This research aims to identify important formulation parameters for the enhancement of nanoparticle (NP) uptake and decreasing the cytotoxicity in macrophages. Fluorescent poly(lactic-co-glycolic acid) (PLGA) nanocarriers were characterized for size distributions, zeta potential and encapsulation efficiency. Incubation time, size class, PLGA derivative and chitosan derivative were assessed for uptake kinetics and cell viability. The major determining factor for enhancing cellular uptake were chitosan coatings, combined with acid-terminated PLGA and small NP size. Moreover, cytotoxicity was more favorable for small, chitosan glutamate-coated, acid-terminated PLGA NPs compared with its plain chitosan-coated counterparts. Chitosan glutamate has been shown to be a valuable alternative coating material for acid-terminated PLGA NPs to efficiently and safely target macrophages.