Recombinant plasmids based on polyethylene glycol-polylactic acid glycolic acid-polylysine composite nanomaterials and their preparation and application

A technology of poly(lactic acid hydroxyethanol) and composite nanomaterials, which is applied in the field of medicine to achieve the effect of delaying progress and reducing apoptosis

Inactive Publication Date: 2019-12-10
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no literature report on the use of mPPP composite nanomaterials to carry recombinant plasmids for targeted therapy of DNP

Method used

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  • Recombinant plasmids based on polyethylene glycol-polylactic acid glycolic acid-polylysine composite nanomaterials and their preparation and application
  • Recombinant plasmids based on polyethylene glycol-polylactic acid glycolic acid-polylysine composite nanomaterials and their preparation and application
  • Recombinant plasmids based on polyethylene glycol-polylactic acid glycolic acid-polylysine composite nanomaterials and their preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of pSNAV2-CXCR3-pIRES-CXCL9

[0043] 1. Using the method of molecular cloning, the internal ribosome entry sites (internal ribosome entrysites, IRES) sequence was used to connect CXCR3 and CXCL9 to jointly clone into the rAAV expression plasmid pSNAV2.0, construct the recombinant plasmid pSNAV2-CXCR3-pIRES-CXCL9 and detect the recombinant Plasmid activity.

[0044] ① Construction of pIRES-CXCL9

[0045] The pCXCL9 N1 plasmid containing CXCL9 (purchased from Prospec, CHM-333) was separated by Sal and Not double-digestion low-melting point gel, and the CXCL9 fragment was recovered, and cloned into pIRES (purchased from Clontech, 631605 ) to construct the pIRES-CXCL9 plasmid in the B multiple cloning site.

[0046] ② Construction of pSNAV2-CXCR3-pIRES-CXCL9

[0047] Using the pSNAV2 cMyc plasmid (purchased from Invitrogen, VPI0017) containing the cMyc tag sequence as a template, the primers were designed as follows:

[0048] 5'-ACTTCTAGGCCTGTACG...

Embodiment 2

[0050] Example 2: mPPP composite nanomaterials entrapped pSNAV2-CXCR3-IRES-CXCL9 (mPPP-pSNAV2-TGFβ1-IRES-TIMP1, PSTIT).

[0051] Disperse 1.0 g of mPPP composite nanomaterials in PBS, adjust the pH to 7.2, filter and sterilize with a 0.22 μm Millipore membrane, and store at 4°C for use. Then 1.5 g of pSNAV2-CXCR3-IRES-CXCL9 was prepared into 50 μL (24-well plate) or 25 μL (96-well plate) DNA dilution solution with PBS (pH=7.2). According to the D / P value of 3, the mPPP composite nanomaterials were diluted in equal volume and slowly added dropwise to the DNA diluent, mixed well and left at room temperature for 30 minutes, which can be used for characterization or cell transfection experiments. Where N represents the number of moles of amino groups in the nanomaterial, and P represents the number of moles of phosphate groups in DNA (1 μg of DNA contains about 3 nmol of phosphate groups). Cell characterization experiment, detected by 90Plus Zeta type Zeta potential and particle ...

Embodiment 3

[0053] Embodiment 3: in vitro experiments, culturing sCS cells, and detecting the curative effect of composite nanomaterials;

[0054] In order to evaluate the effect of the complex mPPP-pSNAV2-TGFβ1-IRES-TIMP1, PSTIT) on cytotoxicity, the present invention uses the MTT method to detect the relative viability of the transfected sCS cells (MTT method, see "New Drug Pharmacology Research" edited by Lu Qiujun) Methods, 2007: 242-243).

[0055] The results of mPPP and PSTIT cell viability detection are as follows: image 3 As shown, both mPPP and PSTIT have no obvious cytotoxicity, and it can be seen that the PEG segment on the surface of nanoparticles can well shield excess positive charges, thereby greatly reducing cytotoxicity. Under the tested N / P ratio conditions, the blank polymer nanoparticle mPPP basically has no toxicity to cells, while PSTIT has obvious cytotoxicity under high N / P conditions.

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Abstract

The invention relates to the technical field of medicine and bioengineering technology, in particular to a recombinant plasmid based on polyethylene glycol-polylactic acid hydroxyl glycollic acid-polylysine composite nanomaterial entrapment and a preparation method and application thereof. The recombinant plasmid entrapped with a composite nanomaterial is mPPP-pSNAV2-TGFbeta1-IRES-TIMP1 (PSTIT). The invention further provides a preparation method of PSTIT and application of PSTIT in preparing medicine for targeted treatment of diabetic foot (DF) and diabetic peripheral neuropathy (DPN). The invention further provides a novel target for DPN treatment. It is proved through cell experiments and animal experiments that PSTIT can effectively relieve apoptosis of nerve cells of mice with DPN and delay progression of DPN by up-regulating expression of Bcl-2 protein and down-regulating expression of Caspase-3 and Bax protein.

Description

Technical field: [0001] The invention relates to the technical fields of medicine and bioengineering, and in particular to a recombinant plasmid based on polyethylene glycol-polylactic acid glycolic acid-polylysine composite nanomaterial and its preparation and application. Background technique: [0002] Diabetic foot (DF), the foot is a complex target organ of diabetes, a multisystem disease. Diabetic neuropathy (DPN) and peripheral vascular disease combine excessive mechanical pressure, which can cause damage and deformity of the soft tissue and bone joint system of the foot, and then cause a series of foot problems, ranging from mild to Neurological symptoms to severe ulcers, infections, vascular disease, Charcot's arthropathy, and neuropathic fractures. If aggressive treatment does not adequately address the symptoms and complications that develop in the lower extremities, the consequences can be disastrous. Clinically, the treatment of diabetic foot mainly focuses on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66A61K48/00A61P3/10A61P17/02A61P25/02
CPCA61K48/0033A61K48/005C12N15/63C12N15/66
Inventor 汤玮石勇铨刘浩琪闾倩
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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