Methotrexate mtx

GE (C₁₇H₂₄O₁₀, molecular weight: 388, Purity >98%, batch number: S25627, structure shown in Figure 1) and Urethane (batch number: 20200710) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methotrexate (MTX) (batch number: B25455) was provided by Yuanye Biotechnology Co., Ltd. (Shanghai, China). Formic acid (batch number: 100264) was obtained from Merck Chemical Technology Co., Ltd. (Shanghai, China). Freund’s Complete Adjuvant (FCA) (batch number: F5881) was obtained from Sigma-Aldrich (St. Louis, MO, United States ). LC-MS grade acetonitrile (batch number: A104443), methyl tert-butyl ether (MTBE, batch number: B108120), Methanol (batch number: M116126), and Isopropanol (batch number: I112020) were provided by Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Ultrapure water was produced by a Milli-Q Gradient A10 Ultrapure Water System (MILLIPORE Co., Ltd., United States ). Dulbecco’s modified Eagle’s medium (DMEM) for MH7A cells culture was purchased from Hy Clone (Logan, UT, United States ). Fetal bovine serum (FBS) was provided by Biological Industries Co., Ltd. (Kibbutz BH, Israel).

Branched polyethylenimine (bPEI, 25 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methotrexate (MTX) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Shanghai Yuanye Biological Technology (Shanghai, China). mPEG-NH₂ (2000 Da) was purchased from Yarebio (Shanghai, China). Linolyl chloride (LC) was obtained from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). Survivin siRNA: Sense (5′–3′): mGCAGGUUCCUmUAUCUGUCAdTdT; Antisense (5′–3′): UGAmCAGAmUAAGGAACCUGmCdTdT; Survivin siRNA negative control: Sense (5′–3′): mUUCUCCGAACmGUGUCACGUdTdT; Antisense (5′–3′): ACGmUGACmACGUUCGGAGAmAdTdT, and Cy3, 5′-FAM and Cy5-labeled survivin siRNA for cellular uptake and biodistribution studies were synthesized by Ribo Biochemistry (Guangzhou, China). HeLa cells were purchased from ATCC (Rockefeller, MD, USA). 4′,6-Diamidino-2-phenylindole (DAPI) and Lyso Tracker™ Green DND-99 were purchased from Invitrogen Co. (Carlsbad, CA, USA). The Dihydrofolate Reductase Assay Kit was also purchased from BioVision (S. Milpitas Blvd., Milpitas, CA, USA). All chemical reagents used were of analytical grade.