# Steering the Clinical Translation of Delivery Systems for Drugs and Health Products
## 3 Poster Presentations
### Cell internalization Kinetics of Biodegradable Nanoparticles Decorated with Hyaluronic Acid  
[PERSON] 1, [PERSON] 1,*, [PERSON] 2, [PERSON] 2 and [PERSON] 1 Dipartimento di Farmacia, Universita di Napoli Federico II, 80138 Napoli, Italy  
2 Consiglio Nazionale delle Ricerche (CNR), Istituto per i Polimeri, Compositi e Biomateriali (IPCB), 80125 Napoli, Italy  
* Correspondence: [EMAIL_ADDRESS]  
Among the strategies to endow nanodevices with active tumor targeting ability, their surface decoration with hyaluronic acid (HA) indisputably plays a major role. Indeed, HA is a negatively charged polysaccharide possessing a strong tropism toward solid tumors due to preferential interaction with CD44 and RHAMM receptors, which are overexpressed in a wide array of cancer cells [50; 51]. Consequently, nanocarriers exposing HA on their surface may be endowed with a specific recognition ability toward tumors [52]. In previous studies, it has been suggested that HA-coated nanoparticles (NPs) can be internalized by a receptor-mediated mechanism [53]. Thus, the main hypothesis of this work is that the mode of ligand presentation can determine the cellular uptake of NPs. Here, poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) have been decorated with HA without chemical reaction, using amphiphilic poloxamers (namely F68 and F127) as bridging moieties between hydrophobic PLGA and hydrophilic HA. Two HA molecular weights were chosen (200 and 800 kDa; NP formulations were named HA2 and HA8, correspondingly) and kinetic internalization studies were carried out on breast carcinoma (HS578T) and healthy mouse fibroblast (L929) cells, used as a control. NPs were produced by a modified nanoprecipitation technique, by forcing a PLGA/F68/F127 solution in acetone (1:0.5:0.5 weight ratio; 5 mL, 3% w/v) through a needle of a syringe at 333 \(\mu\)L/min by a Syringe Pump. The solution was precipitated into 40 mL of an aqueous phase (W1) containing F127 and F68 as surfactants (1:1 w/w ratio; 0.05% w/v) and HA (3.75 and 0.81 mg/ml for HA2 and HA8 NPs). The organic solvent was evaporated overnight, and the obtained NPs washed twice times by centrifugation (10,000 rpm, 20 min) and stored at \(-80^{\circ}\)C. NPs were characterized for their morphology by TEM, while size and z-potential (ZP) by PCS. NP architecture was studied by differential scanning calorimetry (DSC) (10-80 \({}^{\circ}\)C, 5 \({}^{\circ}\)C/min, under inert nitrogen atmosphere). Cellular uptake kinetics of fluorescent NPs, prepared by adding Nile Red (NR) to the organic phase (0.01% w/w NR:polymers), were obtained by performing, at scheduled time points, cell lysis followed by a spectrofluorimetric assay on the lysate to quantify NP-associated fluorescence. Spherical NPs with a \(<\)200 nm mean size was obtained. ZP values are \(<\)-40 mV, indicating the successful HA arrangement on NP surface (Figure 15). DSC results indicated that, for HA2 and HA8 NPs, melting temperature (Tm) and heat (\(\Delta\)Hm) are lower compared to Tm and \(\Delta\)Hm of poloxamers alone, indicating the loss of crystalline poloxamer regions in rubbery PLGA matrix and the formation of a co-amorphous, partially phase-separated system in which the polymers partially behave as separate entities (Table 1). Results of cell internalization showed that both HA2 and HA8 NPs enter more effectively in CD44-overxpressing tumor cells than in healthy cells, indicating that HA-containing NPs do possess a tropism for HS578T cells. More in detail, the uptake of HA8 NPs is higher than HA2 NPs in both cell lines (Figure 16), thereby hinting at a different molecular arrangement of high molecular weight HA which in turn affects the precocity of interaction between HA-decorated nanodevices and the target cells. In conclusion, in this work PLGA-based NPs have been successfully decorated with HA and their tropism for CD44-overex pressing cells has been shown. Moreover, HA8 NPs showed a higher uptake propensity than HA2 NPs.