The objective of this study was to compare the properties of core-shell nanoparticles with a PLGA core and shells composed of different types of polymers; focusing on their structural integrity. Core PLGA nanoparticles were prepared by either the high-pressure homogenization — solvent evaporation technique or nanoprecipitation; using poloxamer 188 (P188); copolymer of divinyl ether with maleic anhydride (DIVEMA); and human serum albumin (HSA) as shell-forming polymers. The shells were formed through adsorption; interfacial embedding; or conjugation. For dual fluorescent labeling; the core and shell-forming polymers were conjugated with Cyanine5; Cyanine3; and rhodamine B. The nanoparticles had negative zeta potentials and sizes ranging from 100 to 250 nm (measured by DLS); depending on the shell structure and preparation technique. The core-shell structure was confirmed by TEM and fluorescence spectroscopy; with the appearance of FRET phenomena due to the donor-acceptor properties of the labels. All shells enhanced cellular uptake of the nanoparticles in Gl261 murine glioma cells. Integrity of the core-shell structure upon their incubation with cells was evidenced by intracellular colocalization of the fluorescent labels using Manders’ colocalization coefficients. This comprehensive approach may be useful for selection of the optimal preparation method already at the early stages of the core-shell nanoparticle development.