Monolayers of endothelial cells (1L-ECs) have been generally used as in vitro vascular wall models to study the vascular mechanisms and transport of substances. However, these two-dimensional (2D-) system cannot represent the properties of native vascular walls which have a 3D-structure and are composed of not only ECs, but also smooth muscle cells (SMCs) and other surrounding tissues. Here in, 5-layered (5L) 3D-arterial wall models (5L-AWMs) composed of EC monolayer and 4-layered SMCs were constructed by hierarchical cell manipulation. We applied the 5L-AWMs to evaluate their barrier function and permeability to nano-materials in order to analyze drug, or drug nanocarrier permeability to the blood vessel in vitro. Barrier property of the 3D-AWMs was confirmed by Zonula occludens (ZO-1) staining and their transendothelial electrical resistance (TEER), which was comparable to 1L-ECs, while the SMCs showed close to zero. The effect of substance size to permeability across the 5L-AWMs was clearly observed from dextrans with various molecular weights, which agreed well with the known phenomena of the in vivo blood vessels. Importantly, transport of nano-materials could be observed across the depth of 5L-AWMs, suggesting the advantage of 3D-AWM5 over general 2D-systems. By using this system, we evaluate the transport of 35 nm phenylalanine-modified poly(gamma-Glutamic Acid) nanoparticles (gamma-PGA-Phe NPs) as a candidate of biodegradable drug carrier. Interestingly, despite of having comparable size to dextran-2000k (28 nm), the gamma-PGA-Phe NPs distinctly showed approximately 20 times faster transport across the 5L-AWM5, suggesting the effect of intrinsic properties of the substance on the transport. This in vitro evaluation system using the 3D-AWM5 is therefore useful for the design and development of nano-drug carriers for treatment of vascular diseases, such as atherosclerosis. (C) 2014 Elsevier Inc. All rights reserved.