Sequential nanoprecipitation for theranostics: Resolving assembly timescale mismatch in composite nanoparticle synthesis

Biomedical applications of colloidal nanocrystals (NC) have focused on nanoscale theranostics, that is, composite nanoparticles (CNP) that function as bioimaging probes while simultaneously delivering therapeutic payloads. Thus, there is a need for controlled CNP manufacturing methods that sufficiently decouple vehicle and cargo properties. Here, we investigate the assembly of poly(ethylene glycol)-b-poly(lactic acid) (PEG–PLA)-based CNPs loaded with PLA co-excipient, NCs of various sizes, and model drug molecule rubrene. We compare the established flash nanoprecipitation (FNP), a single-step method, with the emergent sequential nanoprecipitation (SNaP), a two-step method in which component addition can be temporally modulated. We find that using FNP to co-encapsulate cargo with mismatched assembly timescales yields poor CNP size control and nonuniform populations. In contrast, by delaying the CNP stabilizer addition by a few milliseconds via SNaP, we enable CNP size control, a 3-fold increase in cargo uniformity, and a 10-fold increase in co-encapsulation efficiency with rubrene.