Assembly of drug NPs to erythrocytes and characterization

PLGA NPs encapsulating DOX were prepared using a nanoprecipitation method. Briefly, 5 mg of DOX was dissolved in 500 ul of methanol and 5 ul of triethylamine. This was added to 1 ml of acetone containing 20 mg of PLGA. The mixture was then injected into 10 ml of 1% polyvinyl alcohol solution under constant stirring using a syringe pump at 1 ml/min. The particles were kept under constant stirring overnight before removing the organic solvents using rotary evaporation. The formed particles were centrifuged at 12,000g for 15 min, and the supernatant was analyzed to quantify drug loading. The particles were then resuspended in deionized water and assessed for their size, zeta potential, and polydispersity index using dynamic light scattering (Malvern Zen3600) and SEM (Zeiss FESEM Supra 55VP, Zeiss FESEM Ultra 55). The NPs were washed for a total of two washes with deionized water before their final resuspension in phosphate-buffered saline (PBS). Nanoparticles containing other chemotherapeutic drugs were prepared using the similar nanoprecipitation technique described above with minor modifications (details are shown in the Supplementary Materials).

For the determination of the nanoparticle to erythrocyte ratio, the volume of one nanoparticle was calculated based on the average diameter of the nanoparticle. The mass of one nanoparticle was estimated based on the volume of one nanoparticle and the density of the nanoparticle suspension. The number of nanoparticles per mg of nanoparticles was estimated based on the mass of one nanoparticle. The number of erythrocytes was estimated by the reported values of erythrocytes per uL of blood. 

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