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Compound of poloxamer and/or poloxamine and lipid compound

A technology of poloxamine and poloxamer, which is applied in the preparation of complex nanoparticles, in vivo and in vitro cell gene transfection, and can solve the problems of complex operation, high toxicity and low transfection efficiency

Pending Publication Date: 2020-06-16
SHENZHEN LONGUIDE BIOPHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above non-viral gene delivery methods have the following problems: low transfection efficiency, high toxicity, complicated operation, unfavorable for targeted modification, etc.
At present, scientists around the world have developed a variety of nano-delivery carriers to protect RNAi, DNA, mRNA and other genes from degradation mediated by extracellular DNase or RNase and promote their entry into cells, while improving RNAi, DNA, mRNA and other genes The pharmacokinetic properties of preparations, but most of the current delivery systems still have serious instability, short half-life, high toxicity in vivo, and low transfection efficiency

Method used

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  • Compound of poloxamer and/or poloxamine and lipid compound
  • Compound of poloxamer and/or poloxamine and lipid compound
  • Compound of poloxamer and/or poloxamine and lipid compound

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 Preparation of complex nanoparticles of poloxamer and / or poloxamine combined with lipid

[0041] Prescription 1: T904:DOTAP:DOPE molar ratio is 96:3:1

[0042] First take T904 out of the 4°C refrigerator and equilibrate to room temperature, weigh it at room temperature and add nuclease-free ultrapure water to dissolve it, shake it fully with a mediator for 5 minutes, and let it stand overnight to obtain stock solution A; DOTAP and DOPE from -20 Take it out from the refrigerator at ℃, equilibrate to room temperature and open the seal, weigh it at room temperature with a molar ratio of 3:1, dissolve it with ethanol at 40 °C, add it dropwise to nuclease-free ultrapure water, ultrasonicate for 30 minutes, and transfer it to a medium with a MWCO of 2000. In the dialysis bag, dialyze with nuclease-free ultrapure water for 12 hours, and change the dialysate every 3 hours. After dialysis, filter with a 0.22um water-based filter membrane to obtain stock solution B....

Embodiment 2

[0055] Embodiment two: the characterization of prescription

[0056] The present invention relates to the particle size and potential of nanoparticles, which are tested by Malvern Zetasizer Nano ZSE, and the compound nanoparticles without FLuc-mRNA in prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6 and prescription 7 are made into 1ml For the solution to be tested, the particle size of the dynamic light scattering nanoparticles of the composite nanoparticles without FLuc-mRNA was investigated under the condition of 25°C Size (Intensity Mean), surface potential (ZetaPotential) and polydispersity (PDI).

[0057] According to the optimal mass ratio of nanoparticles and FLuc-mRNA in each prescription (the optimal mass ratio of prescription 1 is 5000, the optimal mass ratio of prescription 2 is 1000, the optimal mass ratio of prescription 3 is 5000, and the optimal mass ratio of prescription 4 is 4500 , the best mass ratio of prescrip...

Embodiment 3

[0061] Example Three: Encapsulation Efficiency of Various Prescriptions of Complex Nanoparticles Combining Poloxamer and / or Poloxamine and Lipid

[0062] Quant-iT RiboGreen RNA Detection Kit (ThermoFische Company) was used to measure the encapsulation rate of FLuc-mRNA in each prescription (prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6 and prescription 7). For specific methods, refer to Kit instructions, the brief processing method of the present invention is:

[0063] According to the optimal mass ratio of nanoparticles and FLuc-mRNA in each prescription (the optimal mass ratio of prescription 1 is 5000, the optimal mass ratio of prescription 2 is 1000, the optimal mass ratio of prescription 3 is 5000, and the optimal mass ratio of prescription 4 is 4500 , the best mass ratio of prescription 5 is 50, the best mass ratio of prescription 6 is 5000 and the best mass ratio of prescription 7 is 2500) Weigh the FLuc-mRNA-free composi...

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Abstract

The invention discloses a poloxamer and / or poloxamine and lipid compound, and relates to the field of gene therapy. The poloxamer and / or poloxamine and lipid compound comprises poloxamer and / or poloxamine and lipid. Wherein the compound is composed of any one of components disclosed in the invention with DOTAP and DOPE; the cost of the compound as a nucleic acid vector is lower than that of a virus vector; compared with a common nano-carrier, the compound is high in transfection efficiency, low in toxicity and good in biocompatibility, liquid does not need to be replaced after administration in an in-vitro experiment, the compound is particularly suitable for transfection of in-vitro cells, in-vivo and in-vitro transfection has high expression, preparation is easy, and the problem that nucleic acid, especially mRNA, is safely and efficiently delivered in vivo and in vitro is creatively solved.

Description

technical field [0001] The present invention relates to the field of gene therapy, in particular to a composite nanoparticle of poloxamer and / or poloxamine combined with lipid for nucleic acid delivery, and a method for preparing the composite nanoparticle, and It is used for gene transfection of cells in vivo and in vitro, and its application in the field of new vaccine drugs. Background technique [0002] Gene therapy refers to the introduction of exogenous therapeutic genes into target cells to correct or compensate diseases caused by gene defects and abnormalities, so as to achieve therapeutic purposes. Gene delivery is the delivery of biologically active exogenous therapeutic genes to the patient's recipient cells through delivery technology, and the translation of exogenous therapeutic genes to produce protein polypeptides to treat certain diseases. Gene transfection is a technology that transfers or transports nucleic acids with biological functions into cells and al...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/51A61K47/18A61K47/10A61K39/00A61P31/16A61P35/00B82Y5/00B82Y30/00C12N15/87
CPCA61K48/0041A61K9/5146A61K39/00A61P31/16A61P35/00B82Y5/00B82Y30/00C12N15/87A61K2039/53Y02A50/30
Inventor 张龙贵刘晨
Owner SHENZHEN LONGUIDE BIOPHARMA CORP
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