The natural turnover of free hyaluronic acid (HA) is predominantly based on its CD44-mediated internalisation in leukocytes. In a phagocytic cell model (RAW 264.7 murine macrophages) we here provide conclusive evidence that this receptor-mediated mechanism endocytosis is responsible also of the uptake of materials where HA is used as a coating agent, in this case chitosan/triphosphate nanoparticles on whose surface HA is electrostatically adsorbed. Alginate-coated nanoparticles were used as a control and they appeared to undergo a qualitatively similar endocytic process, which was mediated by a different scavenging receptor yet to be identified. In this general picture, an important, modulating role appears to be played by how receptors can cluster around individual nanoparticles. The CD44 slow representation (24-48 h) enforces a limit in the amount of available HA internalisation receptors; therefore a higher affinity, and hence a higher degree of clustering, would yield a lower number of internalised nanoparticles. HA presentation can be varied by acting on nanoparticle structure/morphology, and our data suggest that a better presentation may be linked to both higher affinity and lower capacity/uptake rate. Paradoxically, this result would suggest that particles with a lower affinity for CD44 may allow a more efficient HA-mediated delivery of payloads.

The study aimed to explore the influence of the network architecture on the mechanical properties and degradability of HA/PEG gels, and to highlight the relationship between Young's modulus and cell colonization with a selected architecture. Three different families of hyaluronic acid (HA)-based photopolymerized PEG diacrylate (PEGDA) hydrogels were compared, using different concentrations and molecular weights (64 and 234 kDa) of HA: semi-IPNs containing native HA in a PEG network (type I gels); co-networks obtained using thiolated HA as chain transfer agent during PEGDA polymerization (type II gels); co-networks obtained from the in situ preparation of a macromonomer derived from the Michael-type addition of thiolated HA on PEGDA (type III gels). From a comparative study of rheological properties and enzymatic degradability, type II gels were selected for a further study aiming to link their mechanical properties to cell spreading. Employing RGD-functionalized materials, Young's moduli were measured via AFM nanoindentation while the cell spreading behavior was quantitatively evaluated by monitoring morphology and metabolic activity (MTS assay) of L929 fibroblasts. By revealing a clear relation between increasing modulus and increasing cell spreading/proliferation, the study showed the possibility to fine tune the cell/material interactions with appropriate reactive processing techniques.

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We have employed microfluidics (cross-shaped chip) for the preparation of drug-loaded poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles. The polymer precipitates from an acetone solution upon its controlled laminar mixing (flow focusing) with an aqueous solution of a surfactant, allowing for an operator-independent, up-scalable and reproducible preparative process of nanoformulations. Firstly, using PEGylated surfactants we have compared batch and microfluidic processes, and showed the superior reproducibility of the latter and its strong dependency on the acetone/water ratio (flow rate ratio). We have then focused on the issue of purification from free surfactant, and employed advanced characterization techniques such as flow-through dynamic light scattering as the in-line quality control technique, and field flow fractionation (FFF) with dynamic and static light scattering detection, which allowed the detection of surfactant micelles in mixture with nanoparticles (hardly possible with stand-alone dynamic light scattering). Finally, we have shown that the choice of polymer and surfactant affects the release behaviour of a model drug (paclitaxel), with high molecular weight PLGA (RG756) and low molecular weight surfactant (tocopheryl poly(ethylene glycol) 1000 succinate, TPGS) apparently showing higher burst and accelerated release.