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Recomposed tPA gene-chitosan nanoparticle complex and preparation method thereof

A technology of chitosan nanoparticles and complexes, applied in gene therapy, drug combination, pharmaceutical formula, etc., can solve the problems of life-long medication, high cost of treatment, high price, etc., to eliminate or avoid the formation of blood clots , improve bioavailability, reduce the effect of thrombus formation

Inactive Publication Date: 2009-09-23
姬尚义 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing TPA preparations have good clinical curative effect, they are expensive, and patients need life-long medication. The high cost of treatment brings huge economic burden to patients

Method used

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  • Recomposed tPA gene-chitosan nanoparticle complex and preparation method thereof
  • Recomposed tPA gene-chitosan nanoparticle complex and preparation method thereof
  • Recomposed tPA gene-chitosan nanoparticle complex and preparation method thereof

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preparation example Construction

[0031] The preparation method of recombinant tPA gene-chitosan nanoparticle complex of the present invention mainly comprises the following steps:

[0032] 1. Obtain the recombinant tPA gene plasmid pSecTag2B-tPA, which will be described in detail in Example 1 below;

[0033] 2. Using the above-mentioned recombinant tPA gene plasmid and chitosan to prepare the recombinant tPA gene-chitosan nanoparticle complex, the following example 2 will describe in detail.

Embodiment 1

[0034] Example 1: Preparation of recombinant tPA gene plasmid pSecTag2B-tPA

[0035] 1. Main reagents

[0036] A. Plasmid pSecTag2B, purchased from Invitrogen Biological Company, USA,

[0037] B. Competent cells E.Coli JM109, purchased from Promega,

[0038] C. Restriction enzymes HindIII, KpnI, BamHI and XhoI were purchased from Bao Biological Engineering Co., Ltd.,

[0039] D, T4-DNA ligase, purchased from New England Biolabs company,

[0040] E, QIAquick Gel Extraction Kit, QIAGEN PCR Product Purification Kit, purchased from QIAGEN Company.

[0041] 2. Method

[0042] Three ESTs were artificially synthesized: tPA-1 as shown in SEQ ID NO.2, tPA-2 as shown in SEQ ID NO.3 and tPA-3 as shown in SEQ ID NO.4, respectively connected to cloning vectors to prepare plasmids pOTB6, pOTB7 and pCMV-SPORT6.

[0043] Using three pairs of primers tPA-1F (SEQ ID NO.5) and tPA-1R (SEQ ID NO.6), tPA-2F (SEQ ID NO.7) and tPA-2R (SEQ ID NO.8), tPA -3F (SEQ ID NO.9) and tPA-3R (SEQ ID NO....

Embodiment 2

[0046] Embodiment 2: Preparation method of recombinant tPA gene-chitosan nanoparticle complex

[0047] Utilize the recombinant tPA gene plasmid pSecTag2B-tPA and chitosan that embodiment 1 makes to prepare recombinant tPA gene-chitosan nanoparticle complex, comprising the following steps:

[0048] 1) Preparation of chitosan nanoparticles:

[0049] a) prepare 1.5mg / ml chitosan acetic acid solution, wherein the mass ratio of acetic acid to chitosan is 1.5: 1, first measure 2ml analytical pure glacial acetic acid solution, dissolve it in 38ml triple distilled water, and then weigh 60mg of chitosan powder was dissolved in 40ml of the above acetic acid solution to obtain a 1.5mg / ml chitosan acetic acid solution;

[0050] b) 20ml of polyvinyl alcohol aqueous solution with a concentration of 10mg / ml was slowly dripped into 40ml of chitosan acetic acid solution obtained in step a) under the condition of magnetic stirring, and continued magnetic stirring for 1 hour at normal temperature...

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Abstract

The invention discloses a recomposed tPA gene-chitosan nanoparticle complex and a preparation method thereof. The recomposed tPA gene-chitosan nanoparticle complex comprises a chitosan nanoparticle and a recomposed tPA gene plasmid; and the mass ratio between the chitosan nanoparticle and the recomposed tPA gene plasmid is 1 to 1. The recomposed tPA gene-chitosan nanoparticle complex is injected into ventricle muscle corresponding to the valve when in a valve replacement, thus solving the target problem and being capable of obtaining the tPA expression of a plurality of cells including the cardiac muscle cell; the fibrinolytic activity of the cardiac muscle cell is improved, and the occurrence of thrombosis after a valve replacement is greatly reduced; in addition, the invention can eliminate and avoid intravascular agglutination in time, thus avoiding the thrombosis on the periphery of the valve after the valve replacement.

Description

technical field [0001] The invention relates to the field of postoperative thrombosis prevention and treatment drugs, in particular to a recombinant tPA gene-chitosan nano particle complex and a preparation method thereof. Background technique [0002] Under normal physiological conditions, the coagulation-anticoagulation and fibrinolysis-antifibrinolysis mechanisms balance each other to ensure the normal flow of blood in the body without the formation of thrombus or bleeding. The imbalance between thrombus formation and thrombolysis mechanism leads to inappropriate formation of thrombus or failure to effectively remove it after formation, leading to tissue or organ ischemia / necrosis; on the other hand, excessive anticoagulation / fibrinolysis can also lead to bleeding. [0003] Thrombosis is easy to form after artificial heart valve replacement, and patients usually need anticoagulant therapy for life, which brings great inconvenience and danger to patients. How to solve pos...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/14A61K47/36A61P7/02
Inventor 姬尚义季军
Owner 姬尚义
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