The increasing emergence of multidrug resistant (MDR) pathogens, due to antibiotics misuse, translates in obstinate infections, with high morbidity and high-cost hospitalizations. To oppose these MDR superbugs, new antimicrobial options are necessary. Although both quaternary ammonium (QASs) and phosphonium salts (QPSs) possess antimicrobial effects, QPSs have been studied at lesser extent. Recently, we have successfully reported on the bacteriostatic and cytotoxic effects of a triphenyl phosphonium salt against MDR isolates of Enterococcus and Staphylococcus genus. Here, aiming at finding new antibacterial devices possibly bactericidal towards a broader spectrum of clinically relevant bacteria, responsible for severe human infections, we synthesized a water-soluble, sterically hindered quaternary phosphonium salt (BPPB). It encompasses two triphenyl phosphonium groups linked by a C12 alkyl chain, thus embodying the characteristics of molecules known as bola-amphiphiles. BPPB was characterized by ATR-FTIR, NMR and UV spectroscopy, FIA-MS (ESI), elemental analysis and potentiometric titrations. Optical and DLS analyses evidenced BPPB tendency to self-forming spherical vesicles in solution (45 nm by DLS), having a positive zeta potential (+18 mV). The antibacterial effects of BPPB were for the first time assessed against fifty clinical isolates of both Gram-positive and Gram-negative species. Excellent antibacterial effects were observed on all strains tested, involving all the most worrying species included in ESKAPE bacteria. The lowest MICs were 0.250 µg/mL, while the highest ones (32 µg/mL) were observed on MDR Gram-negative metallo-β-lactamase-producing bacteria and/or species resistant also to colistin, carbapenems, cefiderocol, so that intractable with current available antibiotics. Moreover, when administered to HepG2 human hepatic and Cos-7 monkey kidney cell lines, BPPB showed selectivity indices > 10 for all Gram-positive isolated and for clinically relevant Gram-negative superbugs such as those of E. coli species, thus being very promising to be clinically developed.

Neuroblastoma (NB) is a solid tumor occurring in infancy and childhood, whose high-risk form has currently a survival rate < 50 %, despite aggressive treatments. Additionally, drugs-induced secondary tumorigenesis and the emergency of drug resistance worsen the already worrying scenario, calling for an urgent development of new extra-genomic treatments. Quaternary phosphonium salts (QPSs) possess both antimicrobial and anticancer effects. Triphenyl phosphonium salts (TPPs) are mitochondria-targeting compounds, exerting anticancer effects, impairing mitochondria functions and damaging DNA, at the same time. TPP-based bola-amphiphiles, previously proven to self-assemble nanoparticles (NPs) in water and successfully assayed as possible antibacterials, were never tested as suitable antitumor agents. Here, aiming at the development of new antitumor devices to counteract resistant forms of NB, the anticancer effects of a TPP-based amphiphile molecule have been investigated for the first time. To this end, we considered the water-soluble sterically hindered quaternary phosphonium salt (BPPB) encompassing two triphenyl phosphonium groups linked by a C12 alkyl chain, thus embodying both the characteristics of mitochondria-targeting compounds and those of bola-amphiphiles. The anticancer effects of BPPB were assessed against HTLA-230 human stage-IV NB cells and their counterpart resistant to etoposide (ETO), doxorubicin (DOX) and to many other therapeutics (HTLA-ER). Determined IC50 values were very low on both cell populations, already after 24 hours of treatment, when cells viability did not significantly differ from that observed for the longest exposure timing. Moreover, to investigate a putative future inclusion of BPPB in a chemotherapeutic cocktail for NB, its cytotoxic effects were assessed against mammalian cell lines. These included monkey kidney cells (Cos-7), human hepatic cells (HepG2), a lung-derived fibroblast cell line (MRC-5) and red blood cells. From appreciable to very high selectivity indices have been determined after 24 hours treatments, an exposure time after which both NB cell populations tested appeared already fully exterminated.

The absence of highly effective delivery systems is a major challenge for gene therapy. Our work was aimed at the development of novel cationic liposomes possessing high transfection efficiency. For this purpose, a novel disulfide polycationic amphiphile 2S4 was synthesized. Cationic liposomes based on 2S4 and a helper lipid DOPE were formed by the thin film hydration method and exhibited effective pDNA delivery into the HEK293 cells, with a maximal transfection activity superior to that of the commercial agent Lipofectamine^® 2000. Our results suggest that the polycationic amphiphile 2S4 is a promising candidate for in vitro nucleic acid delivery.

Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain DNA of living cells. To do so, it needs to interact with and permeate through cell membranes. The pH of biological media may vary quite significantly and this influences H33342 ionization state, and may change its rate of permeation through biomembranes. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics simulations. While probe insertion occurs for all ionization states, with similar overall equilibrium location, orientation was found to depend on the probe charge, with the neutral form lying mostly parallel and the doubly charged displaying a significant tilt relative to the membrane plane. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy of interaction is negative and is larger at lower pH values. This suggests efficient hydrogen bonding between the probe and the lipid, namely for the protonated +2 form. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from water and octanol partition. This highlights the distinction between lipophilicity and hydrophobicity, and the importance of considering association with lipid bilayers when predicting the affinity for biomembranes.