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Method for Determination of Three-dimensional Morphology and Distribution of Loaded Protein and Polypeptide Drug Microspheres

A measurement method and polypeptide technology, which is applied in the analysis of suspensions and porous materials, measuring devices, particle size analysis, etc., can solve the problems of unable to analyze microspheres, measure closed pores, destroy microsphere structures, etc., and shorten the research and development Cycle, the effect of improving R&D efficiency

Inactive Publication Date: 2016-04-13
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the conventional methods for characterizing the void structure of microspheres have certain limitations, and it is difficult to analyze the structural parameters inside the microspheres.
Such as: Mercury or gas porosity measurement method, nitrogen desorption adsorption method is used to measure the distribution of pores inside microspheres, but it cannot measure the closed pores, and too high pressure may destroy the original structure of microspheres; while scanning Electron microscopy (SEM) can be used to qualitatively analyze the structure of microspheres, and its resolution is high, but only the surface of the microspheres can be observed, and the internal overall structure of the microspheres cannot be analyzed; there are also confocal laser scanning microscopes (CLSM) and Fourier transform infrared spectroscopy can be used to qualitatively or semi-quantitatively observe the distribution of drugs in microspheres, but the resolution is low and cannot satisfy the detailed information of quantitative analysis of drug distribution in microspheres

Method used

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  • Method for Determination of Three-dimensional Morphology and Distribution of Loaded Protein and Polypeptide Drug Microspheres
  • Method for Determination of Three-dimensional Morphology and Distribution of Loaded Protein and Polypeptide Drug Microspheres
  • Method for Determination of Three-dimensional Morphology and Distribution of Loaded Protein and Polypeptide Drug Microspheres

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Embodiment 1

[0047] A method for measuring the three-dimensional shape and distribution of loaded protein and polypeptide drug microspheres, wherein the protein and polypeptide drugs are bovine serum albumin (BSA), and the microsphere skeleton is polylactic acid-polyglycolic acid copolymer (PLGA). The microspheres were prepared by volatilizing the double emulsification solvent. The assay method comprises the following steps:

[0048] 1) Sample preparation

[0049] ①Fixation: Weigh 20mg of microspheres and place them in a 1.5ml centrifuge tube, add 1ml of 2.5% glutaraldehyde fixative solution to each tube, and let stand for 4 hours to ensure that the fixative solution can completely penetrate the microspheres.

[0050] ②Washing: Discard the fixative, and wash the microspheres 3 times with PBS buffer.

[0051] ③Ethanol washing and dehydration: after the third washing, suck out the supernatant with a pipette, wash with gradient ethanol solution, and remove the water in the microspheres. Fi...

Embodiment 2

[0073] A method for measuring the three-dimensional shape and distribution of protein-loaded and polypeptide drug microspheres in this embodiment is basically the same as the method in Example 1, except that:

[0074] 2) Nano computed tomography

[0075] Place the microsphere sample to be tested in the Nano-CT tomography equipment, after pre-calibration, adjust the photon energy to 5keV, scan in the large field of view mode, set the single exposure time to 15 seconds, scan the microsphere, use A 1024-pixel × 1024-pixel CCD image sensor captures two-dimensional images of the microspheres, and collects two-dimensional projection images within an angle of -90°-+90° with a step size of 0.2°.

[0076] Compared with Example 1, comprehensive information is acquired by using the nano-computed tomography parameters of this embodiment, but the amount of information is huge, which increases the difficulty and error of reconstruction.

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Abstract

The invention discloses a method for determination of stereo shape and distribution of protein and polypeptide drug loaded microspheres, and belongs to the technical field of microsphere determination. The method comprises the following steps: 1) sample preparation: fixing a microsphere by a fixing liquid, and adding a coloring agent to combine protein and polypeptide drugs in the microsphere; 2) nano computer tomography: placing the microsphere sample to be detected in a Nano-CT tomography device of microsphere and scanning the microsphere to obtain a two-dimensional projection image of the microsphere; 3) data segmentation: reconstructing to obtain images of different sections of the microsphere, and segmenting the microsphere into a protein and polypeptide drug part and a microsphere skeleton part by preset gray threshold; and 4) construction of three-dimensional structure: conducting three-dimensional reconstruction by using the cross-sectional images of the microsphere to obtain a three-dimensional structure loaded with protein and polypeptide drug, and calculating the structure parameters of the microsphere. By using this method, the three-dimensional structure within the microsphere can be obtained.

Description

technical field [0001] The invention relates to a method for measuring microspheres, in particular to a method for measuring the three-dimensional shape and distribution of loaded protein and polypeptide drug microspheres. Background technique [0002] In recent years, protein peptide drugs have played an important role in the diagnosis and treatment of AIDS, cancer, hepatitis, diabetes and other diseases, or vaccine prevention of diseases. However, due to the poor in vivo and in vitro stability of protein and polypeptide drugs, the drug is quickly inactivated after injection. In order to achieve the curative effect, frequent and high-dose administration is often required, resulting in toxic side effects and tolerance, which severely limits its use in the field of drug therapy. clinical application. [0003] However, biodegradable polymers such as polylactic acid-polyglycolic acid (PLGA) and polylactic acid (PLA) are used to coat biomacromolecular drugs into sustained-relea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N23/04G01N15/08G01N15/02
Inventor 张永明罗宇燕黎呐郭喆霏钟晨
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV