A central challenge in the development of polymeric nanoparticles for various applications is precise engineering of desired physicochemical characteristics in a reproducible manner. The present work concerns the use of microfluidics to control the local polymer concentration inside polymeric nanoparticles. It is demonstrated that the compactness of nanoparticles based on self-assembled hydrophobically modified chitosan (HMCs) biopolymer can be dictated with tunable rapid mixing via hydrodynamic flow focusing in microfluidic channels. It is shown by varying the flow rates, as well as the hydrophobicity of the chitosan chains that the self-assembly behavior of the chains can be controlled by optimizing the size and compactness of the species, along with a more narrow size distribution of the nanoparticles. The size of the particles increased with increasing mixing time, whereas smaller and more compact nanoparticles, comprising of a less number of aggregated chains, are produced for chitosan at higher degrees of hydrophobicity. It was realized that at higher degrees of hydrophobicity and at mixing times longer than the time of aggregation, nanoparticles comprising of almost the same number of hydrophobic stickers were formed. Furthermore, we explored the effectiveness of microfluidic directed to assemble HMCs and to encapsulate paclitaxel (PTX), a common anticancer drug, which revealed remarkably higher encapsulation efficiency compared to the conventional bulk method. The in-vitro release of PTX from the prepared nanoparticles was evaluated to investigate the effect of compactness of the particles on the release profile. The estimated values of the diffusion coefficient of PTX up to 50% release implied controlled sustainability of the drug release with respect to the compactness of the nanoparticles, and a remarkable improvement compared to the uneven bulk mixing method. These results indicate a high potential of the microfluidic approach for precise bottom-up controlling physicochemical properties of polymeric nanoparticles for various applications, such as controlled drug delivery systems. (C) 2013 Elsevier Ltd. All rights reserved.