A remarkable hallmark of Nature is its inherent lack of left-right symmetry across all scales of size and complexity, from elementary particles up to macroscopic systems such as animals, plants and flowers. One of the questions raised by Nature propensity to favour one handedness over the other concerns the correlation of dissymmetry across different levels of complexity. Because Nature's complexity arises through self-assembling of molecules and macromolecules into organisms whose structure, symmetry features and functions are controlled by non-covalent interactions, we employ a dye-surfactant assembly as a model system. Using circular dichroism spectroscopy combined with molecular dynamics simulations we show that the chirality of complex systems can be modulated by non-chiral molecules such as polyamines. This finding provides a different framework for exploring the transmission of chirality, suggesting that non-chiral metabolites may also contribute to chiral shaping.

Second-generation antimicrobial lipopeptides are considered very promising tools to combat the spread of systemic fungal infections caused by fungi multidrug resistance and biofilm-associated infections. The present work reports a study on the lipopeptide Myr-B, derived from myristoylation of the natural antimicrobial peptide Chionodracine. The aim was to exploit its therapeutic potential by developing a suitable procedure for its encapsulation in liposomes, with a view to overcoming its in vivo instability and tendency to aggregation. A systematic investigation allowed to select two optimal liposome formulations, based on dimyristoylphosphatidylcholine, having chains of the same length of the lipopetide, and cholesterol and/or cholesteryl-hemisuccinate. Thin-film hydration and lipid-cake preparation methods were explored, and chemical-physical characterization was integrated with molecular dynamics simulations. The lipid-cake method proved to be the optimal approach, yielding monodisperse, stable liposomes with high encapsulation efficiency. Encapsulation in both liposome formulations markedly enhanced antifungal activity, lowering minimum inhibitory concentrations more than ten-fold against Candida albicans and approximately four-fold against Candida tropicalis compared to free Myr-B. LIVE/DEAD imaging also confirmed a strong reduction in biofilm formation. Liposomes displayed minimal haemolysis, low cytotoxicity toward human fibroblasts, and good in vivo tolerability in Galleria mellonella. Notably, specific interactions between cholesteryl-hemisuccinate and the peptide involved a peculiar liposome structure and a slower release from the liposome. Overall, both Myr-B-loaded liposomes produced by the lipid cake protocol significantly potentiates MYR-B efficacy while maintaining a favourable safety profile. These formulations are not universally applicable to all lipopeptides; however, the underlying criteria presented in this work are.

Purpose: 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. Methods: 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). Results: 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 transresveratrol 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. Conclusion: Our findings support the potential of antioxidant-loaded liposomes as adjuvants in neurodegenerative diseases or sensitizing agents in cancer therapy.

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 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.

Enantiomerically pure activated carboxylic acids (ACAs), (R)- and (S)-2-cyano-2-phenylpropanoic acids, are exploited to program the induction of chirality onto a zinc metal complex over time. NMR analysis shows that binding of the enantiopure ACA conjugate base to the Zn2+ center breaks the symmetry of the complex and induces the formation of a single diastereoisomeric metal complex. Such a diastereoisomer is present only as long as the ACA is found in solution, and the ACA loading determines the time interval in which it persists in solution. At the end of the dissipative ACA cycle, no diastereoisomeric/enantiomeric excess is present anymore in the mixture. For the first time, a coordination process involving an ACA, more precisely its conjugate base, instead of an acid-base reaction, is exploited to drive a dissipative system.

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

IntroductionThis study investigated the interaction with membrane mimetic systems (LUVs), bacterial membranes, the CD spectra, and the bactericidal activity of two designed trematocine mutants, named Trem-HK and Trem-HSK. Mutants were constructed from the scaffold of Trematocine (Trem), a natural 22-amino acid AMP from the Antarctic fish Trematomus bernacchii, aiming to increase their positive charge.MethodsThe selectivity of the designed AMPs towards bacterial membranes was improved compared to Trematocine, verified by their interaction with different LUVs and their membranolytic activity. Additionally, their α-helical conformation was not influenced by the amino acid substitutions. Our findings revealed a significant enhancement in antibacterial efficacy against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae family) pathogens for both Trem-HK and Trem-HSK.ResultsFirstly, we showed that the selectivity of the two new designed AMPs towards bacterial membranes was greatly improved compared to Trematocine, verifying their interaction with different LUVs and their membranolytic activity. We determined that their α-helical conformation was not influenced by the amino acid substitutions. We characterized the tested bacterial collection for resistance traits to different classes of antibiotics. The minimum inhibitory and bactericidal concentration (MIC and MBC) values of the ESKAPE collection were reduced by up to 80% compared to Trematocine. The bactericidal concentrations of Trematocine mutants showed important membranolytic action, evident by scanning electron microscopy, on all tested species. We further evaluated the cytotoxicity and hemolytic activity of the mutants. At 2.5 μM concentration, both mutants demonstrated low cytotoxicity and hemolysis, indicating selectivity towards bacterial cells. However, these effects increased at higher concentrations.DiscussionAssessment of in vivo toxicity using the Galleria mellonella model revealed no adverse effects in larvae treated with both mutants, even at concentrations up to 20 times higher than the lowest MIC observed for Acinetobacter baumannii, suggesting a high potential safety profile for the mutants. This study highlights the significant improvement in antibacterial efficacy achieved by increasing the positive charge of Trem-HK and Trem-HSK. This improvement was reached at the cost of reduced biocompatibility. Further research is necessary to optimize the balance between efficacy and safety for these promising AMPs.

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.

The C1858T variant of the protein tyrosine phosphatase N22 ( PTPN22 ) gene is associated with pathophysiological phenotypes in several autoimmune conditions, namely, Type 1 diabetes and autoimmune thyroiditis. The R620W variant protein, encoded by C1858T, leads to a gain of function mutation with paradoxical reduced T cell activation. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes, LiposiRNA) that selectively inhibit variant allele expression. In this manuscript, we functionalize lipoplexes carrying siRNA for variant C1858T with a high affinity ligand of Siglec-10 (Sig10L) coupled to lipids resulting in lipoplexes (LiposiRNA-Sig10L) that enhance delivery to Siglec-10 expressing immunocytes. LiposiRNA-Sig10L lipoplexes more efficiently downregulated variant C1858T PTPN22 mRNA in PBMC of heterozygous patients than LiposiRNA without Sig10L. Following TCR engagement, LiposiRNA-Sig10L more significantly restored IL-2 secretion, known to be paradoxically reduced than in wild type patients, than unfunctionalized LiposiRNA in PBMC of heterozygous T1D patients.

Glucosylated liposomes composed of the natural saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phos-phocholine (DPPC), cholesterol (Chol) and a cationic amphiphile featuring a glucosyl moiety (GL4), have been developed for delivering the antimicrobial trans-Resveratrol (RSV) to S. epidermidis , characterized by carbohydrate-specific adhesins able to recognize glucose. The cationic derivative of cholesterol, DC-Chol, was also included in liposome formulations, alone or in combination with GL4 , in order to explore the role of both cationic charge and sugar moiety in the interaction of liposomes with bacterial cells. RSV was included inside glucosylated cationic liposomes by the thin film method, coupled with either extrusion or sonication; liposome mean diameter, polydispersity index, surface charge, RSV entrapment efficiency and concentration have been measured by DLS, electrophoretic mobility, and HPLC. The antimicrobial activity of RSV-loaded liposomes was evaluated by monitoring the bacterial growth curves of two cell lines of Staphylococcus epidermidis , a slime positive strain (i.e. a strain able to form a biofilm) and a slime negative one. Results point out that, when the glucosylamphiphile GL4 is included in the formulation, only the extrusion protocol allows obtaining mono disperse liposomes with high RSV entrapment efficiency. The mean diameters of empty and resveratrol-loaded liposomes are all around 120-140 nm and size distribution are narrow, except for samples including GL4 at 5 molar percentage. Here the higher polydispersity index may be the indication of the occurrence of a restructuring phenomenon. The microbiological tests put in evidence a different response of the two bacterial cell lines to liposome treatments, in fact, the slime negative bacterial cells, that are not able to produce the extracellular polymeric substances, are more susceptible to the cationic charge of the liposomes and to the detergent effect of GL4. The most interesting results concern DPPC/Chol/GL4 liposomes on the slime positive strain: this formulation, non-toxic in itself, displays an enhanced antibacterial efficacy with respect to free RSV, killing bacteria even at concentration tenfold under the MIC.

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.

Hypothesis: The positive charge on liposome surface is known to promote the crossing of the Blood brain barrier (BBB). However, when diastereomeric cationic gemini amphiphiles are among lipid membrane components, also the stereochemistry may affect the permeability of the vesicle across the BBB. Experiments: Liposomes featuring cationic diasteromeric gemini amphiphiles were formulated, characterized, and their interaction with cell culture models of BBB investigated. Findings: Liposomes featuring the gemini amphiphiles were internalized in a monolayer of brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPSC) through an energy dependent transport, internalization involving both clathrin- and caveolae-mediated endocytosis. On the same formulations, the permeability was also evaluated across a human derived in vitro BBB transport model. The permeability of liposomes featuring the gemini amphiphiles was significantly higher compared to that of neutral liposomes (DPPC/Cholesterol), that were not able to cross BBB. Most importantly, the permeability was influenced by the stereochemistry of the gemini and pegylation of these formulations did not result in a drastic reduction of the crossing ability. The in vitro iPSC-derived BBB models used in this work represent an important advancement in the drug discovery research of novel brain delivery strategies and therapeutics for central nervous system diseases. (C) 2022 Elsevier Inc. All rights reserved.

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

In this chapter we describe: (1) the procedure for the synthesis of four single chain bolaamphiphiles, displaying chains of 12, 16, 20 and 30 methylene units and triphenylphosphonium moieties as headgroups (TPP1-TPP4); (2) the methods used to characterize TPP1-TPP4 spontaneous aggregation in aqueous solution. We illustrate the determination of Krafft point and cac by conductivity measurements and the procedures used to investigate dimensions, morphology, and stability by dynamic and dielectrophoretic laser light scattering, dialysis, transmission electron microscopy, and Raman spectroscopy measurements.

Novel cationic glycoliposomes, composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and glycoamphiphiles featuring a galactosyl, mannosyl or glucosyl moiety have been investigated for the targeted delivery of trans-resveratrol (RSV), a Quorum Sensing Inhibitor (QSI), to Methicillin Resistant Staphylococcus Aureus (MRSA) biofilms.All the glycosylated formulations show a 10–20 % reduction of their hydrodynamic size and a high positive increase in ζ-potential (20÷27mV), with respect to the almost neutral DOPC/Chol liposomes (-3.7 mV). RSV is entrapped in liposomes with high Entrapment Efficiency (EE%), the formulations containing glycosylated amphiphiles showing higher values of EE% (79–90 %) than those containing DOPC/CHOL (65 %). In all the liposomal formulations, the inclusion of RSV causes a decrease in ζ-potential, which is particularly evident in the negative value of DOPC/Chol liposomes (-14.8 mV). This is probably due to the ionization of a small percentage of RSV molecules that point towards the lipid/water interface, as reported in the literature.Greater antioxidant activity is found when RSV is embedded in glycosylated liposomes, rather than DOPC/CHOL liposomes. This finding suggests a different RSV distribution in the lipid membrane enclosing the glycosylated amphiphiles, which favor an external exposure of RSV.Biological assays carried out to monitor the demolition effect of RSV-loaded liposomes on mature biofilm of MRSA show that the presence of cationic glycoamphiphiles is essential for a demolition effect to take place on the biofilm matrix. In particular, RSV-galactosylated liposomes are the most effective in destroying MRSA biofilm even at a RSV concentration (0.019 mM) sixty times lower than the MIC (1.2 mM). This work demonstrates, for the first time, how the functionalization of liposomes with cationic glycosydic residues can enhance liposome performances as QSI nanocarriers for the treatment of biofilm associated infections.

