N-α-Fmoc protected amino acids, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 2-Cl-trityl chloride resin (100–200 mesh, 1.27 mmol/g) were from EMD Biosciences (San Diego, CA). Papain (EC 3.4.22.2, from papaya latex) and cathepsin B (EC 3.4.22.1, from bovine spleen) were from Sigma-Aldrich (St. Louis, MO). Bis-MAL-dPEG3 (bis-[1,13-(3-maleimidopropionyl)amido]-4,7,10-trioxatridecane, CAS# 756525-89-0) was purchased from Quanta Biodesign (Powell, OH). 1-Hydroxybenzotriazole (HOBt) was purchased from AnaSpec (Fremont, CA). N,N′-Diisopropylcarbodiimide (DIC), 2,2,2-trifluoroethanol (TFE) and all other reagents and solvents were from Sigma-Aldrich (St. Louis, MO). Doxorubicin (DOX) was a kind gift from Meiji Seika Kaisha Ltd. Tokyo, Japan. HPMA,14 4-cyanopentanoic acid dithiobenzoate15 were synthesized according to literature. N-Methacryloylglycylphenylalanylleucylglycyl-doxorubicin (MA-GFLG-DOX) was prepared by the reaction N-methacryloylglycylphenylalanylleucylglycine 4-nitrophenyl ester (MA-GFLG-ONp) with doxorubicin hydrochloride in DMF in the presence of diisopropylethylamine according to the described procedure.16
UV-vis spectra were measured on a Varian Cary 400 Bio UV-visible spectrophotometer. Mass spectra were measured on an FTMS mass spectrometer (LTQ-FT, ThermoElectron, Waltham, MA). 1H-NMR spectra were recorded on a Mercury400 spectrometer using DMSO-d6 as the solvent. Polymerization conversion was determined by the measurement of remaining HPMA monomer concentration at different time points using RP-HPLC (Agilent Technologies 1100 series, Zorbax C8 column 4.6×150 mm) with gradient elution from 2 to 90% of Buffer B within 30 min at flow rate of 1.0 mL/min (Buffer A: deionized water (DI H2O) with 0.1% TFA, Buffer B: acetonitrile with 0.1% TFA). The molecular weight and polydispersity index (PDI) of polymers were measured on an ÄKTA FPLC (fast protein liquid chromatography) system (GE Healthcare, formerly Amersham) equipped with miniDAWN TREOS and OptilabEX detectors (Wyatt Technology, Santa Barbara, CA) using a Superose 6 or 12 HR10/30 column with PBS (pH 7.3) as the mobile phase. Narrow polydispersity polyHPMA fractions prepared by size exclusion chromatography, whose molecular weights were characterized by multiangle light scattering, were used as molecular weight standards. The multiblock polymers were fractionated on the same FPLC system using Superose 6 HR16/60 preparative column. PBS was used as the mobile phase. The flow rate was 1 mL/min. the fraction was collected every 10 min. The salt in the fractions was removed by dialysis. The narrow polydispersity polymer fractions were obtained after freeze-drying.
UV-vis spectra were measured on a Varian Cary 400 Bio UV-visible spectrophotometer. Mass spectra were measured on an FTMS mass spectrometer (LTQ-FT, ThermoElectron, Waltham, MA). 1H-NMR spectra were recorded on a Mercury400 spectrometer using DMSO-d6 as the solvent. Polymerization conversion was determined by the measurement of remaining HPMA monomer concentration at different time points using RP-HPLC (Agilent Technologies 1100 series, Zorbax C8 column 4.6×150 mm) with gradient elution from 2 to 90% of Buffer B within 30 min at flow rate of 1.0 mL/min (Buffer A: deionized water (DI H2O) with 0.1% TFA, Buffer B: acetonitrile with 0.1% TFA). The molecular weight and polydispersity index (PDI) of polymers were measured on an ÄKTA FPLC (fast protein liquid chromatography) system (GE Healthcare, formerly Amersham) equipped with miniDAWN TREOS and OptilabEX detectors (Wyatt Technology, Santa Barbara, CA) using a Superose 6 or 12 HR10/30 column with PBS (pH 7.3) as the mobile phase. Narrow polydispersity polyHPMA fractions prepared by size exclusion chromatography, whose molecular weights were characterized by multiangle light scattering, were used as molecular weight standards. The multiblock polymers were fractionated on the same FPLC system using Superose 6 HR16/60 preparative column. PBS was used as the mobile phase. The flow rate was 1 mL/min. the fraction was collected every 10 min. The salt in the fractions was removed by dialysis. The narrow polydispersity polymer fractions were obtained after freeze-drying.
