-well plate containing type IV collagenase; the spleen was cut into pieces with scissors and placed in a 37°C cell culture incubator for digestion for 40 minutes; After 40 minutes, add MACSbuffer to stop the digestion; then put a 40μm screen on the centrifuge tube, transfer the tissue and all liquids through the screen to the centrifuge tube, and use the flat head of the syringe needle to grind the tissue continuously during the grinding process. Wash repeatedly with MACS buffer. Then put the centrifuge tube into a centrifuge at 1800r / min, centrifuge at 4°C for 5min; discard the supernatant after centrifugation, then add 2ml of red blood cell lysate, and let it stand...

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Abstract

The invention discloses a preparation method of manganese ion-CpG oligonucleotide nanoparticles. The preparation method comprises the following steps of dissolving anhydrous manganese chloride with deionized water to obtain a manganese chloride solution for later use; dissolving CpG oligodeoxynucleotide (CpG ODN) with deionized water to obtain a CpG ODN solution for later use; and mixing the prepared manganese chloride solution and the prepared CpG ODN solution in sterile normal saline, oscillating and uniformly mixing to obtain the manganese ion-CpG oligonucleotide nanoparticles. Compared with single use of manganese chloride or CpG ODN, the manganese ion-CpG oligonucleotide nanoparticles prepared by the method can significantly promote tumor cell necrosis, inhibit tumor cell growth, increase the number of NKT cells in the spleen, significantly enhance the proliferation ability of CD4 + T and CD8 + T cells in the spleen, and show a good anti-tumor effect. In addition, the nanoparticles are relatively low in raw material cost, simple in preparation process and relatively high in biological safety. Good application prospects are realized in tumor immunotherapy.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method and application of manganese ion-CpG oligonucleotide nanoparticles. Background technique [0002] Manganese is one of the trace elements necessary for the body. The latest research has found that manganese ions can activate various links in the innate immune system by transmitting signals of virus invasion, and assist the body to resist virus invasion. It plays the role of "herald" in the body's innate immune response. According to the study, mice lacking manganese ions have significantly weakened resistance to tumors, which proves that manganese plays an important role in the immune surveillance of tumors. [0003] CpG ODN (CpG oligonucleotide, CpG oligodeoxynucleotide) is a synthetic oligodeoxynucleotide (ODN) containing unmethylated cytosine guanine dinucleotide (CpG), which can activate a variety of immune cells , promote tumor antigen presentation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K33/32A61K31/7088A61K9/14A61P35/00
CPCA61K33/32A61K31/7088A61K9/14A61P35/00A61K2300/00
Inventor 唐华张善龙康莉王美祥陈君兰王雪梅
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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