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Method for detecting entrapment rate of protein or polypeptide drugs in lipid vesicles

A technology of lipid vesicles and encapsulation efficiency, which is applied in the direction of measuring devices, biological tests, material inspection products, etc., to achieve the effect of strong light absorption

Inactive Publication Date: 2017-03-15
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the deficiency of the existing method for measuring the encapsulation efficiency of protein or polypeptide drugs in lipid vesicles, and provide a quick and easy encapsulation efficiency calculation method

Method used

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  • Method for detecting entrapment rate of protein or polypeptide drugs in lipid vesicles
  • Method for detecting entrapment rate of protein or polypeptide drugs in lipid vesicles
  • Method for detecting entrapment rate of protein or polypeptide drugs in lipid vesicles

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Prepare liposomes encapsulating bovine serum albumin (BSA) according to the conventional reverse-phase evaporation method, as follows: Weigh 20 mg soybean lecithin and 5 mg cholesterol, dissolve them in 9 mL ether, add them to a round-bottomed flask, add 3 mL 1 mg / mL bovine serum albumin (BSA) solution, sonicated in a water bath for 2 to 5 minutes to form a stable white emulsion suspension, evaporated to dryness with a rotary evaporator to remove the organic solvent, took out the remaining liquid, and ultrasonically treated with an ice-water bath probe until the solution became After clarification, a liposome solution is obtained. The liposome solution was diluted 10 times to a concentration of 0.1 mg / mL. Take a 96-well plate, add the diluted liposome solution at 20 μL per well; take another diluted liposome solution, add Triton X-100 to a final concentration of 0.5%, and add 20 μL per well Add 0.5 mg / mL BSA standard to each well of the standard well, add samples accor...

Embodiment 2

[0037] Prepare the transfer body wrapped with bovine serum albumin (BSA) according to the conventional film dispersion method, as follows: Weigh 30 mg soybean lecithin, 6 mg cholesterol, and 2 mg sodium deoxycholate, dissolve them in an appropriate amount of chloroform, and add them to a round bottom flask , evaporate to dryness with a rotary evaporator to remove the organic solvent, so that a uniform film is formed at the bottom of the flask; place the flask in a vacuum desiccator for at least 3 hours to completely remove the residual organic solvent, and add 5 mL of 1 mg / mL BSA solution , oscillating to hydrate the film, ultrasonic treatment in a water bath for 30 minutes, until the solution becomes clear to obtain the transfer body solution, take an appropriate amount of transfer body solution and dilute it 10 times for later use. Take a 96-well plate, add the diluted transfersome solution at 20 μL per well; take another diluted transfersome solution, add Triton X-100 to a f...

Embodiment 3

[0040] Prepare the liposome solution according to Example 1, take 1 mL of the liposome solution and use an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 kD to remove the free protein by centrifugation, and then use a final concentration of 0.1% Triton X-100 to separate the For liposome demulsification, take 20 μL each, add 200 μL BCA reaction reagent, place it at 50°C for 20 min, place it in a microplate reader quickly, and read the absorbance value at 562 nm. Also scan the plate once every 10 minutes, record continuously for one hour, and then scan again at room temperature, 3, 7, and 24 hours after color development, and record the absorbance changes at different times. Calculate the concentration of each porin according to the standard curve obtained from each measurement, as shown in the attached Figure 4 Shown is the time change curve of the initial total protein concentration (Initial+Triton) and the total protein concentration after ultrafiltrat...

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Abstract

The invention relates to a for detecting the entrapment rate of protein or polypeptide drugs in lipid vesicles, and belongs to the technical field of drug analysis. The method comprises the following steps: diluting a prepared protein or polypeptide wrapped lipid vesicle solution, taking the diluted lipid vesicle solution, adding a nonionic surfactant to carry out demulsification, taking the lipid vesicle solution before and after the demulsification, respectively adding a BCA reaction solution, and carrying out color development at a certain temperature and time; and reading the absorbance at 562 nm, and calculating the protein concentration and the entrapment rate according to a standard curve. The method uses a difference between the reaction rate of free proteins outside the lipid vesicles with a color developer and the reaction rate of the wrapped proteins in the lipid vesicles and the color developer to measure the entrapment rate of the lipid vesicle protein drugs without separating the free proteins, so the method has the advantages of convenience in operation, and good repeatability, solves the problems of tediousness and deviation in the measurement process of the entrapment rate of the lipid vesicle protein drugs, and provides an effective method and basis for relevant production application and research fields of the lipid vesicle protein drugs.

Description

technical field [0001] The invention relates to a method for measuring the encapsulation rate of protein or polypeptide drugs in lipid vesicles, belonging to the technical field of drug analysis. Background technique [0002] The research on lipid vesicles of peptide and protein drugs is a very active field at present. Lipid vesicles are closed vesicles composed of lipids with a water phase inside, and hydrophilic and hydrophobic substances can be encapsulated in the water phase and membrane, respectively. According to different compositions, lipid vesicles include liposomes, transfersomes, plastids, ethosomes, etc., but their structures are all spherical closed vesicles. After oral administration, protein will be degraded and inactivated by acid and alkali conditions in the gastrointestinal tract, body fluids and enzyme damage. After oral administration of lipid vesicles as the carrier, due to the protective effect of the lipid bilayer, it can reduce the degree of degrada...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6833
Inventor 张义浜许化溪曹方引冯雪
Owner JIANGSU UNIV
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