, Anyang, Korea). An isotonic phosphate buffer (25 mM sodium phosphate, 100 mM NaCl; pH = 7.4) was used as mobile phase at a flow rate of 1.0 ml/min. The examination was carried out by UV monitoring at 214 nm. The BSA, GM-CSF, and G-CSF were also dissolved in distilled water and then dispersed in dichloromethane to get controlled water-in-oil (W/O) emulsion. The controlled emulsion and standard protein solutions were also subject to SEC-HPLC for comparing with dextran nanoparticles loaded with proteins. ARN-509 manufacturer Bioactivity assay of proteins during

the formulation steps The GM-CSF, G-CSF, and βcheck details -galactosidase were selected as model proteins to examine the bioactivity during the process. The bioactivity of the GM-CSF recovered during the steps was determined by the proliferation effect induced on TF-1 cell line. The TF-1 cells were grown in a PRMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The cultures were maintained in plastic flasks and incubated in CO2/air (5:95, v/v) at 37°C in a humidified incubator. The bioactivity of the G-CSF recovered LY2874455 chemical structure was determined by the proliferation effect induced on an NSF-60 cell line. The NFS-60 cells were grown in a PRMI 1640 medium supplemented with 10% FBS. The cultures were maintained in plastic flasks and incubated in CO2/air (5:95, v/v) at 37°C in a humidified incubator. The catalysis bioactivity

of the β-galactosidase on o-nitrophenol recovered was determined by the ortho-nitrophenyl-β-galactoside (ONPG) assay. The assay

was carried out according to a protocol from Sigma. Protein activity was determined by the absorbance of the reaction product of ONPG at 420 nm. The β-galactosidase and GM-CSF were also dissolved in distilled water and then were dispersed in dichloromethane to get the controlled W/O emulsion. The controlled emulsion and standard protein solutions were also subject to bioactivity assay for comparing with dextran nanoparticles loaded with proteins. Ability of dextran nanoparticle to overcome acidic microenvironment LysoSensor™ Yellow/Blue dextran (Life Technologies Corporation, Grand Island, NY, USA) was loaded into the dextran nanoparticle to evaluate the ability to attenuate the local acidic microenvironment in the PLGA second microsphere during the in vitro release period. The dextran nanoparticles were encapsulated into composite PLGA microsphere by the solid-in-oil-in-water method [15]. Accordingly, the LysoSensor™ Yellow/Blue dextran solution was encapsulated into the PLGA matrix to act as the controlled sample by the traditional water-in-oil-in-water (W/O/W) double emulsion method [9]. To monitor the change in pH within PLGA microspheres vs. time, 10 mg of dried PLGA microspheres loaded with the LysoSensor™ Yellow/Blue dextran were incubated in tubes containing 1 ml of 20-mM PBS buffer at 37°C under 90 rpm continuously for 12 days.