A kind of multifunctional polymer micelle that jointly transports photosensitizer and gene editing system and its preparation method and application
A technology of gene editing and polymer glue, which is applied in the fields of polymer chemistry and biomedical engineering, can solve the problem of consuming tumor tissue, achieve the effects of promoting apoptosis, avoiding the reduction of tumor treatment effect, and enhancing the anti-tumor effect
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Embodiment 1
[0070] A drug-loaded polymer micelle that jointly transports a photosensitizer and a gene editing system, wherein the loaded photosensitizer is chlorin e6 (Ce6), and the gene editing system is a Cas9 / sgRNA complex, and the synthesis of the polymer micelle route process as figure 1 shown, including the following steps:
[0071] 1. Synthesis of (1S)-N-[5-[(4-Mercaptobutanoyl)amino]-1-carboxypentyl]iminodiacetic Acid (NTA-SH)
[0072] (1)(1S)-N-(5-Carbobenzyloxyamino-1-carboxypentyl)iminodiacetic Acid(N ε -Cbz-NTA) synthesis: take 8.4g N ε -Cbz-Lys (30mmol), add 45mL 2M sodium hydroxide solution to dissolve; then weigh 8.34g bromoacetic acid (60mmol) and dissolve it in 30mL 2M sodium hydroxide solution, add dropwise N ε - Sodium hydroxide solution of Cbz-Lys; react overnight at room temperature, then heat the reaction to 70°C for two hours. After the reaction, the reaction was cooled in an ice bath, and 90 mL of 1N hydrochloric acid solution was added, and a large amount of w...
Embodiment 2
[0093] The structural analysis of embodiment 2 polymer
[0094] Get about 10 mg of each step product of Example 1 and dissolve in an appropriate amount of deuterated solvent, and use 1 H-NMR 400MHz nuclear magnetic resonance spectrometer for testing, detection of polymer structure before and after the reaction 1 The change of H chemical shift, the result is as figure 2 , 4 and 6.
[0095] from figure 2 , Figure 4 and Image 6 It can be seen that all the structural hydrogens of the polymers are well assigned in the H NMR spectra, which proves the successful synthesis of the polymers.
Embodiment 3
[0096] The performance detection of embodiment 3 polymer micelles
[0097] 1. Test of protein loading capacity of NTA@Ce6 micelles
[0098] The amount of 30 μg Cas9 protein was fixed, and an equimolar amount of sgRNA 7.5 μg was added to prepare 37.5 μg of Cas9 / sgRNA complex. Different amounts of NTA@Ce6 micelles were added and incubated at four degrees Celsius for 1 hour. Then ultrafiltration was performed three times with an ultrafiltration tube (MWCO: 300kDa) to remove unbound Cas9 / sgRNA, and the volume was concentrated to 40 μL. Gel electrophoresis was used to detect the Cas9 protein that was not filtered out, that is, the loaded Cas9 protein, and the amount of protein was calculated by the gray value.
[0099] The gel electrophoresis of NTA@Ce6 micelles loaded with Cas9 protein is as follows Figure 7 shown, from Figure 7 It can be seen that when 250ug of NTA@Ce6 micelles was added, the encapsulation efficiency of Cas9 / sgRNA reached 93.1%, and the loading capacity of ...
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