In this work, we report a novel method of layer-by-layer (LbL) deposition using concentration tubes that enables faster process and less damage to fragile nanocores than previously described methods. Such methods are generally based on continuous cycles of centrifugation/resuspension for long times and at high speeds, which may eventually lead to the aggregation of the deflocculated suspension of nanoparticles into a compact, non-resuspendable cake. The new method was applied to the preparation of a doublechambered nanocarrier system, which was successfully loaded with a fluorescently labeled model protein (lysozyme) and a model small molecule (fluorescein) in two defined and separate compartments, namely the poly lactide-co-glycolide (PLGA) core (c.a. 110 nm) and an outer shell obtained by LbL surface coating. The new method yielded stable suspensions of drug-loaded, LbL-coated PLGA nanoparticles, while centrifugation at high speeds and long time intervals leads to a compact cake of non-resuspendable aggregates. These nanocarriers were taken up by MDCK cells in vitro, where a colocalization of both model compounds was shown by confocal imaging.