Hydrogel loaded with OP3-4 polypeptide and anti-CXCL9 antibody as well as preparation method and application of hydrogel
A hydrogel and antibody technology, applied in prostheses, pharmaceutical formulations, drug delivery, etc., can solve the problems of short half-life and limited application of growth factors, and achieve the effects of avoiding inactivation, promoting osteogenesis, and improving the effect of bone repair
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Embodiment 1
[0034] This embodiment provides a hydrogel loaded with OP3-4 polypeptide and anti-CXCL9 antibody, the preparation method of which comprises the following steps:
[0035] 1. Preparation of methacrylamide-modified OP3-4 polypeptide (OP3-MA)
[0036] Take 500mg of OP3-4 (Mw=1449, 0.0345mmol), dissolve it in 10mL of PBS solution with pH=7.4, absorb 29.1μL of methacrylic acid (Mw=86, ~0.0345mmol), dissolve it in 10mL of pH=7.4 Add 13.25mg of EDC (Mw=192, ~0.069mmol) to the PBS solution of 7.4, stir for 15min, add 7.94mg of NHS (Mw=115, ~0.069mmol), continue stirring for 15min, and slowly add it dropwise to the above OP3 -4 solution, reacted for 6 hours, then dialyzed in pure water for 12 hours with a dialysis bag with a molecular weight cut-off of 1000, and lyophilized to obtain a methacrylamide-modified OP3-4 polypeptide, which was designated as OP3-MA.
[0037] NMR test:
[0038] Weigh 3 to 5 mg of OP3-MA, dissolve it in deuterated water, and put it into a clean NMR tube after ...
Embodiment 2
[0052] This example intends to investigate the effect of the modification of the loaded OP3-4 polypeptide and the treatment of the loaded A-CXCL9 on its release in vitro. The specific test method is as follows:
[0053] 1. Sample preparation
[0054] Sample 1 (GelMA / OP3-4)
[0055] Sample 1 is a hydrogel (GelMA / OP3-4) loaded with OP3-4 polypeptide, wherein the OP3-4 polypeptide has not been modified. The preparation method of the hydrogel of this sample is as follows:
[0056] Weigh 0.1g GelMA, dissolve it in 1mL PBS solution, prepare a GelMA solution with a concentration of 0.1g / mL, add 1mg OP3-4 polypeptide to it, weigh 1mg phenyl-2,4,6-trimethyl Lithium benzoyl phosphonate (LAP) was added to the above solution as a photoinitiator, and 500 μL of the solution was pipetted into a 48-well plate, and a stable gel was formed after irradiating with 456 nm blue light for 1 min, which was denoted as GelMA / OP3- 4.
[0057] Sample 2 (GelMA / OP3-MA)
[0058] Sample 2 is a hydrogel ...
Embodiment 3
[0071] The present embodiment explores the cytotoxicity of each hydrogel sample by cell test, and the specific test method is as follows:
[0072] After cultured rat bone marrow mesenchymal stem cells (rBMSCs) were digested and suspended with 0.25% trypsin, the density of each well was 2×10 4 cells / mL of cell suspension were seeded on 48-well plates. After culturing for 12 h, the original culture solution was taken out, and each hydrogel sample was inoculated with 10 5 100 μL / mL of cell suspension. There are at least 5 holes in each group. The medium was changed every 24 hours, and two time points of 1d and 3d were set in the experiment. Each hydrogel sample is GelMA, GelMA / OP3-MA, GelMA / A-CXCL9@PCEC and GelMA / OP3-MA / A-CXCL9@PCEC prepared by referring to the method described in Examples 1 and 2, respectively.
[0073] The specific operation method is as follows: Use CCK8 to quantitatively analyze the survival rate of cells, take out the corresponding well plate at the spec...
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