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Nano granule of polylysine amylum and its preparation method as well as application gene carrier

A poly-lysine and starch nanotechnology, applied in biological nanotechnology and its application fields, can solve the problems of inability to degrade and discharge in time, have immunogenicity, and inorganic nanoparticles cannot be degraded in the body, and achieve DNA loading capacity. Large, low cost, mild effect

Inactive Publication Date: 2003-12-24
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing research on nanoparticles as a gene carrier system has just started, and there are still many problems to be solved in clinical treatment, such as: inorganic nanoparticles cannot be degraded in vivo; organic polymer nanoparticles have immunogenicity in vivo, Moreover, most of them cannot be degraded and excreted in time in human cells.

Method used

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  • Nano granule of polylysine amylum and its preparation method as well as application gene carrier
  • Nano granule of polylysine amylum and its preparation method as well as application gene carrier
  • Nano granule of polylysine amylum and its preparation method as well as application gene carrier

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Experimental program
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Effect test

Embodiment 1

[0035] (1): Preparation of anionic starch nanoparticles

[0036] 1. Preparation of water phase: Prepare starch into a 10% aqueous solution, heat and decompose it in boiling water at 100°C until the solution is clear, then place it at room temperature to cool.

[0037] 2. Oil phase preparation: Take 15ml of toluene and 5ml of chloroform, add 2% of the total volume of surfactant SPan80, stir and mix at a speed of 500 rpm.

[0038] 3. Take 1ml of starch hydrolyzate and add it to the high-speed stirring oil phase, and continue stirring for 20 minutes until a tiny emulsion is formed.

[0039] 4. Add the crosslinking agent POCl accounting for about 0.01% of the starch mass 3 , continue to stir and react for about 30 minutes, then wash three times with 95% alcohol, and freeze-dry to obtain anionic starch nanoparticles with a size of 30-50mm and a potential value lower than -5mV in a neutral pH environment. (2): Preparation of polylysine starch nanoparticles

[0040] 1. Weigh 1 mg ...

Embodiment 2

[0045] Transduction and expression of polylysine starch nanoparticles loaded with green fluorescent protein gene (pEGFP) in human tumor cells

[0046] 1. Construction of nano-gene carrier: Take 10 μl of poly-lysine starch nanoparticle aqueous suspension with a concentration of 1 μg / μl, add 3 μg pEGFP plasmid DNA, mix well, and place at room temperature for 30 minutes.

[0047] 2. Dilution of the nanogene carrier: add the above-mentioned DNA / nanoparticle complex into 100 μl of serum-free medium, mix well, and let stand for 30 minutes.

[0048] 3. Co-cultivation and transformation: Add the medium containing the DNA / nanoparticle complex dropwise to the culture dish in which the human breast cancer cells are cultured, co-cultivate for 5-8 hours, and then add complete medium for co-cultivation.

[0049] 4. Detection of transgenes: After 24-48 hours of co-cultivation, observe the fluorescence of the cells under a fluorescence microscope. see Image 6 , fluorescence indicates that ...

Embodiment 3

[0051] Transduction of polylysine starch nanoparticles loaded with green fluorescent protein gene (pEGFP) in rice callus cells.

[0052] 1. Construction of nano-gene carrier: Take 10 μl of polylysine starch nanoparticle aqueous suspension with a concentration of 1 μg / μl, add 3 μg of pEGFP plasmid DNA, mix well, and place at room temperature for 30 minutes to obtain DNA / nanoparticle complexes.

[0053] 2. Establishment of rice suspension cell line: Transplant well-growing rice callus into fresh medium of MS+BA 0.2 mg / l+2,4-D 1 mg / l. One week later, the uppermost callus was selected and transferred to the culture medium, and cultured in the dark for 48 hours on a shaker at 110 rpm.

[0054] 3. Co-cultivation: Add acetosyringone to the above-mentioned culture medium with a final concentration of 100 μM, sonicate for 15 seconds, add DNA / nanoparticles, and co-cultivate on a shaker at 110 rpm.

[0055] 4. Detection of transgene: after 36 hours of co-culture, observe whether there i...

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Abstract

A polylysine-starch nanoparticle composed of the anionic starch nanocore and the cationic polylysine shell layer is prepared through adding the hydrolytic starch liquid to oil phase, stirring, addingcross-linking agent, reaction while stirring, washing with alcohol, drying to obtain said nanocores, dispersing them in buffering phosphoric acid solution, adding the aqueous solution of polylysine, waving and centrifugal separation. It can be used as gene carrier with advantages of high transduction efficiency, no immunogenicity, no cell toxic, and good biodegradability.

Description

Technical field: [0001] The method mainly relates to the field of biological nanotechnology and its application, especially the application of biological nanomaterials in the research and development of molecular biology, genetic engineering technology, biomedicine and other fields. Background technique: [0002] The gene carrier system is the key to the realization of gene transduction and gene therapy. A suitable gene carrier can introduce therapeutically valuable genes into human cells in a safe, efficient, controllable, and easy way, and realize its expression, so as to achieve the purpose of treating diseases . At present, there are mainly two kinds of gene carrier systems: virus-mediated and non-viral-mediated, and virus-mediated is the main one. However, due to the unavoidable shortcomings of viral vector systems, such as the immune response and toxicity of the virus itself, more and more researchers have turned their attention to non-viral vector systems. With the ...

Claims

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Application Information

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IPC IPC(8): A61K47/36C08J3/12C08L3/02
Inventor 刘选明肖苏尧刘斌刘俊唐冬英
Owner HUNAN UNIV
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