Nanotechnology offers advanced biomedical tools for diagnosis and drug delivery, stressing the value of investigating the mechanisms by which nanocarriers interact with the biological environment. Herein, the cellular response to CD44-targeted nanoparticles (NPs) was investigated. CD44, the main hyaluronic acid (HA) receptor, is widely exploited as a target for therapeutic purposes. HA NPs were produced by microfluidic platform starting from HA with different molecular weights (Mw, 280, 540, 820 kDa) by polyelectrolyte complexation with chitosan (CS). Thanks to microfluidic technology, HA/CS NPs with the same physical features were produced, and only the effects of HA Mw on CD44-overexpressing cells (human mesenchymal stem cells, hMSCs) were studied. This work provides evidence of the HA/CS NPs biocompatibility regardless the HA Mw and reveals the effect of low Mw HA in improving the cell proliferation. Special attention was paid to the endocytic mechanisms used by HA/CS NPs to enter hMSCs. The results show the notable role of CD44 and the pronounced effect of HA Mw in the NPs’ internalization. HA/CS NPs uptake occurs via different endocytic pathways simultaneously, and most notably, NPs with 280 kDa HA were internalized by clathrin-mediated endocytosis. Instead, NPs with 820 kDa HA revealed a greater contribution of caveolae and cytoskeleton components.

Collagen, thanks to its biocompatibility, biodegradability and weak antigenicity, is widely used in dressings and scaffolds, also as electrospun fibers. Its mechanical stability can be improved by adding polycaprolactone (PCL), a synthetic and biodegradable aliphatic polyester. While previously collagen/PCL combinations were electrospun in solvents such as hexafluoroisopropanol (HFIP) or trifluoroethanol (TFE), more recently literature describes collagen/PCL nanofibers obtained in acidic aqueous solutions. A good morphology of the fibers represents in this case still a challenge, especially for high collagen/PCL ratios. In this work, thanks to preliminary rheological and physicochemical characterization of the solutions and to a Design of Experiments (DOE) approach on process parameters, regular and dimensionally uniform fibers were obtained with collagen/PCL ratios up to 1:2 and 1:1 w/w. Collagen ratio appeared relevant for mechanical strength of dry and hydrated fibers. WAXS and FTIR analysis showed that collagen denaturation is related both to the medium and to the electrospinning process. After one week in aqueous environment, collagen release was complete and a concentration dependent stimulatory effect on fibroblast growth was observed, suggesting the fiber suitability for wound healing. The positive effect of collagen on mechanical properties and on fibroblast biocompatibility was confirmed by a direct comparison of nanofiber performance after collagen substitution with gelatin.

Adipose tissue comprises both adipose and non-adipose cells such as mesenchymal stem cells. These cells show a surface antigenic profile similar to that of bone-marrow-derived MSC. The cells derived from the dedifferentiation of mature adipocytes (DFAT) are another cell population with characteristics of stemness. The aim of this study is to provide evidence of the stemness, proliferation, and differentiation of human adipose stem cells (hASC) and DFAT obtained from human subcutaneous AT and evaluate their potential use in regenerative medicine. Cell populations were studied by histochemical and molecular biology techniques. Both hASC and DFAT were positive for MSC markers. Their proliferative capacity was similar and both populations were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. DFAT were able to accumulate lipids and their lipoprotein lipase and adiponectin gene expression were high. Alkaline phosphatase and RUNX2 gene expression were greater in hASC than in DFAT at 14 days but became similar after three weeks. Both cell populations were able to differentiate into chondrocytes, showing positive staining with Alcian Blue and gene expression of SOX9 and ACAN. In conclusion, both hASC and DFAT populations derived from AT have a high differentiation capacity and thus may have applications in regenerative medicine.

Chitosan nanoparticles (CS NPs) showed promising results in drug, vaccine and gene delivery for the treatment of various diseases. The considerable attention towards CS was owning to its outstanding biological properties, however, the main challenge in the application of CS NPs was faced during their size-controlled synthesis. Herein, ionic gelation reaction between CS and sodium tripolyphosphate (TPP), a widely used and safe CS cross-linker for biomedical application, was exploited by a microfluidic approach based on a staggered herringbone micromixer (SHM) for the synthesis of TPP cross-linked CS NPs (CS/TPP NPs). Screening design of experiments was applied to systematically evaluate the main process and formulative factors affecting CS/TPP NPs physical properties (mean size and size distribution). Effectiveness of the SHM-assisted manufacturing process was confirmed by the preliminary evaluation of the biological performance of the optimized CS/TPP NPs that were internalized in the cytosol of human mesenchymal stem cells through clathrin-mediated mechanism. Curcumin, selected as a challenging model drug, was successfully loaded into CS/TPP NPs (EE% > 70%) and slowly released up to 48 h via the diffusion mechanism. Finally, the comparison with the conventional bulk mixing method corroborated the efficacy of the microfluidics-assisted method due to the precise control of mixing at microscales.

Hyaluronic acid-based nanoparticles (HA NPs) can be used to deliver a protein cargo to cells overexpressing HA receptors such as CD44 since they combine the low toxicity of the carrier and the retention of the protein integrity with the receptor-mediated internalization. HA properties play a crucial but sometimes unclear role in managing the formation and stability of the meshwork, cell interactions, and ultimately the protein entrapment efficacy. Nowadays, microfluidic is an innovative technology that allows to overcome limits linked to the NPs production, guaranteeing reproducibility and control of individual batches. Taking advantage of this technique, in this research work, the role of HA weight average molecular weight (Mw) in NPs formation inside a microfluidic device has been specifically faced. Based on the relationship between polymer Mw and solution viscosity, a methodological approach has been proposed to ensure critical quality attributes (size of 200 nm, PDI ≤ 0.3) to NPs made by HA with different Mw (280, 540, 710 and 820 kDa). The feasibility of the protein encapsulation was demonstrated by using Myoglobin, as a model neutral protein, with an encapsulation efficiency always higher than 50%. Lastly, all NPs samples were successfully internalized by CD44-expressing cells.