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Kidney-targeting DNA nanometer raft-IL-33 as well as preparation method and application thereof

A kidney-targeted, SH-DNA technology, applied in the field of kidney-targeted DNA nano-raft-IL-33 and its preparation, can solve problems such as complexity, poor accumulation, poor cytokine targeting performance, etc. Achieve the effects of relieving pain, reducing drug costs, and improving drug targeting efficiency

Pending Publication Date: 2021-12-24
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, exosomes are primarily targeted to and enriched in the liver, leading to suboptimal accumulation of cytokine-encapsulated exosomes in the kidney
The technology of using genetic engineering and protein engineering to improve the targeting of cytokines is very complicated, and the targeting performance of the modified cytokines is not good

Method used

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  • Kidney-targeting DNA nanometer raft-IL-33 as well as preparation method and application thereof
  • Kidney-targeting DNA nanometer raft-IL-33 as well as preparation method and application thereof
  • Kidney-targeting DNA nanometer raft-IL-33 as well as preparation method and application thereof

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Experimental program
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preparation example Construction

[0037] The invention discloses a method for synthesizing cytokine drugs based on DNA nano rafts, which comprises the following steps:

[0038] (1) Preparation: Prepare DNA single strands (M 13mp 18DNA single strands, staple strands, capture strands), thiol-modified DNA (SH-DNA), recombinant interleukin 33, 3-(2-pyridyl dithio) N-Hydroxysuccinimide Propionate (SPDP), 1xTAE-Mg 2+ buffer.

[0039] (2) The preparation of DNA-IL-33 was separated and purified by ultrafiltration, and the preparation efficiency was characterized by ultraviolet-visible mass spectroscopy;

[0040] (3) Synthesis of DNA origami rafts, by PCR synthesizing a capture strand complementary to the DNA in step (1). It was separated and purified by ultrafiltration, and its morphology was characterized by atomic force microscopy (AFM).

[0041] (4) The assembly and synthesis of DNA nano-rafts, the DNA-IL-33 in step (1) and the DNA origami rafts in step (2) were mixed, assembled and synthesized DNA nano-rafts, s...

Embodiment 1

[0062] Example 1 Preparation of DNA-IL-33

[0063] Dissolve 100 μg of IL-33 powder in 50 μL of NaCl solution. React 50 μL of 6.6 μM IL-33 solution with 50-fold excess of SPDP in 1x PBS buffer (pH 8.5) for 2 hours. Excess SPDP was removed by Zeba spin desalting column (7KMWCO, 0.5 mL). Then, IL-33 was coupled to sulfhydryl-modified DNA strands (3-fold excess) in 1×PBS buffer (pH 7.4) for 8 hours at room temperature. The efficiency of HS-DNA modification of IL-33 was assessed by monitoring the net increase in UV-vis spectrum at 343 nm due to the generation of pyridine-2-thione (extinction coefficient: 8080 M-1 cm-1). In 1x PBS (pH=7.4), ultrafiltration (3000 g, 10 minutes) was performed 3 times with a 30 kD cut-off filter membrane (Amicon) to remove excess SH-DNA strands.

Embodiment 2

[0064] Example 2 Synthesis of DNA origami rafts

[0065] In 1x TAE-Mg 2+ In buffer, 10 nM of M 13mp 18 DNA single strand was mixed with 50-fold excess of staple strand and 100-fold excess of capture. The mixture was annealed from 95 °C to 4 °C.

[0066] Table 3 Annealing program from 95℃ to 4℃

[0067] temperature gradient 95℃ 2min 95-60℃ -0.1℃ / 12s 60℃ 12min 60-25℃ -0.1℃ / 12s 4℃ Keep

[0068] In 1xTAE-Mg 2+ Excess staple strands were removed by ultrafiltration (3000g, 10min) twice in buffer and once (3000g, 10min) in 1x PBS (pH=7.4) and capture chains, and were characterized by atomic force microscopy (AFM).

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Abstract

The invention relates to the field of biological medicines, in particular to a kidney-targeting DNA nanometer raft-IL-33 as well as a preparation method and application thereof. According to the kidney-targeting DNA nanometer raft-IL-33 as well as the preparation method and application thereof, a DNA nanometer technology is adopted to accurately and quantitatively load the IL-33 on a DNA nanometer raft, such that the drug use amount and the drug administration frequency of the IL-33 are reduced, the drug cost is reduced, the pain caused by multiple drug administration is reduced, the drug targeting efficiency is improved, and the side effect is reduced.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the kidney-targeted DNA nano-raft-IL-33 and its preparation method and application. Background technique [0002] Numerous studies have shown that the cytokine interleukin 33 (IL-33) has a positive role in the prevention of ischemic acute kidney injury (AKI) and renal protection. The IL-33-induced increase in ILC2 prompts ILC2 to produce large amounts of Th2 cytokines (IL-4 and IL-13), thereby suppressing the pro-inflammatory activity of the type 1 immune response (TNF-α, IL-1b, IL-6, CXCL1 and CXCL2 significantly decreased). In addition, IL-33-induced ILC2 directly promotes tubule repair by producing Areg to attenuate renal injury. IL-33 can also promote the increase of anti-inflammatory macrophages (M2). M2 produces anti-inflammatory cytokines and secretes HO-1 anti-inflammatory enzyme, thereby inhibiting inflammation and tissue damage. IL-33 has a positive preventive and protect...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K47/54A61K38/20A61P13/12B82Y5/00B82Y30/00B82Y40/00
CPCA61K38/20A61K47/549A61K47/6949A61P13/12B82Y5/00B82Y30/00B82Y40/00
Inventor 李威
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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