A double aptamer-modified targeted drug delivery system and its preparation method
A technology of targeted drug delivery and aptamer, which can be used in pharmaceutical formulations, medical preparations with non-active ingredients, and medical preparations containing active ingredients, etc. It can solve the problems of tumor recurrence and metastasis, limiting the efficacy of chemotherapy, etc.
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Embodiment 1
[0032] Example 1: Synthesis of PEG-Ad5
[0033] First, take 100 μL of COOH-PEG2000-COOH solution with a concentration of 6.05 μM, and then add 100 μL of NHS (N-hydroxysuccinimide) solution with a concentration of 6.05 μM and EDC (carbodiimide hydrochloride) with a concentration of 12.1 μM salt) solution 100 μL, adjusted the pH to 6.0 with MES buffer, stirred gently at room temperature, and incubated for 1 h to randomly activate the carboxyl groups in COOH-PEG2000-COOH. Then add 20 μL of adenovirus stock solution, dilute to 400 μL with water, stir and react at 37° C. for 2 h (300 r / min) to prepare PEG-Ad5. Finally, ultrafiltration centrifugation was performed with an ultrafiltration centrifuge tube with a molecular cut-off of 100KD, and refined PEG-Ad5 was obtained after washing. The modified PEG-Ad5 was detected by SDS-PAGE electrophoresis. Coomassie brilliant blue was used for protein staining, and 0.1mol / L iodine solution was used for PEG staining.
[0034] The pretreated...
Embodiment 2
[0036] Example 2: Synthesis of DUP-1-PEG-Ad5
[0037] Take the purified PEG-Ad5, add 50 μL of NHS solution with a concentration of 6.05 μM and 50 μL of EDC solution with a concentration of 12.1 μM, adjust the pH to 6.0 with MES buffer, and incubate for 1 h at room temperature to activate the refined PEG-Ad5. Another unreacted carboxyl group in Ad5. Then, 100 μL of DUP-1 aptamer solution with a concentration of 3.03 μM was added to the reaction solution, and the volume was adjusted to 400 μL with water, and stirred at 37°C, and reacted in the dark for 2 hours (300r / min) to prepare DUP-1-PEG- Ad5. Finally, an ultrafiltration centrifuge tube with a molecular cut-off of 100KD was used for ultrafiltration and centrifugation, and refined DUP-1-PEG-Ad5 was obtained after washing. Dilute the refined DUP-1-PEG-Ad5 with water to 1mL, detect the fluorescence intensity of the DUP-1 aptamer in the solution, and then use the standard curve of the fluorescence intensity of the DUP-1 aptame...
Embodiment 3
[0038] Example 3: Synthesis of A10-3.2 / DUP-1-PEG-Ad5
[0039] Take the refined DUP-1-PEG-Ad5, add 50 μL of NHS solution with a concentration of 6.05 μM and 50 μL of EDC solution with a concentration of 12.1 μM, adjust the pH to 6.0 with MES buffer, and incubate for 1 h at room temperature with gentle stirring. Activation of remaining unreacted carboxyl groups in purified DUP-1-PEG-Ad5. Then use MES buffer to adjust the pH of the reaction solution to 7.0, add 100 μL of A10-3.2 aptamer solution with a concentration of 3.03 μM, dilute to 400 μL with water, and react at 37 ° C for 2 h (300 r / min) in the dark to prepare A10-3.2 / DUP-1-PEG-Ad5 was obtained. Finally, an ultrafiltration centrifuge tube with a molecular cut-off of 100KD was used for ultrafiltration and centrifugation, and refined A10-3.2 / DUP-1-PEG-Ad5 was obtained after washing. Dilute the refined A10-3.2 / DUP-1-PEG-Ad5 with water to 1 mL, detect the fluorescence intensity of the A10-3.2 aptamer in the solution, and th...
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