CpG medicine as well as preparation method and application thereof
A drug and composition technology, applied in the field of CpG drugs and their preparation, can solve the problems of limited application, lack of further docking, lack of further development of CpG-related applications, etc., to achieve good killing, enhanced immune monitoring, high-efficiency and low-toxic delivery effects Effect
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Embodiment 1
[0079] This embodiment prepares CpG medicine through the following steps
[0080] (1) Polypeptide synthesis: synthesize MMP-2 substrate polypeptides with different hydrophilic and hydrophobic properties, and adjust the hydrophilic and hydrophobic properties of the polypeptide by adjusting the number of hydrophilic amino acid serine at the C-terminal of the polypeptide. The synthetic sequences are GPLGVRGSSS, GPLGVRGSSSSS, and GPLGVRGSSSSSSS. segment polypeptide.
[0081] (2) Synthesis of poly-N-isopropylacrylamide-b-methyl methacrylate and poly-N-hydroxyethylacrylamide-b-methyl methacrylate
[0082] Poly-N-isopropylacrylamide-b-methyl methacrylate: Add N-isopropylacrylamide to a round bottom flask, then add 2-(dodecyltrithiocarbonate)-2 -N-hydroxysuccinimide methylpropionate and azobisisobutyronitrile, dissolved in N,N-dimethylformamide (DMF) at a concentration of 1.5g / mL, stirred and dissolved, sealed the system, Introduce nitrogen gas for 30 minutes, and react at a constant ...
Embodiment 2
[0095] This example uses the co-assembled micelles obtained in Example 1 to test the induction of autophagic death of TLR9-positive tumor cells
[0096] First, TLR9-positive B16 tumor cells and TLR9-negative LLC1 tumor cells were used as research objects to observe the different tumor cell killing effects of different molecules on TLR9 receptor expression at different doses. Divide B16 and LLC1 cells into 6 × 10 3 Seed in 96-well plates at 37 °C and 5% CO 2 The drug and different molecules were prepared according to a certain concentration gradient and added to the medium (drug molecule CpG concentration 0.4μg / mL, 1μg / mL, 2μg / mL, 4μg / mL, 10μg / mL, 20μg / mL), cultured for another 24 hours; removed the medium containing the drug and different drug molecules, and washed the cells twice with phosphate buffered saline (PBS) to remove the residues of the medium and drug molecules, and then CCK The -8 reagent was diluted in the culture medium at a concentration of 10% (v / v), and aft...
Embodiment 3
[0102] This example uses the CpG drug prepared in Example 1 for the treatment test of TLR9 positive tumor cells
[0103] Inoculate the B16 tumor tissue block and LLC1 tumor tissue block on the right hind limb of the mouse. After about 5 days, the tumor to be inoculated in the mouse grows to a size of about 200mm 3 Start cancer treatment. Intravenous administration and detection of tumor size at intervals of one day, the given drug is molecule 4 that meets the implementation and preparation of the above method, and the control drug molecule is given to other groups of mice at the same time (the control drug molecule includes normal saline shown in C in the figure, CpG alone). drug molecule, molecule 1, molecule 2, molecule 3, and molecule 4), for about 2 weeks of treatment; observe the size of the tumor after the treatment ( Figure 6A and Figure 6B shown), and keep recording the tumor volume (such as Figure 7A and Figure 7B shown) and survival (as shown in Table 1). It...
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