Intracellular Trafficking and Uptake of Hyaluronan-Doxorubicin Conjugates in Vitro

The purpose of this study was to determine the mechanism of uptake and release of doxorubicin from HA nanoparticle. We used human head and neck squamous cell carcinoma MDA-1986 as a model cell line. MDA-1986 was imaged after incubation with HA-DOX (NPs), doxorubicin, HA-CY7 or HA-DOX-CY7 at 15 minutes, 1, 6, 24, and 48 hours' time points using an inverted fluorescence microscope. Also, the cell nucleus was stained with DAPI stain, and the cells were imaged after incubation with HA-DOX conjugates, doxorubicin, HA-CY7 or HA-DOX-CY7 for 6 hours. Furthermore, the lysosomes were stained to determine if the lysosomal pathway is the major degradation pathway of the nanoparticles and the carrier. To determine the internalization mechanism, four inhibition conditions were used: excess HA to block HA receptors, anti CD44 antibody (Hermes-1) to block the CD44 receptor, chlorpromazine to inhibit clathrin mediated endocytosis, and reduced temperature of 4°C. The fluorescence intensities of the cells co-incubated with the nanoparticle or the carrier with different inhibitors were compared using the inverted fluorescence microscope. The quantitative analysis of cells treated with HA-DOX and co-incubated with different inhibition conditions (10 mg/mL HA, chlorpromazine, Hermes-1 and 4°C) showed significant decrease in the fluorescence intensity except in cells pretreated with 5 mg/mL HA. Also, the quantitative analysis of cells treated with HA-CY7 and co-incubated with different inhibition conditions (excess HA, chlorpromazine and Hermes-1) showed significant decrease in the fluorescence intensity. However, the quantitative analysis of cells treated with free doxorubicin or HA-DOX-CY7 showed significant decrease in the fluorescence intensity in only cells pretreated with 25- µM chlorpromazine or cells incubated at 4 °C. The suggested internalization mechanism of HA-DOX conjugates is an active transport mechanism mediated mainly by the CD44, one of the HA receptors, through a clathrin-dependent endocytic pathway. On the other hand, a sulfated-HA derivative has similar uptake profile as the parent HA-CY7, and it is mainly localized in the cytosol

Cellular Uptake and Internalization of Hyaluronan-based Doxorubicin and Cisplatin Conjugates

Background Hyaluronan (HA) is a ligand for the CD44 receptor which is crucial to cancer cell proliferation and metastasis. High levels of CD44 expression in many cancers have encouraged the development of HA-based carriers for anti-cancer therapeutics. Purpose The objective of this study was to determine whether HA conjugation of anticancer drugs impacts CD44-specific HA-drug uptake and disposition by human head and neck cancer cells. Methods The internalization and cellular disposition of hyaluronan-doxorubicin (HA-DOX), hyaluronan-cisplatin (HA-Pt), and hyaluronan-cyanine7 (HA-Cy7) conjugates were investigated by inhibiting endocytosis pathways, and by inhibiting the CD44–mediated internalization pathways that are known to mediate hyaluronan uptake in vitro. Results Cellular internalization of HA was regulated by CD44 receptors. In mouse xenografts, HA conjugation significantly enhanced tumor cell uptake compared to unconjugated drug. Discussion The results suggested that the main mechanism of HA-based conjugate uptake may be active transport via CD44 in conjunction with a clathrin–dependent endocytic pathway. Other HA receptors, hyaluronan–mediated motility receptor (RHAMM) and lymphatic vessel endothelial hyaluronan receptor (LYVE-1), did not play a significant role in conjugate uptake. Conclusions HA conjugation significantly increased CD44 mediated drug uptake and extended the residence time of drugs in tumor cells

In Vitro Safety Assessment of In-House Synthesized Titanium Dioxide Nanoparticles: Impact of Washing and Temperature Conditions

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO2 NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO2 NPs synthesized via the Stöber method under different washing and temperature conditions. TiO2 NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO2 NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO2 NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO2 NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO2, indicating that toxicity is partially driven via non-oxidative pathways. TiO2 NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions