Biologically mineralized insulin protein nano particle and preparation method and application thereof
A biomineralization and nanoparticle technology, applied in chemical instruments and methods, peptide/protein components, medical preparations containing active ingredients, etc., can solve the problems of high cost, low efficiency, poor protein biocompatibility, etc. Low cost, simple and easy preparation method, good biocompatibility
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Embodiment 1
[0052] Example 1 Preparation and Characterization of Biomineralized Insulin Protein Nanoparticles (BIP)
[0053] Human insulin protein was prepared with Tris-HCl (1.0mol / L, pH7.4) at a concentration of 5mg / mL;
[0054] Calcium chloride was prepared with triple distilled water to a final concentration of 1.0 mmol / L.
[0055] Add 8 μL of 5 mg / mL human insulin protein solution and 5 μL of 1 mmol / L calcium chloride solution to 1 mL of high-glucose DMEM medium in sequence. 2 The cells were cultured in a cell culture box for 4 hours, centrifuged at 8000g for 10 minutes, and biomineralized insulin protein nanoparticles were separated.
[0056] The resulting biomineralized insulin protein nanoparticles were analyzed by SEM (scanning electron microscope), TEM (transmission electron microscope), DLS (dynamic light scattering) and TGA / DSC (thermogravimetric / differential scanning calorimetry), XRD ( X-ray diffraction) and FTIR (Fourier transform infrared) and other technical means to ch...
Embodiment 2
[0063] Example 2 MTT method detects the cytotoxicity of insulin and biomineralized insulin protein nanoparticles
[0064] Hep-G2 cells in good growth state were digested with trypsin digestion solution, dispersed into a single cell suspension with DMEM medium containing 10% fetal bovine serum, and divided into 5×10 3 seeded in 96-well plates at 37°C, 5% CO 2 Conditioned for 24 hours. After the cells adhered to the wall, add 1 × 10 -6 mol / L dexamethasone solution induced by high glucose DMEM medium for 60 hours to form insulin resistant cell model.
[0065] After the resistance model was established, the medium was discarded, and different concentrations of Ca were added to the cell culture plate. 2+ and high glucose DMEM medium with different concentrations of insulin protein. At 37°C, 5% CO 2 After culturing for 4 hours under conditions, replace with 100 μL fresh DMEM medium containing 10% fetal bovine serum and culture for 48 hours. Then add 10 μL of 5 mg / mL MTT soluti...
Embodiment 3
[0067] Example 3 Delivery of Biomineralized Insulin Protein Nanoparticles in Insulin-Resistant Cells
[0068] Referring to the method in Example 2 "Measurement of Cytotoxicity of Insulin and Biomineralized Insulin Protein Nanoparticles by MTT Method", an insulin-resistant Hep-G2 cell model was established. After the resistance model is established, discard the medium in the cell culture plate, add biomineralized insulin protein solution (insulin protein is labeled with FITC) to the cell culture plate, and place at 37°C, 5% CO 2 Incubate in a cell incubator, and in the same way, add unmineralized insulin (that is, add insulin protein solution in DMEM medium, insulin protein is labeled with FITC, and the final concentration is the same as that of biomineralized insulin protein solution, Both were 40mg / L) as a control.
[0069] After incubation for 4 hours, the liquid in the cell culture plate was sucked off, rinsed twice with PBS buffer, and rinsed with 0.03% EDTA solution. Ce...
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