Triphenyl-phosphonium (TPP)-Based Nanovesicles Induce ROS Increase, Eradicate MDR Melanoma and Neuroblastoma Cells, While Exerting Low Toxicity Towards Red Blood Cells (RBCs), Human Not Tumorigenic Cells and Mice Primary Cell Cultures

Neuroblastoma (NB) and metastatic cutaneous melanoma (MCM) are a severe children malignant solid tumour and the most aggressive skin cancer existing respectively, with low survival rate and poor prognosis, due to therapy-induced secondary tumorigenesis and emerging multidrug-resistance (MDR). Recently, we obtained in water self-forming TPP-based bola amphiphilic nanovesicles (BANs), deriving by the quaternary phosphonium salt, 1,1-(1,12-dodecanediyl)-bis-[1,1,1]-triphenyl phosphonium di-bromide (BPPB), synthesized by reacting 1,12-dibromo-dodecane with triphenylphosphine in ethanol, refluxing for 48 h (Alfei et al. 2024a). ATR-FTIR, NMR, UV, FIA-MS (ESI) and elemental analysis confirmed its structure. DLS analyses showed spherical vesicles of 45 nm, having positive ζ =f +18 mV (Alfei et al. 2024a). Extensively employed as drug delivery systems, no report existed on the per se cytotoxic activity of any BAN towards any cancer cell line, before our studies. Notably, BANs provided excellent outcomes, when tested on drug-sensitive and MDR MCM and HR-NB cells, as well as on non-tumorigenic human cells, mice primary cells and RBCs to assess their cytotoxicity and their biocompatibility, respectively (Alfei et al. 2024b; Alfei et al. 2024c; Alfei et al. 2025a; Alfei et al. 2025b). Specifically, experimented against PLX-sensitive BRAF mutants MCM cells (MeOV and MeTRAV), BANs showed a ROS-dependent cytotoxicity, displaying nanomolar IC50 of 49 (MeOV) and 171 nM (MeTRAV) and inducing a ROS production 16-fold higher than in the control (72h treatments). Conversely, BANs elicited low cytotoxicity against various non-tumorigenic human cells and RBCs, allowing selectivity index (SI) values of 5-19 (MRC-5, 72h), 8-11 (HepG2, 24h) and 87-229 (RBCs, 72h). Regardless of the acquired resistance, PLX-resistant MCM cells exposed to BANs provided nanomolar IC50 of 88 nM (PLX-R-MeOV, 48h) and 81 nM (PLX-R-MeTRAV, 72h), as well. ROS increase was 25- and 15-fold higher than in the control (MeOV and MeTRAV respectively), after 72h exposure. Anyway, a ROS-dependent cytotoxicity was established only in MeTRAV for 72h treatments. Forecasting a future development of BANs as topical device to treat skin MCM lesions, its biocompatibility was essayed on human keratinocytes (HaCaT), observing SI values up to 12. Furthermore, RBCs were exposed to BANs, high HC50 values were observed allowing SI = 16–173 and 4–192 (PLX-R MeOV and MeTRAV, respectively). Recently, BANs were tested against drug-sensitive HTLA 230 human stage-IV and MDR HTLA ER high-risk (HR)-NB cells, observing apoptotic death at IC50 of 0.4-0.9 and 1.20-1.35 µM, respectively, sustained by moderate ROS overproduction (ROS levels 5-6-fold higher than in the control). Here, by an analytical approach, a correlation study was carried out to verify if BANs cytotoxicity depended on their concentrations, exposure times and/or ROS overproduction and if ROS increase depended on BANs concentrations and/or time exposure. To better support the potential clinical development of BANs as a new therapeutic option to treat MDR HR-NB, their effects were tested on primary astrocytes and neurons. Appreciable and good tolerability was observed in neurons and astrocytes respectively, allowing SI values up to 24. Collectively, these in vitro results pave the way for future in vivo investigations using BANs and for the possible development of BANs as a novel therapeutic strategy to treat MDR MCM and HR-NB. REFERENCE Alfei, S. et al. 2024a. Synthesized Bis-Triphenyl Phosphonium-Based Nano Vesicles Have Potent and Selective Antibacterial Effects on Several Clinically Relevant Superbugs. Nanomaterials 14(16), p. 1351. doi: 10.3390/nano14161351. Alfei, S. et al. 2024b. The Remarkable and Selective In Vitro Cytotoxicity of Synthesized Bola-Amphiphilic Nanovesicles on Etoposide-Sensitive and -Resistant Neuroblastoma Cells. Nanomaterials 14(18), p. 1505. doi: 10.3390/nano14181505. Alfei, S. et al. 2025a. TPP-Based Nanovesicles Kill MDR Neuroblastoma Cells and Induce Moderate ROS Increase, While Exert Low Toxicity To-wards Primary Cell Cultures: an in Vitro Study . IJMS 26(16), p. 4991. doi: https://doi.org/10.3390/ijms26114991. Alfei, S., Torazza, C., Bacchetti, F., Signorello, M.G., Passalacqua, M., Domenicotti, C. and Marengo, B. 2025b. Tri-Phenyl-Phosphonium-Based Nano Vesicles: A New In Vitro Nanomolar-Active Weapon to Eradicate PLX-Resistant Melanoma Cells. International Journal of Molecular Sciences 26(7), p. 3227. doi: 10.3390/ijms26073227. Alfei, S., Zuccari, G., Athanassopoulos, C.M., Domenicotti, C. and Marengo, B. 2024c. Strongly ROS-Correlated, Time-Dependent, and Selective Antiproliferative Effects of Synthesized Nano Vesicles on BRAF Mutant Melanoma Cells and Their Hyaluronic Acid-Based Hydrogel Formulation. International Journal of Molecular Sciences 25(18), p. 10071. doi: 10.3390/ijms251810071.