A preparation method for actively targeting biofilm nano-preparation

A nano-preparation and active targeting technology, applied in the field of brain-targeted molecularly modified biofilm nano-preparations, preparation of active-targeted biofilm nano-preparations, and DCDX-modified erythrocyte membrane preparations, can solve limitations, limit modifications, and progress problems such as slowness, to achieve good application prospects and increase the effect of intracerebral delivery

Active Publication Date: 2020-12-08
SHANGHAI WHITTLONG PHARMA INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason for its slow progress is that as an endogenous biomimetic membrane, various chemically sensitive biological characteristics of the biofilm surface limit the application of conventional chemical modification methods on the biofilm surface.
The preparation of active targeting biofilm preparations in the prior art is mainly realized by the lipid intercalation method, that is, the targeting molecules with lipid intercalation function are directly inserted into the lipid layer of the biomembrane by means of co-incubation, but this method It has its limitations: 1) The surface of the biomembrane has a strong negative charge, which limits the modification of the positively charged targeting molecules due to electrostatic interaction; 2) Since the biomembrane is a lipid structure with a bilayer , which also limits the modification of lipid-soluble targeting molecules

Method used

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  • A preparation method for actively targeting biofilm nano-preparation
  • A preparation method for actively targeting biofilm nano-preparation
  • A preparation method for actively targeting biofilm nano-preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Streptavidin-PEG 3400 -DSPE with Biotin-PEG 3500 - D Synthesis and characterization of CDX

[0052] Streptavidin-PEG 3400 - The synthesis of DSPE is as follows: 20 mg of streptavidin was dissolved in 2 mL of borate buffer (pH 7.6), and a 10-fold excess of Traut’s reagent was added to react at room temperature for 2 h, and purified by ultrafiltration at 4°C to obtain thiolated streptavidin white. 5mg Mal-PEG 3400 - DSPE was dissolved in 1mL of dichloromethane, rotary steamed to form a film, added the thiolated streptavidin solution obtained in the above reaction, reacted at 37°C for 1 hour, purified by dialysis and freeze-dried to obtain the product;

[0053] Biotin-PEG 3500 - D CDX was synthesized as follows: 0.41 mmoL biotin was dissolved in 1 mL dichloromethane, 0.615 mmoL N-hydroxysuccinimide and 0.615 mmoL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added, Stir at room temperature for 12 h under the protection of nitrogen, and drop the reaction soluti...

Embodiment 2

[0054] Example 2 D Preparation and Characterization of CDX-RBCNP

[0055] 1) D Preparation of CDX-RBCNP

[0056] Take whole blood from male ICR mice, centrifuge (1000g / min, 4°C) for 5min, discard the upper layer of serum and white blood cell layer, wash the lower layer of red blood cells with 1×PBS, resuspend in 0.25×PBS for 30 minutes at 4°C, and centrifuge (15000g / min, 4°C) for 7 minutes to remove hemoglobin, and the obtained light red erythrocyte membrane was resuspended and stored in double distilled water;

[0057] Weigh 10 mg of polylactic-glycolic acid (PLGA), dissolve it in 1 mL of acetone, add 3 mL of double-distilled water dropwise, and dry in vacuum for 2 hours to obtain PLGA nanoparticles (NP);

[0058] The erythrocyte membrane suspension was sonicated at 100 W for 3 minutes to obtain erythrocyte membrane vesicles. 40 μL Streptavidin-PEG 3400 -DSPE PBS solution (5mg / mL) was incubated with erythrocyte membrane vesicles obtained from 100μL whole blood in a water...

Embodiment 3D

[0063] Example 3 D In vitro targeting verification of CDX-RBCNP

[0064] 1) D CDX-RBCNP / DiI with D Preparation of CDX-RBCNP / DiD

[0065] Drug loading of DiI or DiD Prepare DiI or DiD-loaded PLGA nanoparticles by dissolving DiI or DiD and PLGA in acetone, and the other preparation processes are the same as blank D CDX-RBCNP;

[0066] 2) D In Vitro Targeting Validation of CDX-RBCNP to Primary Brain Capillary Endothelial Cells

[0067]The brains of 4-week-old SD rats were decapitated, and the cerebral cortex was quickly separated in pre-cooled D-Hanks solution. The meninges and large blood vessels of the brain were removed, then cut into pieces, and collagenase and DNase were added to digest at 37°C for 90 minutes. Centrifuge at 1000rpm for 8min, discard the supernatant, transfer to DMEM solution of 20% BSA, centrifuge (1000g / min, 4°C) for 20min, discard the upper liquid, transfer the bottom microvessels to DMEM culture medium, centrifuge at 1000rpm for 5min, and use Resus...

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Abstract

The invention belongs to the field of pharmacology and biology and relates to a preparation method of an active target biological membrane nano preparation and a constructed brain target molecule modified biological membrane nano preparation, in particular to a method of preparing an active target biological membrane nano preparation by modifying a target molecule to the surface of a biological membrane through a membrane insertion method and a streptavidin-biotin strategy, a prepared DCDX modified red blood cell membrane preparation, and an application of the preparation in brain disease imaging and target treatment. Test results show that the active target biological membrane nano preparation prepared by the method has the advantage of universality; the amount of the constructed DCDX modified red blood cell membrane preparation entering the brain can be increased; and the preparation has a good application prospect in the diagnosis and treatment of brain diseases.

Description

technical field [0001] The invention belongs to the fields of pharmacy and biology, and relates to a preparation method of an active targeting biofilm nano-preparation and a constructed brain-targeting molecularly modified biofilm nano-preparation. In particular, it relates to the method of combining membrane insertion method and "streptavidin-biotin" strategy to modify targeting molecules to the surface of biofilms to prepare active targeting biofilm nano-preparations, and the prepared D CDX-modified erythrocyte membrane preparations and their applications in brain disease imaging and targeted therapy. Background technique [0002] In recent years, the nano-preparation of biofilm-coated drugs has been widely studied and reported in the field of pharmacy as a new type of nano-drug delivery system. As a new type of biomimetic preparation, the nano-preparation of biofilm-coated drugs not only has the advantages of traditional nano-drugs, but also significantly enhances the sa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/51A61K47/10A61K47/24A61K31/704A61P35/00
CPCA61K9/5123A61K9/5146A61K9/5153A61K9/5169A61K31/704
Inventor 陆伟跃柴芝兰胡雪峰魏晓丽
Owner SHANGHAI WHITTLONG PHARMA INST
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