Multifunctional polypeptide/liposome/hyaluronic acid assembled viroid nucleic acid carrier

A nucleic acid carrier and hyaluronic acid technology, applied in the fields of genetic material components, recombinant DNA technology, and other methods of inserting foreign genetic materials, etc., can solve the problems of complicated problems, no new advantages in shielding charges, and less than ideal effects. , to avoid toxicity and break through the long-term circulation in the body

Active Publication Date: 2015-11-04
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that in addition to the high toxicity of polycations, this type of carrier still achieves long-term circulation in the body and targets specific tissues by chemically grafting polyethylene glycol (PEG) and targeting groups on the peripheral cationic liposomes. Receptors on the cell surface, a large number of experimental results show that the effect of this type of carrier in vivo is not ideal
Among them, the multifunctional receptor-targeted nanoparticles prepared by Professor Hart, S.L. use polypeptides and liposo

Method used

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  • Multifunctional polypeptide/liposome/hyaluronic acid assembled viroid nucleic acid carrier
  • Multifunctional polypeptide/liposome/hyaluronic acid assembled viroid nucleic acid carrier
  • Multifunctional polypeptide/liposome/hyaluronic acid assembled viroid nucleic acid carrier

Examples

Experimental program
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Example Embodiment

[0048] The present invention relates to a preparation method of the above-mentioned nucleic acid carrier, comprising the following steps:

[0049] a. Preparation of cationic liposomes by thin film dispersion method: using cationic lipids ((2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP)) and neutral lipids (1,2-dioleyloxypropyl) A chloroform solution with a mass ratio of oleoyl-sn-glycerol-3-phosphoethanolamine (DOPE)) was rotary evaporated to remove the solvent, then hydrated overnight, and sonicated for 30-60 minutes.

[0050] b. Preparation of nanocarriers: use the above-prepared liposomes to dilute to a certain concentration, mix them with a certain concentration of polypeptides at a mass ratio of 0.75:4 and 1:4, respectively, and add them to DNA and siRNA. Nanoparticles with positive charges on the surface were prepared, wherein the mass ratio of liposome, polypeptide and nucleic acid of the two types of nanoparticles was 0.75:4:1 and 1:4:1, and incubated at room ...

Example Embodiment

[0052] Embodiment 1, the preparation method of Q-complexes

[0053] like figure 1 As shown, the system for measuring physicochemical properties of Q-complexes was performed in aqueous solution. After mixing the above cationic liposomes and polypeptides in different mass ratios, add them into the nucleic acid aqueous solution and continue to mix them evenly. After incubating at room temperature for 30 minutes, polyanions in different proportions were added to the mixture and mixed well and allowed to stand for 15 minutes. Serum-free medium can be used in the carrier system for cell experiments instead of ultrapure water.

Example Embodiment

[0054] Example 2. Gel electrophoresis of Q-complexes

[0055] Prepare a mass ratio of 1.0% agarose solution, heat and dissolve in a microwave oven, take 40 mL of the solution, pour it into a special EB-contaminated beaker, add about 4 μL of EB solution, stir well, pour it into the mold, insert a comb, about 30min After that, the gel will solidify, add an appropriate amount of TAE buffer to the electrophoresis tank, put the agarose gel into the electrophoresis tank and wait for sample loading. Then, complex solutions with different mass ratios were prepared and incubated at room temperature for 30 min. The marker for loading is a 1000-10000kb plasmid maker. When loading, first take 1 μL of loading buffer, add 5 μL of sample sample, mix well, and add it to the gel well. Add a voltage of 120 volts, the blue bromophenol blue will quickly move to the bottom of the gel, about 20 minutes later, take a picture with a UV gel imaging system. Gel electrophoresis results of complexes ...

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Abstract

The invention discloses a multifunctional polypeptide/liposome/hyaluronic acid assembled viroid nucleic acid carrier. The specific building method of the carrier comprises the steps that multifunctional polypeptide and cationic liposome are mixed, and then nucleic acid is added in the mixture for self-assembling to form nanoparticles with positive charge on the surface; polyanions are wrapped on the surfaces of the nanoparticles through the electrostatic interaction, and therefore the viroid nucleic acid carrier is assembled. The carrier has the advantages that by means of the human body safe biological materials, the novel non-viral carrier is built, wherein the novel non-viral carrier can tightly wrap nucleic acid and in-vivo long-term cycle and efficient targeting cancer cells, smoothly break through a cytomembrane and an endosome membrane to enter cytoplasm through the lipidosome membrane melting effect, release nucleic acid in the subacid cytoplasm through disassembling and self-degradation or enter a cell nucleus in a nanoparticle mode by changing a polypeptide sequence.

Description

technical field [0001] The present invention relates to a drug delivery technology in the field of drug technology, in particular to a construction and preparation technology of a non-viral vector system that can be used for in vivo system delivery of nucleic acids (DNA, siRNA, microRNA, shRNA, etc.), and specifically relates to a Multifunctional polypeptide / liposome / hyaluronic acid-assembled virus-like nucleic acid vector. Background technique [0002] In gene therapy, the key bottleneck for nucleic acid drugs from conception to clinical application is the development of its in vivo delivery system. Among them, non-viral vectors are mainly polycationic and cationic liposomes. Compared with viral vectors, they have low cost, high gene loading density and molecular weight, and simple preparation, except that the carrier itself has no biological safety hazards. It has the advantages of being convenient for chemical modification, but also has the disadvantages of high chemical...

Claims

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Application Information

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IPC IPC(8): C12N15/87A61K48/00A61K9/51
Inventor 杜子秀徐宇虹谢舫胡凌云李臻博金博
Owner SHANGHAI JIAO TONG UNIV
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