Francesca Ceccacci

We aimed to develop mitochondriotropic liposomes (TPP3-liposomes) formulated with a phospholipid (PC) and the triphenylphosphonium bolaamphiphile TPP3, and encapsulate two antioxidants, trans-resveratrol (hydrophobic) and Trolox (hydrophilic), for mitochondrial therapy of neurodegeneration and drug-resistant tumors. The influence of the PC (saturated or unsaturated) and TPP3/PC ratio on liposome physico-chemical properties (diameter, polydispersity, charge, transition temperature, and stability over time) were investigated by Dynamic and Dielectrophoretic Light Scattering measurements. Evaluation of cytotoxicity, mitochondrial targeting and effect on mitochondrial membrane potential of TPP3-liposomes were conducted utilizing an MTT assay, laser scanning confocal microscopy and flow cytometry on drug-resistant human breast cancer cells (MDA-MB231) and murine skeletal muscle cells (C2C12): MDA-MB231 cells have been selected as a model for studying multiple drug resistance (MDR) in cancer; C2C12 cells have been chosen to investigate the oxidative stress associated with the ageing process and neurodegenerative muscle diseases. Two different strategies were explored for antioxidant loading: active loading into the liposome aqueous cavity (resveratrol, Trolox) and passive loading inside the lipid bilayer membrane (resveratrol). The amount of TPP3 bolaamphiphile and lipid composition affect liposomes’ physicochemical properties, liposome bilayer organization, and antioxidant loading efficiency. TPP3 confers the ability to reach mitochondria even in low amounts (2.5%); liposomes with 2.5% of TPP3 are non-toxic and capable of encapsulating the antioxidants. TPP3-liposomes encapsulating trans-resveratrol in the lipid bilayer membrane or in the aqueous cavity were developed, with high entrapment efficiency in both cases. Trolox was encapsulated in the aqueous cavity of liposomes, without precedents in literature, with very high entrapment efficiency and enhanced stability following encapsulation. TPP3-liposomes can deliver resveratrol to the mitochondria in the MDA-MB231 cells, exerting a protective activity on the mitochondrial structure. Our findings support the potential of antioxidant-loaded liposomes as adjuvants in neurodegenerative diseases or sensitizing agents in cancer therapy.

Eugenia Belcastro was not included as an author in the original publication

The chirality transfer from chiral domains to achiral molecules is an important theoretical and applicative issue. In this work, we have investigated the interaction between two anionic chiral surfactants bearing a proline residue as hydrophilic head and the cationic, achiral porphyrin Zn(II) [5-{4-(3-trimethylammonium)propyloxyphenyl}-10,15,20-triphenylporphyrinyl]chloride to assess the effects of the structural variations in both units on the chirality transfer efficiency and amplification. We showed that the efficiency of transferring molecular information depends on the surfactant’s features, namely the chiral configuration of the polar head, the length of the aliphatic chain, and the aggregation state. At the same time, the presence of a coordinated metal and the peripheral charged group on the porphyrin macrocycle are key factors. In detail, the study of the hetero-aggregates formed at a surfactant concentration below the critical micellar concentration (cmc) indicates that the chirality depends on the synergy of hydrophobic effect, coordination interaction, and electrostatic forces. If the surfactant concentration is higher than the cmc, at a low concentration, porphyrins are included in micelles as monomers. Under these conditions, no chirality transfer is evident. When the porphyrin is in excess with respect to the micelles, an efficient asymmetry induction is again observed, transmitted from the chiral polar head to the porphyrin oligomers included in the micelle, through the polar heads and the hydrocarbon chains of the surfactants.

Background: The C1858T PTPN22 variant is strongly associated with type 1 diabetes and autoimmune thyroid disease. Current treatment is substitutive hormonal administration, which does not target the disease pathogenetic mechanism. We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes, in order to halt autoimmune disease progression. Objectives: The objective of this study was to optimize lipoplex formulations functionalized with F9-PEG (a Siglec-10’s ligand) to facilitate targeted delivery by investigating their physical and chemical properties to warrant the best performance in in vivo studies. Methods: The effectiveness of siRNA liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to the methods of F9-PEG addition and ATTO740 fluorescent labeling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS and DELS), and high-performance liquid chromatography (HPLC). Results: The optimal charge ratio of +2/−1 (lipid/siRNA) ensured a greater stability of lipoplexes and complete complexation of siRNA. Stability was improved by selecting a protocol of preparation that envisages functionalization with F9-PEG and the addition of ATTO740 lipid in the lipid film preparation step. HPLC confirmed the integrity of siRNA after preparation. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients.

The C1858T PTPN22 variant is strongly associated with Type 1 diabetes and autoimmune thyroid disease. Current treatment is the substitutive hormonal administration which does not target the disease pathogenetic mechanism. Background/Objectives: We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes in order to halt autoimmune disease progression. The objective of this study was to optimize lipoplexes formulations functionalized with F9-PEG (a Siglec-10's ligand) to facilitate targeted delivery, by testing their physical and chemical properties to warrant best performance in in vivo studies. Methods: The effectiveness of siRNA-liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to methods of F9-PEG addition and ATTO740 fluorescent labelling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS, DELS), and high-performance liquid chromatography (HPLC). A preliminar investigation of lipoplexes biodistribution was conducted by fluorescent tagging in C57BL/6 mice. Results: The optimal charge ratio of +2 /-1 (lipid/siRNA) ensured greater stability of lipoplexes. Stability was improved by functionalization with F9-PEG further by the addition of ATTO740-dye. HPLC confirmed the integrity of siRNA after preparation. Injection of PKH26-labelled lipoplexes in mice showed selective accumulation in target tissues within 48 hours. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients.

Antibiotic resistance due to bacterial biofilm formation is a major global health concern that makes the search for new therapeutic approaches an urgent need. In this context,, trans-resveratrol (RSV), a polyphenolic natural substance, seems to be a good candidate for preventing and eradicating biofilm-associated infections but its mechanism of action is poorly understood. In addition, RSV suffers from low bioavailability and chemical instability in the biological media that make its encapsulation in delivery systems necessary. In this work, the anti-biofilm activity of free RSV was investigated on Staphylococcus aureus and, to highlight the possible mechanism of action, we studied the anti-adherence activity and also the cell wall damage on a MRSA strain. Free RSV activity was compared to that of RSV loaded in liposomes, specifically neutral liposomes (L = DOPC/Cholesterol) and cationic liposomes (LG = DOPC/Chol/GLT1) characterized by a galactosylated amphiphile (GLT1) that promotes the interaction with bacteria. The results indicate that RSV loaded in LG has anti-adherence and anti-biofilm activity higher than free RSV. On the other side, free RSV has a higher bacterial-growth-inhibiting effect than encapsulated RSV and it can damage cell walls by creating pores; however, this effect can not prevent bacteria from growing again. This RSV ability may underlie its bacteriostatic activity.

Biofilm-associated bacterial diseases are a major health problem due to the high antibiotic resistance of biofilm infections

The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.

Lipid stereochemistry was shown to drive cell internalization of liposomes through either clathrin or caveolae. Molecular dynamic simulations highlighted molecular interactions that affect physicochemical and biological features of liposomes.