Double-targeting compound nano-gel, and preparation method and application thereof
A nanogel, dual-targeting technology, applied in nanotechnology, nanotechnology, nanomedicine, etc., can solve problems such as incompatibility, achieve effective treatment, increase concentration, and break through the bottleneck of treatment
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[0041] A method for preparing a dual-target composite nanogel, comprising the steps of:
[0042] a. Preparation of nanogels
[0043] Herceptin and hyaluronic acid are cross-linked by carboxyl and amino groups to generate nanogels with a particle size in the range of 500-5000 nanometers, in which the molar mass ratio of HA (hyaluronic acid) and antibody Herceptin (Herceptin) is 20:1-1:1.
[0044] b. Preparation of positively charged nanoparticles: Mix the cationic liposome prepared by the film dispersion method with the aqueous solution of the cationic polypeptide, and then add it to siRNA to prepare positively charged nanoparticles on the surface, wherein the cationic liposome of the nanoparticle, The mass ratios of polypeptides and nucleic acids are 1:8:1, 1:4:1 and 1:2:1 respectively, and incubated at room temperature for 30-60 minutes. Then correspondingly add different proportions of nanogels, wherein the mass ratios of HA and siRNA in the nanogels are 20:1:4:1, 25:1:4:1...
Embodiment 1
[0046] Embodiment 1, preparation and characterization of nanogel Herceptin-HA
[0047] Such as figure 1 As shown, Herceptin and hyaluronic acid were cross-linked in different mass ratios. Negatively charged micron-sized nanogels were prepared. SDS-PAGE to verify the connection of antibodies: Mix the connected Herceptin-HA, negative control group and Herceptin with Loading buffer (v / v=5), heat in a water bath at 95°C for 5 minutes to completely denature the protein. Prepare the SDS-PAGE protein gel with a mass percentage concentration of 10% according to the kit instructions. SDS buffer) in 80V for 15min, then 120V for 45min. Remove the protein glue, stain with Coomassie Brilliant Blue staining solution on a shaker for 1 hour, and decolorize with pure water ( figure 2 ). Zetasizer2000 was used to measure the surface charge and particle size of the complex, the sample measurement time was set to automatic, each sample was measured 3 times, and the average value was plotte...
Embodiment 2
[0048] Embodiment 2, composite nanogel ALPR assembly and characterization
[0049] Composite nanogels were prepared by incubating the nanogels with positively charged nanoparticles in aqueous solution for 20-40 minutes ( Figure 5 ). Zetasizer 2000 was used to measure the surface charge and particle size of the complex, the sample measurement time was set to automatic, each sample was measured 3 times, and the average value was plotted ( Figure 6 and Figure 7 ). Add the sample dropwise to the copper grid, then dropwise add a 4% phosphotungstic acid solution by mass, blot the liquid on the surface of the copper grid with filter paper, then place it overnight for transmission electron microscope photography. The experimental results obtained are as Figure 8 : Micelle in regular spherical shape. The size is between 30-60 nanometers. Cryo-TEM morphology observation: 10mg / mL DOTAP / DOPE liposomes were prepared, and then LPR and ALPR with a liposome concentration of 3.33ug / u...
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