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Neutral complex nanoparticles composed of poloxamer and/or poloxamine and lipid

A technology of poloxamine and poloxamer, which is applied in the field of neutral complex nanoparticles, in vivo and in vitro cell gene transfection, and can solve the problems of high toxicity in vivo, unfavorable targeted modification, inactivation, etc.

Active Publication Date: 2020-06-09
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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  • Neutral complex nanoparticles composed of poloxamer and/or poloxamine and lipid
  • Neutral complex nanoparticles composed of poloxamer and/or poloxamine and lipid
  • Neutral complex nanoparticles composed of poloxamer and/or poloxamine and lipid

Examples

Experimental program
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Effect test

Embodiment 1

[0054] Example 1: Preparation of neutral complex nanoparticles of poloxamer and / or poloxamine combined with lipids

[0055] Recipe 1: T304:17R4:PC molar ratio is 172:105:1

[0056] First, take T304 and 17R4 out of the refrigerator at 4°C and equilibrate to room temperature, weigh them at room temperature according to the molar ratio of 172:105, add nuclease-free ultrapure water to dissolve, fully shake for 5 minutes with a mediator, and obtain stock solution A; Take PC out of the refrigerator at -20°C, equilibrate to room temperature, unseal, weigh at room temperature, dissolve with ethanol at 40°C, add nuclease-free ultrapure water drop by drop, ultrasonicate for 60 minutes, and transfer to a dialysis bag with MWCO of 10,000 Dialysis was performed with nuclease-free ultrapure water for 24 hours, and the dialysate was changed every 6 hours. After dialysis, filter with a 0.22um water-based filter membrane to obtain stock solution B. Mix stock solution A and stock solution B at...

Embodiment 2

[0069] Embodiment two: the characterization of prescription

[0070] The particle size and potential of the neutral complex nanometer involved in the present invention are tested by Malvern Zetasizer Nano ZSE, and the neutrality of prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6 and prescription 7 without FLuc-mRNA The complex nanoparticles were made into 1ml of the test solution, and the neutral complex nanoparticles without FLuc-mRNA were investigated under the condition of 25°C for prescriptions 1, 2, 3, 4, 5, 6 and 7. The particle size (Intensity Mean), surface potential (Zeta Potential) and polydispersity (PDI) of the dynamic light scattering nanoparticles.

[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 400, the optimal mass ratio of prescription 3 is 2000, the optimal mass ratio of ...

Embodiment 3

[0076] Example 3: Encapsulation efficiency of various formulations of neutral complex nanoparticles of poloxamer and / or poloxamine combined with lipids

[0077] 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:

[0078] 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 400, the optimal mass ratio of prescription 3 is 2000, the optimal mass ratio of prescription 4 is 5000, The optimal mass ratio of prescription 5 is 2000, the optimal mass ratio of prescription 6 is 100 and the optimal mass ratio of prescription 7 is 400) Weigh the neutral comp...

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Abstract

The invention provides neutral complex nanoparticles composed of poloxamer and / or poloxamine and lipid, and relates to the field of gene therapy. The nanoparticles comprise the poloxamer and / or poloxamine and the lipid, and contain PC or DSPC. The complex provided by the invention can be used for gene transfection of cells in vivo and in vivo, can deliver extracellular genes to specific cells to realize gene expression, can be used for scientific research, especially for preparing a gene transfection kit or a cell labeling kit, and can be used for developing novel drugs for humans, especiallyfor developing mRNA tumor vaccines and mRNA influenza virus vaccines.

Description

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

Claims

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Application Information

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