Amicon regenerated cellulose centrifuge filter units

Prior to drug encapsulation, DOX-HCl was desalted to generate DOX following reported procedures ^{21 (link)}. NPs were then formulated following a nanoprecipitation method ^{22 (link)}. Briefly, an acetone/DMSO (1:1, v/v) solution of the block copolymer (10 mg/ml, 1ml) was added dropwise to a stirred aqueous phase (5 ml DI water). The mixture was stirred on a magnetic stir plate at 900 rpm for 2h at ambient temperature to obtain blank NPs. DOX, NR or DiR dye-loaded NPs were similarly prepared using an acetone/DMSO (1:1, v/v) solution of the block copolymer (10 mg/ml, 1ml) containing 2 mg/ml DOX, 0.1 mg/ml NR or 0.036 mg/ml DiR, respectively. The NP suspensions were subsequently centrifuged (2,880 g for 10min) to remove the large aggregates formed from polymers. The supernatant containing the NPs was then transferred to Amicon regenerated cellulose centrifuge filter units (MWCO=30KDa, EMD Millipore) and centrifuged (4500 g for 15min) to remove the free drug or dye and organic solvent. Subsequently, the drug or dye-loaded NPs were collected after thorough washing (three times) with PBS (pH 7.4) using centrifuge filters and immediately used for characterization and biological studies.

Drug or dye-loaded NPs with immobilized avidin-PA were prepared following the procedure described previously, with the addition of avidin-PA (0.125 mg/ml) in the stock polymer solution. Following centrifugation to remove the large polymer aggregates, un immobilized avidin-PA, free drug or dye, and organic solvent was removed by transferring the supernatent containing the NPs to Amicon regenerated cellulose centrifuge filter units (MWCO=100KDa, EMD Millipore). The purified avidin-PA-NPs were then re-suspended in PBS at a concentration of 10 mg/ml. Avidin-PA incoporation in the NPs was quantified by a bicinchoninic acid (BCA) assay following the manufacturer's protocols. The concentration of immobilized avidin-PA was derived from a series of standards at an avidin concentration of 6.25-100 μg/ml. NPs without avidin-PA was used as the control. Finally, biotinylated anti-CD19Abs (20 ul, 0.5 mg/ml) were added to avidin-PA-NP suspension (0.5ml, 10 mg/ml) in PBS, and the mixture was incubated at 37°C for 2h. Free Abs were removed by ultracentrifugation (162,635 g for 30min) and pure NPs were obtained by repeated washing with PBS (3 times). The resultant CD19-targeting NPs (CD19-NPs) was resuspended in PBS at desired concentrations for in vitro and in vivo testing.