A kind of NO-loaded targeting microbubble and preparation method and application thereof

A technology of targeting microbubbles and microbubbles, applied in the field of NO-loaded C4d targeting microbubbles and its preparation, can solve the problems of severe infection, toxicity, and high cost, and achieve high safety and good clinical application prospects

Active Publication Date: 2022-03-04
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are systemic, effective or short-term effective in some patients, costly, and may cause complications such as serious infection and toxicity 6

Method used

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  • A kind of NO-loaded targeting microbubble and preparation method and application thereof
  • A kind of NO-loaded targeting microbubble and preparation method and application thereof
  • A kind of NO-loaded targeting microbubble and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation of Example 1 Loaded NO Microbubbles (NO-MBs)

[0037] 18mg 1,2-dipalmitoyl-rac-glycero-3-phosphocholine, 3.5mg

[0038] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],

[0039] 1 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphate and 0.72 μg of biotin-DSPE-PEG2000 were dissolved in 4 ml of chloroform, and the chloroform was evaporated by a rotary evaporator until a film appeared at the bottom of the bottle. Then, 4 ml of phosphate-buffered saline (containing 0.72 mg of streptavidin) was added to hydrate the film, and placed in a constant temperature shaker at 60° C. for 1 hour to obtain liposomes. 4ml liposomes are packed into 1.5ml centrifuge tubes per 0.5ml volume. Under vacuum conditions, different proportions of perfluoropropane (C 3 f 8 ) and NO (0, 25%, 50%, 75% and 100%) were continuously injected into 0.5 ml of liposome suspension. Finally, the centrifuge tube was vibrated mechanically for 45 seconds using a Vial...

Embodiment 2

[0040] Example 2 NO-MB C4d and control microbubbles NO-MB Con preparation of

[0041] When the content gas of NO-MBs is 75%C 3 f 8 and 25% NO, its stability and contrast are the best, so C 3 f 8 :NO=3:1 NO-MBs. Obtaining NO-MB using streptavidin-biotin coupling Con and NO-MB C4d . For binding to streptavidin MBs, a C4d antibody (1 mg / mL, anti-Rat C4dCat. No. HP8034; Hycult Biotech Inc., Plymouth Meeting, PA) was biotinylated. Immunoglobulin G (IgG) antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as specificity comparison. Incubate 4.5 μg of antibody with NO-MBs at room temperature for 30 minutes, then centrifuge and wash (2000rpm, 2min), use a needle to suck off the lower layer of liquid to remove unbound antibody, resuspend the upper layer of microbubbles with 500ul of phosphate-buffered saline, NO-MB Con and NO-MB C4d .

[0042] NO-MB was measured using a Coulter counter (Multisizer 3 Coulter counter, Beckman Coulter, Inc.) Con and NO-MB...

Embodiment 3

[0043] Example 3 Modeling and Contrast

[0044] (1) Establishment of heart transplantation AMR model

[0045] Adult male (200g-250g) BN and Lewis rats were purchased from Weitong Lihua, and placed in the animal room of Sun Yat-sen University for breeding. Animal experiments were approved by the Animal Committee of Sun Yat-Sen University. Two weeks before heart transplantation, the skin of BN rats was transplanted into the dorsal region of Lewis rats. During heart transplantation, the aorta and pulmonary artery of the donor heart are anastomosed with the recipient's aorta and inferior vena cava, respectively.

[0046] (2) Image acquisition for C4d deposition assessment and quantitative analysis

[0047] When performing contrast-enhanced echocardiography for detection of transplanted hearts, the cross-section was fixed on the long axis of the left ventricle. 100 μl NO-MB C4d and NO-MB Con with the same microbubble concentration (1.31±0.13×10 6 Contrast 1.29±0.14×10 6 ,P>...

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Abstract

The invention discloses a targeting microbubble loaded with NO and a preparation method thereof. The microbubble is a hollow liposome, streptavidin is combined on the surface of the liposome, and C4d modified by streptavidin and biotin / C3d antibody coupling, the liposome is filled with a mixed gas of NO / CO and polymer inert gas. The present invention successfully prepared NO‑MB C4d This new type of microbubbles, and using the microbubbles, has successfully realized the targeted and precise treatment of AMR in grafts. The treatment process is safe and has a good clinical application prospect.

Description

technical field [0001] The invention relates to the technical field of heart transplantation rejection treatment, in particular to a C4d targeting microbubble loaded with NO (nitric oxide) and its preparation method and application. Background technique [0002] Heart transplantation is the best treatment for end-stage heart disease, however, the incidence of antibody-mediated rejection (AMR) in heart transplantation remains high and is considered an important factor in graft loss 1 . [0003] AMR is caused by anti-donor specific antibody (DSA). After DSA binds to the antibody on the surface of the graft vascular endothelial cells, it activates the complement system through the classical pathway and damages the graft. At the same time, chemokines such as C3a and C5a can recruit immune cells such as macrophages, T cells, and B cells to migrate to the graft, thereby damaging the graft. 2 . C4d is a cleavage fragment activated by complement, which can be covalently bound to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K33/00A61K47/54A61K47/68A61K47/69A61P37/06
CPCA61K33/00A61K47/557A61K47/68A61K47/6913A61P37/06A61K2300/00
Inventor 孙启全廖涛
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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