Mcf 7 cells

Poly(ethylene glycol) (PEG) methyl ether, molecular weight [MW] 5,000 Da), L-lactide (3,6-dimethyl-1,4-dioxite-2,5-dione), stannous octoate (Sn[Oct]₂, Tin[II]2-ethylhexanoate), 4-(dimethylamino)pyridine (DMAP), pyren, succinic anhydride, pyridine, triethylamine (TEA), N-hydroxysuccinicimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), anhydrous 1,4-dioxane, N,N-dimethylformamide (DMF), D₂O-d₆, and CDCl₃ were purchased from Sigma-Aldrich (St Louis, MO, USA). Triphosgene and branched PEI (MW 10,000 Da) were purchased from Alfa Aesar^® Johnson Matthey Korea (Seoul, South Korea). Dichloromethane (DCM), methanol (MeOH), ethanol (EtOH), and toluene were purchased from Honeywell Burdick & Jackson^® (Muskegon, MI, USA). Dox⋅HCl was purchased from Borung Co. (Seoul, South Korea). All other chemicals used were of analytical grade. For cell culture, human breast cancer MCF7 cells and cervical cancer KB cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, South Korea). RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Welgene (Seoul, South Korea). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Tokyo, Japan). P(Asp) was prepared as previously reported.20 The MW of P(Asp) was ~4,000 Da (degree of polymerization =35).

ZP was purchased from TCI, and E2, used for the positive control, was purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO), used as a solvent, was purchased from Life Technologies (Carlsbad, CA, USA). MCF-7 cells (Passage No. 176) were purchased from the Korean Cell Line Bank (No. 30022), and were used in the experiment after serial subculture at 37°C, 5% carbon dioxide (CO₂).
After sufficient culturing, the MCF-7 cells were counted, and then distributed into a 96-well plate (100 μL per well, 5 × 10⁴ cell/ mL). The plate was lightly shaken to evenly distribute the cells, and then incubated at 37°C, 5% CO₂ for 24 hours. In the subsequent steps, Dulbecco’s modified Eagle’s medium (DMEM) containing 5% charcoal stripped fetal bovine serum 5% and 1% penicillin streptomycin glutamine was used to eliminate cell proliferation factors. E2 and other test substances were diluted using DMSO as a solvent in the culture medium. Afterward, 100 µL of test substance was added to each group: control group, 0.1% DMEM; E2 positive control group, 1 × 10^-9 M E2 [19 (link)]; and ZP group, 1 × 10^-9 to 1 × 10^-6 M ZP. The final concentration of DMSO was maintained at 0.1%. The plate was incubated at 37°C, 5% co₂ for 6 days (144 hours). Then, cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay.

HEK293 and 3T3-L1 cells were purchased from ATCC (Manassas, VA, USA), and MCF-7 cells were purchased from KCLB (Korea Cell Line Bank, Seoul), and Rat-1 cells overexpressing human IR were kindly provided by Dr Nicholas J. G. Webster from the University of California, San Diego. HEK293, Rat-1/hIR and MCF-7 cells were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) with 10% (vol/vol) fetal bovine serum (FBS, Lonza) and 3T3-L1 pre-adipocytes were maintained in high glucose DMEM with 10% bovine serum (BS, Gibco) at 37°C under a humidified atmosphere containing 5% CO₂. For adipocyte differentiation, 3T3-L1 pre-adipocytes were cultured for 2 days past confluence. Differentiation was initiated by changing the medium to DMEM containing 1 μM dexamethasone, 500 nM IBMX, 850 nM insulin and 10% FBS. After 2 days, the medium was replaced with DMEM containing 850 nM insulin and 10% FBS and then incubated for two additional days. Finally, the medium was changed to DMEM containing only 10% FBS and incubated for 4–5 days until at least 90% of the cell population exhibited accumulation of lipid droplets.