Nanoparticle synthesis

PLGA nanoparticles were synthesized using a nanoprecipitation technique based on modifying previously described methods [31] for passive PEGylation with Pluronic® F-127 [1]. PLGA was dissolved in acetone at 20 mg/mL and added to 0.1% (w/v) Pluronic® F-127 at a 1:11 (v/v) ratio with a syringe pump at a flow rate of 1 mL/min. Acetone was evaporated from the aqueous phase over 4–6 h in a fume hood. Particles were then washed by centrifugation at 10,000g x 20 min and resuspended in water using alternating vortexing and water bath sonication as needed. Aliquots of NP suspension were lyophilized and analyzed for fluorescence or drug content. Remaining suspensions were stored at 4°C until further use.

For fluorescent rhodamine-conjugated PLGA nanoparticles (Rho-NP), we first synthesized a rhodamine-B-PLGA conjugate using methods described by Costantino et al [32]. We determined the conjugation efficiency to be 15.7% (loading of 0.169% (w/w) rhodamine B:PLGA) by dissolving polymer conjugate and measuring fluorescence. Rhodamine-B-PLGA conjugate was dissolved in acetone and NP formed as described above. To synthesize ETR-loaded nanoparticles (ETR-NP), we dissolved ETR at 10% (w/w ETR/PLGA) theoretical loading in the PLGA/acetone solution. ETR was passively loaded in NP during NP synthesis, not directly conjugated to PLGA like rhodamine-B. ETR-NP were synthesized as described above, except that the NP suspension was filtered after evaporation using 2 micron syringe filter to remove any drug precipitate before washing. Characterization of ETR-NP in terms of size, zeta potential, polydispersity index (PDI), drug loading, and encapsulation efficiency is described in the Supporting Information (Supplementary Table S1).