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Complex nanoparticles of poloxamer and/or combination of poloxamer amine and PEG lipids

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

Active Publication Date: 2020-12-25
SHENZHEN LONGUIDE BIOPHARMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DNA vaccines have the following serious safety issues: whether the exogenous DNA integrates into the host genome after entering the body, resulting in the activation of oncogenes or the inactivation of tumor suppressor genes; For example, it leads to low immune function of the body; whether the vaccine DNA, as a foreign substance, will cause the body to produce anti-DNA antibodies; whether the CTL response induced by the DNA vaccine will have a killing effect on other cells
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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  • Complex nanoparticles of poloxamer and/or combination of poloxamer amine and PEG lipids
  • Complex nanoparticles of poloxamer and/or combination of poloxamer amine and PEG lipids
  • Complex nanoparticles of poloxamer and/or combination of poloxamer amine and PEG lipids

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Preparation of various prescriptions of cationic complex nanoparticles LLLRNA1003

[0046] Prescription 1: The molar ratio of T304:PEG2000-C-DMG:DSPC:mPEG2000-C-CLS is 100:3:20:80

[0047] First take T304 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; mix PEG2000-C-DMG, DSPC and mPEG2000-C-CLS were taken out from the -20°C refrigerator, equilibrated to room temperature and opened, weighed at room temperature with a molar ratio of 3:20:80, and dissolved in chloroform in a round bottom flask. Use a rotary evaporator to evaporate and remove chloroform, pour the stock solution A into a round-bottomed flask, and continue to sonicate for 60 minutes at 40 degrees Celsius with a 2-second pause and a 2-second pause, transfer it to a dialysis bag with a MWCO of 5000, and...

Embodiment 2

[0062] Example 2: Characterization of the cationic complex nanoparticle formulation of poloxamer and / or poloxamine combined with lipid

[0063] The present invention relates to the particle size and potential of nanoparticles. Malvem Zetasizer Nano ZSE is used to test the cationic complexes containing FLuc-mRNA in prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6, prescription 7 and prescription 8. granules were made into 1ml of the solution to be tested, and the effects of cationic complex nanoparticles without FLuc-mRNA were investigated under the condition of 25°C. The particle size (IntensityMean), surface potential (Zeta Potential) and polydispersity (PDI) of the dynamic light scattering nanoparticles are shown in Table 1.

[0064] 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 5000, the optima...

Embodiment 3

[0069] Example 3: Testing the loading efficiency of various formulations of poloxamer and / or poloxamine and lipid-combined cationic complex nanoparticles

[0070] Quant-iT RiboGreen RNA Detection Kit (ThermoFischer Company) was used to measure the inclusion of FLuc-mRNA by each prescription of LLLRNA-1003 (prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6, prescription 7 and prescription 8). Sealing rate, the specific method refers to the kit instructions, the brief processing method of the present invention is:

[0071]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 5000, the optimal mass ratio of prescription 3 is 3500, the optimal mass ratio of prescription 4 is 100, The optimal mass ratio of prescription 5 is 1500, the optimal mass ratio of prescription 6 is 5000, the optimal mass ratio of prescr...

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Abstract

The invention discloses cationic complex nanoparticles of poloxamer and / or a combination of poloxamer amine and PEG lipids, and relates to the field of gene therapy. The cationic complex nanoparticlescomprise poloxamer and / or poloxamer amine and PEG lipids, wherein the cationic complex nanoparticles comprise one or more of formulas as shown in the specifications. As a nucleic acid carrier, the cationic complex nanoparticle is lower in cost than a virus carrier, convenient for quality control, higher in transfection efficiency than a common nano carrier, low in toxicity, good in biocompatibility, free of liquid change after administration in an in-vitro experiment, particularly suitable for transfection of isolated cells, effective in in-vivo and in-vitro transfection, and simple to prepare, so that the problem in that nucleic acid, especially mRNA, is safely and efficiently delivered in vitro and vivo is creatively solved.

Description

technical field [0001] The present invention relates to the field of gene therapy, in particular to a cationic complex nanoparticle of poloxamer and / or poloxamine combined with lipid for nucleic acid delivery, and a method for preparing the cationic complex nanoparticle, Its use in in vitro and in vivo cell gene transfection, 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K9/51A61K47/10A61K47/18A61K47/24A61K47/28A61K39/00A61P35/00A61P31/16B82Y5/00B82Y30/00C12N15/88
CPCA61K48/0041A61K9/5146A61K39/00A61P31/16A61P35/00B82Y5/00B82Y30/00A61K2039/53C12N15/88Y02A50/30
Inventor 张龙贵刘晨
Owner SHENZHEN LONGUIDE BIOPHARMA CORP
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