Effective and safe preparation method of oral recombinant human lactoferrin sericin nanoparticles and application

A technology of human lactoferrin and nanoparticles, applied in the field of biomedicine, can solve problems such as complex preparation process, low drug load clinical application development, etc.

Pending Publication Date: 2020-07-07
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Drug delivery systems currently used for UC treatment include liposomes, micelles, mesoporous silica nanoparticles, and hydrogels. These systems have their own characteristics, such as improving drug solubility by enhancing epithelial cel

Method used

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  • Effective and safe preparation method of oral recombinant human lactoferrin sericin nanoparticles and application
  • Effective and safe preparation method of oral recombinant human lactoferrin sericin nanoparticles and application
  • Effective and safe preparation method of oral recombinant human lactoferrin sericin nanoparticles and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of sericin nanoparticles

[0036] rhLF silkworm cocoons and WT silkworm cocoons are obtained by breeding rhLF silkworm strains and D9L silkworm strains in our laboratory. The cocoons are stored at room temperature before use. WT and rhLF silkworm strains were reared with fresh mulberry leaves to obtain WT and rhLF cocoons, and the cocoons were stored at room temperature before being used. The cocoons are frozen with liquid nitrogen and then crushed into silkworm cocoon powder with a high-speed pulverizer. Weigh a certain amount of silkworm cocoon powder, extract with pH8.0 8M urea containing 50mM Tris-HCl at a bath ratio of 10mg / mL in a water bath at 80℃ for 1h, then filter with gauze to remove undissolved silk fibroin to obtain sericin The solution and sericin solution were centrifuged at 10000 rpm and 25°C, and the supernatant was taken for later use. The WT sericin solution and rhLF sericin solution were dialyzed with a molecular weight of 10kDa...

Embodiment 2

[0037] Example 2: Characteristic analysis of sericin nanoparticles

[0038] See attached figure 1 , The pore size, secondary structure, and release characteristics of recombinant human lactoferrin sericin nanoparticles were tested. The specific testing methods and results are as follows:

[0039] 1. Fourier transform infrared absorption spectrometer (FTIR) analysis

[0040] The secondary structure of sericin nanoparticles was analyzed by FTIR (Nicolet iN10 instrument, Thermo Fisher, USA), and the spectrum was collected in the range of 800-4000 cm-1, and then the secondary structure was analyzed by the amide I method, 1640-1610 cm -1 , 1650-1640cm -1 , 1660-1650cm -1 , 1700-1660cm -1 Representing β-sheets, random coils, α-helices and β-turns, respectively, more than 30 independent samples were used for FTIR analysis.

[0041] See attached figure 1 A, 1F, the prepared sericin nanoparticles are measured, the average particle size of WT-nano and rhLF-nano are both within 200nm, the average...

Embodiment 3

[0048] Example 3: Detection of uptake of rhLF cells in rhLF-nano

[0049] RAW264.7 macrophages were added to a 96-well plate at a density of 5×104 cells / well and cultured in DMEM medium containing 10% FBS for 12 hours. After the cells adhered to the wall, the medium was replaced with 90μl / well, and then 10μl WT-nano was added separately. , RhLF-nano and rhLF-STD solutions, remove the medium at a certain time point (1, 2 and 4h), wash three times with cold PBS, then add 4% paraformaldehyde for fixation for 10 minutes, Triton X100 for cell punching -RhLF antibody and immunofluorescence secondary antibody are used to label cells with rhLF. After the labeling is completed, observe and take pictures under a fluorescence microscope. Three repeats per group.

[0050] In order to explore whether rhLF can be endocytosed by macrophages and exert its activity, the cells were co-cultured with WT-nano, rhLF-nano and rhLF-STD, and the effect of rhLF endocytosis into cells was detected with anti...

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Abstract

The invention provides an effective and safe preparation method of oral recombinant human lactoferrin sericin nanoparticles and an application. Uptake efficiency of rhLF cells can be improved, LPS induced macrophage inflammation can be inhibited, and DSS-induced acute colitis can be treated. The sericin nanoparticles containing rhLF are prepared from rhLF transgenic silkworm cocoons as a raw material, by sericin extraction, dialysis and ethanol induction, and are directly used for oral administration treatment of colitis. The sericin nanoparticles are suitable for large-scale production, simple to operate, and high in application value.

Description

Technical field [0001] The invention is applied to the technical field of biomedicine, and specifically relates to a preparation method and application of oral recombinant human lactoferrin sericin nanoparticles. Background technique [0002] Lactoferrin is a multifunctional innate immune glycoprotein, and its biological activities have been found in terms of re-immunization, antibacterial, anti-tumor, anti-oxidation, and stimulation of cell growth. Lactoferrin is widely present in exosomes such as milk, tears, pancreatic juice, bile and semen, etc. It is mainly stored by secondary granules of neutrophils. The content of lactoferrin is very low under normal physiological conditions and will increase significantly with infection. Researchers have detected abundant lactoferrin receptors in activated B lymphocytes and T lymphocytes, monocytes / macrophages, testicular brush border cells, platelets and newborn cells, which means that lactoferrin has a variety of organisms. Learn func...

Claims

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Application Information

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IPC IPC(8): A61K38/40A61K9/51A61K47/46A61P1/00
CPCA61K38/40A61K9/5176A61K9/0053A61P1/00Y02A50/30
Inventor 王峰夏庆友许胜赵萍
Owner SOUTHWEST UNIVERSITY
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