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2 protocols using succinic anhydride

1

Succinic anhydride-conjugated PEI for P-glycoprotein silencing

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Succinic anhydride, b-PEI (MW 1,800), b-PEI (MW 250,000), 2-(7-Azabenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU), N-ethyldiisopropylamine (DPIEA) et.al was purchased from Adamas-beta (Shanghai, China). All of solvent were purchased from Macklin (Shanghai, China). Chloroquine Phosphate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used for Western Blotting and immunofluorescence including rabbit anti-LC3B, anti-p62, anti-Pgp, goat anti-rabbit IgG (H + L) (HRP, Cora Lite 594) were obtained from Proteintech (Wuhan, China). Alexa fluor 647-labeled goat anti-rabbit IgG (H + L), rabbit anti-CD44, anti-ki67, Ad-GFP-LC3B, Lyso-Tracker Red, TUNEL Apoptosis Assay Kit, acid phosphatase assay kit et.al were purchased from Beyotime (Shanghai, China). All other reagents for western blotting and gel electrophoresis were obtained from Solarbio (Beijing, China).
Targeting human P-gp siRNA sequences:
Sense: 5’-AAGAAGGAAAAGAAACCAACUdTdT-3’;
Anti-sense: 5’-AGUUGGUUUCUUUUCCUUCUUdTdT-3’.
All of siRNA were obtained by GenePharma Co. Ltd. (Shanghai, China).
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2

Synthesis and Characterization of PEGylated Nanoparticles

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PTX was purchased from Beijing Huafeng United Technology Co., Ltd (Beijing, People’s Republic of China). DSF was obtained from Aladdin (Shanghai, People’s Republic of China). Succinic anhydride (SA) and DMA were purchased from Adamas Reagent Co., Ltd (Shanghai, People’s Republic of China) and used as received. N,N-dimethylformamide (DMF) was stored over calcium hydride (CaH2) and purified by vacuum distillation with CaH2. Amino-terminated methoxy poly(ethylene glycol) (PEG-NH2; molecular weight [MW] =5,000) was purchased from Shanghai Yare Biotech, Co., Ltd (Shanghai, People’s Republic of China) and used after dried under vacuum. ε-(Benzyloxycarbonyl)-l-lysine N-carboxyanhydride (Lys(Z)-NCA) were synthesized by the Fuchs-Farthing method using bis(trichloromethyl)carbonate (triphosgene) according to the literature.29 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Hoechst 33342 were purchased from Beyotime Institute of Biotechnology and used without further purification.
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