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Ultrasonic contrast agent for stem cell ultrasonic tracing and preparation method thereof

An ultrasound contrast agent and stem cell technology, which can be used in echo/ultrasound imaging agents, preparations for in vivo experiments, pharmaceutical formulations, etc. problem, to achieve the effect of high biological safety, high stability and long circulation time in vivo

Active Publication Date: 2018-10-12
GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, for ultrasonic tracer imaging of stem cells, there are very few similar studies in other research groups in China, such as using micron-scale ultrasonic microbubbles to label stem cells to form stem cell-microbubble communities, but ordinary lipid microbubbles Most of the diameters are micron-scale and the stability is poor, which makes the tracking time of the system short and fails to meet the clinical needs of stem cell transplantation therapy; and we use CD90 to modify the lipid PLGA nano-microbubble contrast agent, so that it can be specifically identified by stem cells Endocytosis assists in enhancing the imaging ability of stem cells, therefore, the present invention has higher practicability and imaging effect

Method used

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  • Ultrasonic contrast agent for stem cell ultrasonic tracing and preparation method thereof
  • Ultrasonic contrast agent for stem cell ultrasonic tracing and preparation method thereof
  • Ultrasonic contrast agent for stem cell ultrasonic tracing and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of lipid PLGA nano-microbubble-stem cell complex:

[0042] (1) Dissolve PLGA and DSPE-PEG2000-biotin in dichloromethane.

[0043] (2) Add an appropriate concentration of 6% NH 4 HCO 3 The aqueous solution is emulsified in the above-mentioned PLGA phospholipid mixture using an ultrasonic breaker.

[0044] (3) Add the emulsion into the PVA solution with a concentration of 4%. After the homogenization is completed, stir magnetically at room temperature for 2.5-3 hours to obtain biotinylated lipid PLGA nano-microbubbles.

[0045] (4) Lipid-PLGA nano-microbubbles targeted to CD90 were prepared by linking biotinylated lipid PLGA nano-microbubbles with CD90 antibody through biotin-streptavidin reaction.

[0046] (5) CD90-targeted lipid PLGA nano-microbubbles and rat bone marrow mesenchymal stem cells extracted from the femoral and tibial bone marrow cavity of SD rats by the whole bone marrow adherent culture method mediated endocytosis of stem cells th...

Embodiment 2

[0047] Embodiment 2 Contrast effect maintenance time:

[0048] Add a certain amount of the lipid PLGA nano-microbubble-stem cell complex prepared by the method in Example 1 into the ultrasonic gel imitation body prepared by 1% agarose solution, and observe it with a 16MHz ultrasonic probe at 2 hours, 4 hours, and 36 hours respectively. Imaging effects in vitro in two-dimensional and contrast modes. see results figure 2 . This example is to illustrate the length of time that the ultrasound contrast agent can maintain the contrast ability in vitro. It can be seen that it can still provide good ultrasound imaging and contrast effects stably at 36 hours. This result shows that the complex is helpful for observation The distribution of stem cells near the designated injection area after transplantation.

Embodiment 3

[0049] Example 3 Ultrasound contrast agent guides the fixed-point injection of stem cells in animals:

[0050] Centrifuge the prepared lipid PLGA nanobubble-stem cell complex at a speed of 1000rpm for 4 minutes, collect the cells, and dilute the cells to 2×10 with PBS buffer. 7 pieces / ml.

[0051] The complex was orthotopically injected into mice bearing C6 xenograft tumors subcutaneously ( image 3 ) or inside the tumor ( Figure 4 ), a 16MHz small animal ultrasound probe was placed on the surface of the observation site, and it could be observed that the local injection area had a more pronounced ultrasound enhancement effect than the non-injection area.

[0052] According to the comparison of the ultrasound imaging effects between the non-antibody group and the antibody group, it can be seen that the targeted lipid PLGA microbubbles added with antibodies can specifically bind to the surface molecules of stem cells, and the specific endocytosis of stem cells can enhance an...

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Abstract

The invention discloses an ultrasonic contrast agent for stem cell ultrasonic tracing and a preparation method thereof. The ultrasonic contrast agent is constructed from stem cells modified by lipid PLGA nano-micro-bubbles,wherein the complex comprises CD90-targeted lipid PLGA nano-micro-bubbles and stem cells which are interacted with each other. According to the ultrasonic contrast agent,the lipid PLGA nano-micro-bubble and stem cell complex is successfully constructed by using modified nano-micro-bubble groups and an abundantly expressed CD90 antigen molecular reaction,endocytosis of the stem cells on the lipid PLGA nano-micro-bubbles can be enhanced,the activity and dryness of the stem cells are kept,ultrasonic real-time imaging guide stem cell transplantation therapy is realized,thenenhancement of the stem cell transplantation treatment effect is further assisted,and the ultrasonic contrast agent has certain significance in the field of stem cell tracing.

Description

technical field [0001] The invention relates to an ultrasonic contrast agent for ultrasonic tracing, in particular to an ultrasonic contrast agent for ultrasonic tracing of stem cells and a preparation method thereof. Background technique [0002] Stem cells (stem cells) are a kind of multi-potential cells with self-replication ability, and its types include embryonic stem cells, bone marrow mesenchymal stem cells, hematopoietic stem cells, neural stem cells, tendon stem cells and so on. At present, stem cells have been widely used in many fields of medicine, such as cell replacement therapy, gene therapy, tissue engineering, regenerative medicine, etc., and the development of stem cell regenerative medicine is bringing a revolution to modern medicine. For stem cell transplantation therapy, how to make the transplanted stem cells accurately distribute to the injury site and maintain its high survival rate to ensure the efficacy of stem cell therapy, and how to ensure the saf...

Claims

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Application Information

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IPC IPC(8): A61K49/22
CPCA61K49/223
Inventor 刘红梅刘赛石辅天王宇
Owner GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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