Though liposome-based drugs are in clinical use, the mechanism of cell internalization of liposomes is yet an object of controversy. The present experimental investigation, carried out on human glioblastoma cells, indicated different internalization routes for two diastereomeric liposomes. Molecular dynamics simulations of the lipid bilayers of the two formulations indicated that the different stereochemistry of a lipid component controls some parameters such as area per lipid molecule and fluidity of lipid membranes, surface potential and water organization at the lipid/water interface, all of which affect the interaction with biomolecules and cell components.

Antimicrobial peptides (AMPs) are short peptides active against a wide range of pathogens and, therefore, they are considered a useful alternative to conventional antibiotics. We have identified a new AMP in a transcriptome derived from the Antarctic fish Trematomus bernacchii. This peptide, named Trematocine, has been investigated for its expression both at the basal level and after in vivo immunization with an endemic Antarctic bacterium (Psychrobacter sp. TAD1). Results agree with the expected behavior of a fish innate immune component, therefore we decided to synthesize the putative mature sequence of Trematocine to determine the structure, the interaction with biological membranes, and the biological activity. We showed that Trematocine folds into a α-helical structure in the presence of both zwitterionic and anionic charged vesicles. We demonstrated that Trematocine has a highly specific interaction with anionic charged vesicles and that it can kill Gram-negative bacteria, possibly via a carpet like mechanism. Moreover, Trematocine showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against selected Gram-positive and Gram-negative bacteria similar to other AMPs isolated from Antarctic fishes. The peptide is a possible candidate for a new drug as it does not show any haemolytic or cytotoxic activity against mammalian cells at the concentration needed to kill the tested bacteria.

Antimicrobial peptides have been identified as one of the alternatives to the extensive use of common antibiotics as they show a broad spectrum of activity against human pathogens. Among these is Chionodracine (Cnd), a host-defense peptide isolated from the Antarctic icefish Chionodraco hamatus, which belongs to the family of Piscidins. Previously, we demonstrated that Cnd and its analogs display high antimicrobial activity against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). Herein, we investigate the interactions with lipid membranes of Cnd and two analogs, Cnd-m3 and Cnd-m3a, showing enhanced potency. Using a combination of Circular Dichroism, fluorescence spectroscopy, and all-atom Molecular Dynamics (MD) simulations, we determined the structural basis for the different activity among these peptides. We show that all peptides are predominantly unstructured in water and fold, preferentially as α-helices, in the presence of lipid vesicles of various compositions. Through a series of MD simulations of 400 ns time scale, we show the effect of mutations on the structure and lipid interactions of Cnd and its analogs. By explaining the structural basis for the activity of these analogs, our findings provide structural templates to design minimalistic peptides for therapeutics.

Hypothesis: Bolaamphiphiles are characterized by wide polymorphism of their aggregates, due to the connection of the headgroups that renders their investigation very intriguing in several technological applications. Some bolaamphiphiles displaying the triphenylphosphonium motif (TPPbolaamphiphiles) were previously explored for their ability in crossing the mitochondria] membranes but their colloidal features, which are crucial for the potential development of an effective drug delivery system, were never investigated. Experiments: Single chain TPP-bolaamphiphiles, featuring chains of 12, 16, 20 and 30 methylene units, were synthesized and their aggregation features (Krafft point, cac, dimensions, morphology, stability) were investigated by conductivity, dialysis, transmission electron microscopy, Raman spectroscopy, dynamic and dielectrophoretic laser light scattering measurements. Findings: All the TPP-bolaamphiphiles spontaneously self-assemble into vesicles, independently of the chain length. The bolaamphipile with the longest chain forms monodispersed vesicles whereas for the other bolaamphiphiles two distinct populations of vesicles are observed. All vesicles are not equilibrium systems, in particular vesicles formed by the bolaamphiphiles featuring 20 and 30 methylene units result notably stable to dilution thanks to both the tightening of molecular packing at increasing chain length and the progressive reduction of the monomer percentage in U-shaped conformation. These features make these TPP-bolaamphiphiles very attractive as minor components for the development of novel mitochondriotropic liposomes. (C) 2018 Elsevier Inc. All rights reserved.