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Apolipoprotein modified fusion type multifunctional nano vesicle, and preparation method and application thereof

An apolipoprotein and nanovesicle technology, applied in the fields of nanotechnology, nanotechnology, nanomedicine, etc., can solve the problem of lack of research, and achieve poor targeting, improved therapeutic effect, and improved encapsulation effect.

Pending Publication Date: 2021-11-09
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, there is currently no relevant research on how to combine nanozymes and photosensitizers in a reproducible, efficient and quantifiable manner through the action of lipoproteins and exosomes.

Method used

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  • Apolipoprotein modified fusion type multifunctional nano vesicle, and preparation method and application thereof
  • Apolipoprotein modified fusion type multifunctional nano vesicle, and preparation method and application thereof
  • Apolipoprotein modified fusion type multifunctional nano vesicle, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of high-density lipoprotein nanoparticles loaded with nanozymes

[0047] Preparation of oleic acid-modified Fe by pyrolysis 3 o 4 (OA-Fe 3 o 4 ) nanoparticles. First, by FeCl 3 React with sodium oleate to synthesize iron oleate complex. 1.184g (4.38mmol) of FeCl 3 ·6H 2 O was dissolved in 6 mL of ultrapure water to obtain FeCl 3 Clear solution. Then, 3.653 g of sodium oleate (12 mmol) was added to the above clear solution. In addition, a mixed solvent consisting of 14 mL of n-hexane and 8 mL of ethanol was injected into the mixture, and the mixed solution was stirred and heated to 70 °C for 4 h. After the reaction was completed, the temperature was naturally cooled to room temperature, and the upper organic layer (ferric oleate) was separated with a separatory funnel, and washed three times with ultrapure water. Finally, n-hexane was evaporated by slow heating to give a waxy reddish-brown product (iron oleate). Using the newly synthesi...

Embodiment 2

[0049] Example 2: Preparation process of DC-derived exosomes loaded with ALA-loaded antigen

[0050] Take 1mL with a density of 1×10 6 Cells / mL mouse glioma GL261 cells were frozen in liquid nitrogen for 3 minutes, and then thawed in a water bath at 56°C to obtain tumor lysates. Repeat freeze-thaw 5 times. Lysates were centrifuged at 100 x g for 10 min to remove cell debris and passed through a 0.22 μm filter. Samples were stored at -20°C.

[0051] Exosomes were obtained from bone marrow-derived dendritic cells (BMDCs). Bone marrow was isolated from femurs and tibias of male C57BL / 6 mice aged 6-8 weeks and washed with RPMI 1640 medium. Erythrocytes were lysed and remaining cells were cultured in complete medium (RPMI 1640 containing 10% FBS, 1% penicillin-streptomycin, 20 ng / mL murine GM-CSF and IL-4). Change the medium in half every 2 days. On day 5, tumor lysates (50 μg / mL) were added to BMDCs, and cells were incubated for an additional 24 h for antigen loading. To ge...

Embodiment 3

[0053] Example 3: Investigation of the properties of fusion-type multifunctional nanovesicles modified by apolipoprotein

[0054] 1.1 Fusion study of fusion-type multifunctional nanovesicles modified by apolipoprotein

[0055] HDL and Dex were fused by sonication, and the specific operation was to mix 200 μg of protein-equivalent Dex (the tumor-associated antigen-loaded Dex prepared in Example 2) and 1 mg of lipid-equivalent HDL (prepared in Example 1) into 1 mL of Final volume, ultrasonic crushing 5min, power 195W. Then place it at 37°C for 1 hour to promote the closure of the phospholipid membrane to obtain Dex-HDL / ALA-Fe 3 o 4 .

[0056] 1.1.1 FRET fluorescence spectrum verification fusion

[0057] FRET is energy transfer based on the spatial distance between the donor and acceptor molecules. Fluorescent NBD and Rho lipids are selected as the FRET pair. The donor HDL dilutes its lipid on the non-fluorescent acceptor Dex membrane during the fusion process. This increase...

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Abstract

The invention discloses an apolipoprotein modified fusion type multifunctional nano-vesicle and a preparation method and application thereof. The multifunctional fusion type nano-vesicle is mainly formed by fusing bionic lipoprotein and exosome, wherein the bionic lipoprotein is mainly formed by apolipoprotein biological peptide, phospholipid and nano-enzyme, and the exosome is impacted by an antigen and is loaded with a photosensitizer. The preparation method comprises an incubation method, an extrusion method, a freeze thawing method, a polyethylene glycol induction method and an ultrasonication method. The preparation method disclosed by the invention has a simple condition and low cost, and the prepared apolipoprotein modified fusion type multifunctional nano-vesicle has the advantages of high endogenous properties, biological safety, a strong drug loading capacity, specificity, focus site targeting properties, drug loading mode diversity and the like. The nano-drug delivery system can realize "diagnosis and treatment integration" and "multi-mode treatment", and can realize early diagnosis and targeted efficient treatment of complex progressive diseases such as tumors, neurodegenerative diseases and the like.

Description

technical field [0001] The invention belongs to the technical field of nano-preparation, and in particular relates to a fusion-type multifunctional nano-vesicle modified by apolipoprotein and its preparation method and application method. Background technique [0002] Exosomes are lipid bimolecular membrane vesicles with a diameter of 40nm-160nm. They are widely distributed in various body fluids and secreted by almost all living cells, including stem cells, immune cells, and tumor cells. Exosomes are formed by inward budding of endosomal membranes to form multivesicular bodies (MVBs). Subsequently, exosomes are released into the extracellular space by fusion of MVBs with the plasma membrane. After being released from the cell surface, exosomes can interact with the extracellular matrix or be taken up by distant or neighboring cells, thus performing the function of intercellular communication. This intercellular vesicle trafficking pathway plays an important role in many a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/69A61K47/64A61K33/26A61K9/51A61K47/24A61K39/00A61K47/46A61K41/00A61K47/54A61K49/00A61P35/00B82Y5/00B82Y20/00B82Y40/00
CPCA61K47/6911A61K47/64A61K33/26A61K47/542A61K9/5123A61K47/6901A61K39/0011A61K41/0057B82Y5/00B82Y20/00B82Y40/00A61P35/00A61K49/0084A61K49/0056A61K2300/00
Inventor 王若宁王笑红李俊松狄留庆
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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