The study evaluated physicochemical properties of eight different polymeric nanoparticles (NPs) and their interaction with lung barrier and their suitability for pulmonary drug delivery. Eight physiochemically different NPs were fabricated from Poly lactic-co-glycolic acid (PLGA, PL) and Poly glycerol adipate-co-ω-pentadecalactone (PGA-co-PDL, PG) Eight NPs were successfully formulated from two polymers using emulsion-solvent evaporation; 200, 500 and 800 nm, negatively-charged and positively-charged. All different NPs did not alter tight junctions and PG NPs showed similar behavior to PL NPs, indicating its suitability for pulmonary drug delivery. Active endocytosis uptake mechanisms with physicochemical dependent manner were observed. In addition, NPs internalization and co-localization with lysosomes were visually confirmed indicating their vesicular transport. PG and PL NPs had shown no or low harmful effects on the barrier integrity, and with effective internalization and vesicular transport, thus, prospectively can be designed for pulmonary delivery applications.