Characterization of nanoparticles Particle size and zeta potential Particle size and size distribution of nanoparticles were PF-01367338 measured using dynamic light scattering on a Malvern Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK). The lyophilized nanoparticles were diluted with
DI water before measurement. Surface charge of the nanoparticles was determined by laser Doppler anemometry using a Zetasizer Alvocidib Nano Series (Malvern Instruments). All measurements were done in triplicate. Surface morphology The morphology of nanoparticles was characterized by field emission scanning electron microscopy (FESEM; ZEISS 77 SUPRA 40VP, Carl Zeiss, Co., Ltd., Shanghai, China) at 5.0 kV electron high tension. To prepare samples for the FESEM observations, a drop of the particle suspension was placed on a grid or a stud, and the supernatant liquid was removed with a capillary after the particles were allowed to settle. The particles were then coated with platinum layer for 30 s. Drug loading and encapsulation efficiency The encapsulation efficiency (EE) and the actual drug loading of the nanoparticles were measured PCI-32765 clinical trial by high-performance liquid chromatography (LC 1100, Agilent
Technologies, Santa Clara, USA) as described before [31, 32]. In short, dried nanoparticles (5 mg) were dissolved in 1 ml of methylene chloride under vigorous vortex. The organic solution was transferred to 5 ml of mobile phase consisting of acetonitrile and deionized water (50:50, v/v). Methylene chloride was evaporated under a nitrogen stream until a clear solution obtained. The samples were then used for high-performance liquid chromatography (HPLC) analysis. The column effluent was monitored at 227 nm with a UV–vis detector. The standard size HPLC column (4.6 × 250 mm) is run at a flow rate of 1 mL/min. The drug encapsulation efficiency was defined as the percentage of the drug loaded in the final product. All these experiments were done in triplicates. In vitro drug release Accurately weighted aliquots of drug-loaded nanoparticles (15 mg) were suspended in 5 ml release medium (PBS pH Erlotinib nmr 7.4 containing 0.1% w/v
Tween 80). The use of Tween 80 in the release media was able to increase the solubility of drug in the PBS and avoided the binding of drug to the tube wall. The nanoparticle suspension was transferred into a dialysis tubing membrane which is sealed at one end with a clamp. The sealed dialysis bag was placed into a centrifuge tube and immersed in 15-ml release medium. The centrifuge tube was placed in an orbital water bath shaking at 130 rpm at 37.0°C. A 10 ml aliquots of samples was periodically removed for HPLC analysis and replaced with fresh medium. The samples were extracted with 2 ml methylene chloride and reconstituted in 5 ml mobile phase. Methylene chloride was evaporated under a nitrogen stream until a clear solution was obtained.
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