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
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[0034] Example one
[0035] (1): Preparation of anionic starch nanoparticles
[0036] 1. Water phase preparation: mix the starch into a 10% aqueous solution, place it in boiling water at 100°C and heat it to decompose until the solution is clear, and cool it at room temperature.
[0037] 2. Oil phase preparation: Take 15ml of toluene and 5ml of chloroform, add 2% of the total volume of the surfactant SPan80, stir and mix at a speed of 500 rpm.
[0038] 3. Take 1ml of starch hydrolysate 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 cross-linking agent POCl which accounts for about 0.01% of starch mass 3 , Continue to stir and react for about 30 minutes, then wash with 95% alcohol three times, freeze-dry, and obtain anionic starch nanoparticles with a size of 30-50mm and a potential value of less than -5mV in a neutral pH environment. (2): Preparation of polylysine starch nanoparticles
[0040] ...
Example Embodiment
[0044] Embodiment two:
[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 10μl of polylysine starch nanoparticle suspension with a concentration of 1μg / μl, add 3μg of pEGFP plasmid DNA, mix well, and place at room temperature for 30 minutes.
[0047] 2. Dilution of nano gene carrier: add the above-mentioned DNA / nanoparticle complex to 100 μl serum-free medium, mix well, and place for 30 minutes.
[0048] 3. Co-cultivation and transformation: Add the DNA / nanoparticle complex-dissolved medium dropwise to the culture dish for culturing human breast cancer cells for 5-8 hours, and then add complete medium for co-cultivation.
[0049] 4. Detection of transgene: After co-cultivating for 24 to 48 hours, observe the fluorescence of the cells under a fluorescence microscope. See Figure 6 , Fluorescence indicates that the gene is expressed in tumo...
Example Embodiment
[0050] Embodiment three:
[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 10μl of polylysine starch nanoparticle suspension with a concentration of 1μg / μl, add 3μg pEGFP plasmid DNA, mix well, and leave it at room temperature for 30 minutes to obtain a DNA / nanoparticle complex.
[0053] 2. Establishment of rice suspension cell line: transfer the well-growing rice callus to MS+BA0.2mg / l+2,4-D 1mg / l fresh medium. One week later, the uppermost callus was selected and transferred to the culture medium, and cultured in a dark box on a shaker at 110 rpm for 48 hours.
[0054] 3. Co-cultivation: Add acetosyringone to the above medium to a final concentration of 100 μM, ultrasonicate for 15 seconds, add DNA / nanoparticle particles, and then co-culture on a shaker at 110 rpm.
[0055] 4. Detection of transgene: After 36 hours of co-cultivation, observe whether...
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