On-Chip Synthesis of Hyaluronic Acid-Based Nanoparticles for Selective Inhibition of CD44+ Human Mesenchymal Stem Cell Proliferation

In this study, an innovative microfluidics-based method was developed for one-step
synthesis of hyaluronic acid (HA)-based nanoparticles (NPs), by exploiting polyelectrolytic interactions
between HA and chitosan (CS), in order to improve reliability, reproducibility and possible scale-up
of the NPs preparation. The on-chip synthesis, using a staggered herringbone micromixer, allowed to
produce HA/CS NPs with tailored-made size and suitable for both parenteral (117.50 4.51 nm) and
loco-regional (349.15 38.09 nm) administration, mainly composed by HA (more than 85% wt) with
high negative surface charge (< 20 mV). HA/CS NPs were successfully loaded with a challenging
water-insoluble molecule, Everolimus (EVE), an FDA- and EMA-approved anticancer drug able to
lead to cell cycle arrest, reduced angiogenesis and promotion of apoptosis. HA/CS NPs resulted to
be massively internalized in CD44+ human mesenchymal stem cells via CD44 receptor-mediated
endocytosis. HA/CS NPs selectiveness towards CD44 was highlighted by blocking CD44 receptor
by anti-CD44 primary antibody and by comparison to CS-based NPs cellular uptake. Eventually,
high eectiveness in inhibiting cell proliferation was demonstrated on-chip synthetized EVE loaded
HA/CS NPs by tracking in vitro DNA synthesis.