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Method for preparing protamine-deoxycholic acid conjugate with heparin transfer function

A technology of deoxycholic acid and protamine, which is applied in the field of preparation of protamine-deoxycholic acid conjugates, can solve the problem of lack of amphiphilicity, difficulty in passing through cell membranes, and low transfection efficiency of protamine, etc. question

Inactive Publication Date: 2014-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore protamine has the potentiality of becoming a non-viral gene carrier, but it is rarely reported as a gene carrier independently, which is related to the low transfection efficiency when protamine is used alone, and the low transfection efficiency caused by protamine There are two possible reasons: First, protamine is highly hydrophilic and does not have amphipathic properties, which makes it difficult for it to pass through the cell membrane, because the main component of the cell membrane is the amphiphilic phospholipid bilayer
Second, the complex structure formed by protamine and DNA is too tight, which makes it difficult for DNA to be released from the protamine / DNA complex

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Preparation of DOCA-NHS

[0012] Mix 3.54g deoxycholic acid (DOCA), 2.40g N, N'-dicyclohexylcarbodiimide (DCC) and 1.48g N-hydroxysuccinimide (NHS), add 30ml tetrahydrofuran to make a suspension, DOCA The molar ratio of , NHS, and DCC is: 1:1.2:1.2, and the concentration of NHS and DCC should be slightly higher than that of DOCA to make the activation complete. The mixed solution was magnetically stirred and reacted for 12 hours in a nitrogen atmosphere at room temperature, and the resulting white dicyclohexylurea precipitate was removed by filtration. Add excess n-butane to the filtrate, unreacted DCC, and NHS are dissolved in n-butane, and the white precipitate is succinylated DOCA. The precipitate is obtained by suction filtration, fully washed with n-butane, and vacuum-dried at room temperature to obtain deoxycholorol Acid active ester (DOCA-NHS). Infrared absorption spectrum analysis shows that the product DOCA-NHS is at 2933cm -1 and 2862cm -1 A str...

Embodiment 2

[0013] Example 2: Preparation of protamine-deoxycholic acid conjugate

[0014] Dissolve protamine sulfate (SP) in a boric acid buffer solution containing 8M urea at pH 9.0, magnetically stir at room temperature and dissolve completely, then add THF solution containing NHS and DOCA-NHS, and finally form a solution containing 30% (v / v ) THF water / tetrahydrofuran mixed reaction system. Reacted at 25°C for 1.5 hours, dialyzed overnight at 4°C with a dialysis bag (molecular weight cut-off: 3500), and the dialysate was pH7.4 phosphate buffer containing 150mM NaCl. Subsequently, the crude product was adjusted to pH 5.0 with 1.0 M HCl, then washed 5 times with ether to remove the water-insoluble deoxycholic acid produced by the decomposition of unreacted active ester, and then dialyzed with ultrapure water for 72 hours and then freeze-dried to obtain Protamine-deoxycholic acid conjugate. pass 1 H-NMR detection shows that 0.67ppm, 0.90ppm, and 0.97ppm correspond to the chemical shif...

Embodiment 3

[0015] Example 3: Preparation of protamine-deoxycholic acid conjugate / heparin nanocomposite

[0016] Take a certain amount of heparin sodium solution, slowly add it dropwise to a certain amount of protamine-deoxycholic acid conjugate solution, shake while adding dropwise, shake immediately after the dropwise addition for 5-10 minutes, and let it stand for 30 minutes. The apparent morphology of the particles was observed using a transmission electron microscope. Prepare protamine-deoxycholic acid conjugates and heparin complexes with a weight ratio (w / w) of 0.1 to 20 respectively according to the above method, and control the final concentration of heparin sodium to 0.04mg / ml, determine and heparin The particle size and distribution of the nanocomposites obtained by combining different mass ratios and the Zeta-potential of nanoparticles. When the ratio of protamine-deoxycholic acid conjugate / heparin is 1.5:1, the particle size is relatively stable, the particle size is about 1...

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Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a method for preparing a protamine-deoxycholic acid conjugate with a heparin transfer function. The amidogen of protamine and the carboxyl of deoxycholic acid form an amide bond through teh effect of a cross-linking agent to prepare an amphipathic conjugate, and then the amphipathic conjugate and heparin form a self-assembled aggregation in order to conveniently release the heparin in cells. The hydrophobic modification of the conjugate can enhance the self-aggregation stability of nano-composites; the cationic performance of the conjugate entraps the heparin, so that the distribution capability of the heparin in the cells is increased, and the heparin is prevented from, degradation caused by the effect of heparinase and further the transfer of the heparin into cancer cells and the release and biological effect of the heparin in the cells are realized.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a preparation method of a protamine-deoxycholic acid conjugate with heparin transmission function. Background technique [0002] Heparin, as a highly sulfated polysaccharide, has long been widely used as an anticoagulant drug with good results. In recent years, studies have confirmed that heparin can prolong the survival time of patients with malignant tumors. Because heparin has many advantages such as good biocompatibility, water solubility and biodegradability, there are many studies on the application of heparin in antitumor. Existing studies have shown that the anti-tumor effect of heparin can be divided into extracellular effect and intracellular effect. Extracellularly, heparin can inhibit the proliferation of vascular endothelial cells stimulated by vascular endothelial growth factor and fibroblast growth factor, and inhibit the formation of neovascularization in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/42A61K9/14A61K31/727A61P35/00
Inventor 滕丽萍张艺壤付海田王敏陈敬华
Owner JIANGNAN UNIV
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