Using the fluorescent probe dye 1,1'-dioctadecy1-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) as a surrogate for hydrophobic drugs, we investigate the effects of water content and on-chip flow rate on the multiscale structure, loading and release properties of Dil-loaded poly(epsilon-caprolactone)-block-poly (ethylene oxide) (PCL-b-PEO) nanoparticles produced in a gas-liquid segmented microfluidic device. We find a linear increase in PCL crystallinity within the nanoparticle cores with increasing flow rate, while mean nanoparticle sizes first decrease and then increase with flow rate coincident with the disappearance and reappearance of long filament nanoparticles. Loading efficiencies at the lower water content (cwc + 10 wt%) are generally higher (up to 94%) compared to loading efficiencies (up to 53%) at the higher water content (cwc + 75 wt%). In vitro release times range between similar to 2 and 4 days for nanoparticles produced at cwc + 10 wt% and >15 days for nanoparticles produced at cwc + 75 wt%. At the lower water content, slower release of DiI is found for nanoparticles produced at higher flow rate, while at high water content, release times first decrease and then increase with flow rate. Finally, we investigate the effects of the chemical and physical characteristics of the release medium on the kinetics of in vitro Dil release and nanoparticle degradation. This work demonstrates the general utility of dye-loaded nanoparticles as model systems for screening chemical and flow conditions for producing drug delivery formulations within microfluidic devices. (C) 2016 Elsevier Inc. All rights reserved.