Poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were prepared by an O/W emulsion-solvent evaporation method with polyethyleneimine (PEI) in the water phase as a stabiliser. Layer-by-Layer (LbL) assembly was used to engineer the surface of the NPs. Then, the multilayer coated PLGA NPs were further modified via self-assembly with lipid vesicles composed of 1,2-dioleoyl-sn-glycero-3-choline (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) at different molar ratios: 65 : 35, 75 : 25, 85 : 15 and 95 : 5. The influence of the lipid composition of the NPs on cellular uptake and uptake pathways for the HepG2 cell line was studied by means of flow cytometry and confocal laser scanning microscopy (CLSM). Macropinocytosis, clathrin-mediated and caveolae-mediated endocytosis are the main internalisation pathways of the lipid coated PLGA NPs. Lipid coated PLGA NPs tend to form vesicle-like aggregates in close proximity to the nucleus. Co-localisation studies indicate that lipid coated NPs could be associated with the endoplasmic reticulum (ER). Decreasing the proportion of negatively charged DOPS in the lipid coating results in a decrease in NP uptake and an increase in the presence of vesicle-like aggregates with an apparently higher association with the ER.