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Reconstituted apolipoprotein b lipoparticle, a preparation method and uses thereof

a technology of apolipoprotein and lipoprotein, which is applied in the field of reconstituted apolipoprotein b lipoprotein, can solve the problems of lipophilic drugs, difficult to be transported in the physiological condition of the human body, and the synthesis pathway of ldl still remains unknown

Inactive Publication Date: 2015-07-02
NAT CHIAO TUNG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a method for making a reconstitutable apolipoprotein B lipoparticle. It involves dissolving apolipoprotein B and lipid in a buffer containing amphiphilic compounds and urea. After dialyzing the mixture against another buffer containing no urea and less amphiphilic compounds, the concentration of urea and amphiphilic compounds is reduced. The technical effect is a simpler and more efficient way to prepare apolipoprotein B lipoparticles.

Problems solved by technology

However, some synthesis pathways for LDL still remained unknown.
Moreover, there are a lot of drugs that are lipophilic and hard to be transported in the physiological condition of the human body.

Method used

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  • Reconstituted apolipoprotein b lipoparticle, a preparation method and uses thereof
  • Reconstituted apolipoprotein b lipoparticle, a preparation method and uses thereof
  • Reconstituted apolipoprotein b lipoparticle, a preparation method and uses thereof

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example 1

Materials

[0044]All chemicals, unless otherwise noted, were purchased from Merck (Rahway, N.J.) or Sigma (St. Louis, Mo.). Human serum was obtained from a healthy volunteer using a protocol approved by the Mackay Memorial Hospital Institutional Review Board (IRB No. 10MMHIS082). Specific pathogen-free, 4-5 weeks old BALB / c mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All experiments were carried out in accordance with the Academia Sinica Animal care and Use Committee guideline. Non-coating 2 nm, superparamagnetic iron oxide nanoparticles (hereinafter referred to SPIONPs) can be synthesized or purchased. M4N can be synthesized or purchased. Anti-apoB (C1.4, monoclonal antibody), anti-LDLR, anti-p53 and anti-Erk antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.); anti-apoB polyclonal antibody was purchased from Roche (Branford Conn.); cy3-conjugated secondary antibody was purchased from Jackson Immuno Research Laborator...

example 2

Purification of Low Density Lipoprotein

[0045]Whole bloods were centrifuged at 5000×g for 10 mins for separating blood cells and plasma. After collecting plasma, very low density lipoprotein was removed by centrifugation at 45000×g for 16 hrs. Since the density of LDL in a human body ranges from 1.060 to 1.063 g / ml, LDL was then adjusted to such density range using potassium bromide (KBr) and purified by centrifugation at 45000×g for 20 hrs.

example 3

Isolation of Lipoprotein B-100

[0046]The purified LDL was delipidated by an ice-cold methanol / ether mixture (methanol: ether=3:1, −80° C.), followed by mixed. The mixture was centrifuged at 3000×g for 5 mins and the precipitants were the isolated lipoprotein B-100.

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Abstract

The present invention provides a method for preparing a reconstituted apolipoprotein B lipoparticle and the method comprises steps of (a) dissolving an apolipoprotein B and a lipid in a first buffer containing 2 M to 8 M urea and 1 wt % to 15 wt % amphiphilic compounds to form a mixture; and (b) dialyzing the mixture against a second buffer containing 0 M to 2M urea and 0 wt % to 0.5 wt % amphiphilic compounds for 1 to several times for lowering concentrations of the urea and the amphiphilic compounds in the mixture. The present invention further provides an apolipoprotein B lipoparticle and a use for the production of an apolipoprotein B lipoparticle used for delivering a hydrophobic substance.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to a reconstituted apolipoprotein B lipoparticle, and a preparation method thereof, and more particularly to a reconstituted apolipoprotein B lipoparticle for delivering hydrophobic substances.[0003]2. Description of Related Art[0004]Low density lipoprotein is composed of a single apolipoprotein B and several lipids (including cholesteryl ester (CE)), free cholesterol (FC), triglyceride (TG), and phospholipid (PL), and its size is approximately 18 to 25 nanometer (nm). Apolipoprotein B not only synthesizes low density lipoproteins (hereinafter referred to LDL), it is also the major protein of very low-density lipoproteins (VLDL) and chylomicrons. In the human body, apolipoprotein B has two major forms: apolipoprotein B-100 (apoB-100) of full length, consisting of 4563 amino acids, and apolipoprotein B-48 with 48% N-terminal sequence of the apolipoprotein by mRNA modification contains 2153 a...

Claims

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Application Information

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IPC IPC(8): A61K9/50A61K49/18A61K9/14
CPCA61K9/5052A61K49/1866A61K9/146A61K9/5123A61K31/09A61K9/5169
Inventor CHANG, CHIA-CHINGCHEN, GONG-SHENCHENG, TSAI-MUCHU, HSUEH-LIANG
Owner NAT CHIAO TUNG UNIV
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