ABSTRACT
ABSTRACT:Objective To prepare polymeric nanomicelles capable of simultaneously loading doxorubicin (DOX) and Bcl‐2 small interfering RNA (Bcl‐2 siRNA ) , and to explore their in vitro cytotoxicity and cellular uptake in MCF‐7 human breast cancer cells .Methods Copolymer poly (ethylene glycol )‐g‐polyethylenimine‐g‐poly(γ‐benzyl‐L‐glutamate) was synthesized by the combination of reductive amination and carbodiimide methods , and its chemical structure was verified by 1 H NMR .Empty and drug‐loaded copolymeric nanomicelles were prepared by dialysis method and characterized by transmission electron microscope and dynamic light scattering .The ability of the nanomicelles to compress Bcl‐2 siRNA was measured by by agarose gel electrophoresis method . The release profiles of DOX and Bcl‐2 siRNA from the nanomicelles were explored by means of fluorescence spectrometry and dialysis method .The in vitro cytotoxicity and cellular uptake of DOX and Bcl‐2 siRNA co‐loaded nanomicelles in MCF‐7 human breast cancer cells were characterized by MTT assay and confocal laser scanning microscopy , respectively .Results The critical micelle concentration of the copolymer was about 4 mg/L ,and the sizes of self‐assembled empty and drug‐loaded nanomicelles were smaller than 200 nm .The drug‐loading efficiency and drug‐loading content of DOX in the nanomicelles were 88 .7% and 15 .1% ,respectively .The DOX‐loaded nanomicelles could efficiently compress Bcl‐2 siRNA when an N/P ratio was ≥64 .The zeta potential of DOX and Bcl‐2 siRNA co‐loaded nanomicelles was +30 mV .The release behavior of the cargoes from the nanomicells was pH‐sensitive , and the release of Bcl‐2 siRNA was more sensitive to acidic pH than that of DOX . The nanomicelles could simultaneously deliver DOX and Bcl‐2 siRNA into MCF‐7 cells , and the co‐delivered DOX and Bcl‐2 siRNA significantly increased the cytotoxicity of DOX (P<0 .05) .Conclusion The polymeric nanomicelles can co‐load DOX and Bcl‐2 siRNA and deliver them into MCF‐7 cells , and DOX in combination with Bcl‐2 siRNA can synergistically inhibit the growth of MCF‐7 cells and promote cell apoptosis ,suggesting that the nanomicells may be a promising carrier for the co‐delivery for chemotherapeutics and genes .
ABSTRACT
Objective To evaluate the performances of glucose oxidase immobilized on hydrophilic-hydrophobic silica gel, which may be employed to prepare glucose sensor for the determination of glucose in body fluids. Methods The silica gel was prepared from precursors ?-aminopropyltrimethoxysilane (APTMOS) and methyltrimethoxysilane (MTMOS) by sol-gel technique. Glucose oxidase (GOD) was covalently attached to the silica gel via carbodiimide coupling reaction between a carboxylic acid group on enzyme and an amine group of the silica gel under the participation of the linking reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide. The performances of immobilized GOD were explored. Results The optimum conditions were obtained as follows: volume fraction of APTMOS 70%, enzyme content given 16 800 U, the temperature of 35 ℃ and buffer pH 5.5. The decrement in the activity of immobilized GOD for the first 2 weeks was less than 10% of its original activity, and the activity of immobilized GOD retained more than 75% of its original activity after 1 month of testing. Six independently prepared immobilized GOD on the silica gel resulted in an average bioactivity of 1 290.9 ?mol?min -1?g -1 with an R.S.D. of 3.4%. The Michaelis constant (K m) of immobilized GOD was 9.1 mmol?L -1. Conclusion Immobilizing GOD on the silica gel via the formation of peptide bonds is an outstanding enzyme immobilization method.