Resveratrol and Resveratrol-Loaded Galactosylated Liposomes: Anti-Adherence and Cell Wall Damage Effects on Staphylococcus aureus and MRSA.

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.

Quantification of particle number concentration in liposomal suspensions by Laser Transmission Spectroscopy (LTS).

Laser Transmission Spectroscopy (LTS) is an experimental technique able to determine the particle number concentration and the size of colloidal suspensions by a single measurement of the transmittance of a laser beam through the suspension of particles as a function of the wavelength. In this protocol, we show that LTS represents a unique and powerful tool to investigate suspensions of liposomes, where the precise quantification of the number concentration is particularly relevant for the complete definition of the colloidal properties of the suspension. We study a model formulation of Soy-PC:Chol liposomes and we validate LTS results by comparison with High-Performance Liquid Chromatography determination of lipid mass. Then LTS protocols is applied to state-of-art liposomal nanocarrier suspensions. We explain details of data analysis to obtain the particle number concentration by using the Lambert-Beer law and by calculating the extinction cross section, within the framework of Mie theory for spherical vesicles. We also determine the liposome radius and compare it with the hydrodynamic radius measured by Dynamic Light Scattering. As future perspective, we aim to extend LTS analysis to other nanostructures with different geometries and to contribute to the development of new quantitative strategies for the accurate characterization of nanocarriers and other nanoparticles.

Electrochemical Bottom-Up Synthesis of Chiral Carbon Dots from L-Proline and Their Application as Nano-Organocatalysts in a Stereoselective Aldol Reaction.

Chirality is undoubtedly a fundamental property of nature since the different interactions of optically active molecules in a chiral environment are essential for numerous applications. Thus, in the field of asymmetric synthesis, the search for efficient, sustainable, cost-effective and recyclable chiral catalysts is still the main challenge in organic chemistry. The field of carbon dots (CDs) has experienced tremendous development in the last 15 years, including their applications as achiral catalysts. Thus, understanding the implications of chirality in CDs chemistry could be of utmost importance to achieving sustainable and biocompatible chiral nanocatalysts. An efficient and cost-effective electrochemical synthetic methodology for the synthesis of L-Proline-based chiral carbon dots (CCDs) and EtOH-derived L-Proline-based chiral carbon dots (CCDs) is herein reported. The electrochemical set-up and reaction conditions have been thoroughly optimised and their effects on CCDs size, photoluminescence, as well as catalytic activity have been investigated. The obtained CCDs have been successfully employed to catalyze an asymmetric aldol reaction, showing excellent results in terms of yield, diastereo- and enantioselectivity. Moreover, the sustainable nature of the CCDs was demonstrated by recycling the catalysts for up to 3 cycles without any loss of reactivity or stereoselectivity.

Transport of cationic liposomes in a human blood brain barrier model: Role of the stereochemistry of the gemini amphiphile on liposome biological features.

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.

Design and Characterization of Myristoylated and Non-Myristoylated Peptides Effective against Candida spp. Clinical Isolates.

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.

Resveratrol loaded in cationic glucosylated liposomes to treat Staphylococcus epidermidis infections.

Glucosylated liposomes composed of the natural saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (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 monodisperse 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.

How stereochemistry of lipid components can affect lipid organization and the route of liposome internalization into cells.

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.

Synthesis and Characterization of Mitochondria-Targeted Triphenylphosphonium Bolaamphiphiles.

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.

Time-programmable pH: decarboxylation of nitroacetic acid allows the time-controlled rising of pH to a definite value.

In this report it is shown that nitroacetic acid 1 (O2NCH2CO2H) can be conveniently used to control the pH of a water solution over time. Time-programmable sequences of the kind pH1(high)-pH2(low)-pH3(high) can be achieved, where both the extent of the initial pH jump (pH1(high)-pH2(low)) and the time required for the subsequent pH rising (pH2(low)-pH3(high)) can be predictably controlled by a judicious choice of the absolute and relative concentrations of the reagents (acid 1 and NaOH). Successive pH1(high)-pH2(low)-pH3(high) sequences can be obtained by subsequent additions of acid 1. As a proof of concept, the method is applied to control over time the pH-dependent host-guest interaction between alpha-cyclodextrin and p-aminobenzoic acid.

The Interaction between Amyloid Prefibrillar Oligomers of Salmon Calcitonin and a Lipid-Raft Model: Molecular Mechanisms Leading to Membrane Damage, Ca2+-Influx and Neurotoxicity.

To investigate the interaction between amyloid assemblies and "lipid-rafts", we performed functional and structural experiments on salmon calcitonin (sCT) solutions rich in prefibrillar oligomers, proto- and mature-fibers interacting with liposomes made of monosialoganglioside-GM1 (4%), DPPC (48%) and cholesterol (48%). To focus on the role played by electrostatic forces and considering that sCT is positive and GM1 is negative at physiologic pH, we compared results with those relative to GM1-free liposomes while, to assess membrane fluidity effects, with those relative to cholesterol-free liposomes. We investigated functional effects by evaluating Ca2+-influx in liposomes and viability of HT22-DIFF neurons. Only neurotoxic solutions rich in unstructured prefibrillar oligomers were able to induce Ca2+-influx in the "lipid-rafts" model, suggesting that the two phenomena were correlated. Thus, we investigated protein conformation and membrane modifications occurring during the interaction: circular dichroism showed that "lipid-rafts" fostered the formation of ß-structures and energy filtered-transmission electron microscopy that prefibrillar oligomers formed pores, similar to Aß did. We speculate that electrostatic forces between the positive prefibrillar oligomers and the negative GM1 drive the initial binding while the hydrophobic profile and flexibility of prefibrillar oligomers, together with the membrane fluidity, are responsible for the subsequent pore formation leading to Ca2+-influx and neurotoxicity.

Aggregation behaviour of triphenylphosphonium bolaamphiphiles.

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 (TPP-bolaamphiphiles) were previously explored for their ability in crossing the mitochondrial 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.

Monosialoganglioside-GM1 triggers binding of the amyloid-protein salmon calcitonin to a Langmuir membrane model mimicking the occurrence of lipid-rafts.

GM1 ganglioside is known to be involved in the amyloid-associated diseases and it is a crucial factor for the assembly of amyloid proteins on lipid-rafts, which are lipid structures located on the synaptic plasma membranes. Due to its slow aggregation rate, we employed salmon calcitonin (sCT) as a suitable probe representative of amyloid proteins, to study the interaction between this class of proteins and a membrane model. Here, we prepared a neuronal membrane model by depositing onto mica two Langmuir-Blodgett films in liquid-condensed phase: the outer monolayer was characterized by high content of GM1 (50%) and minority parts of cholesterol and POPC (25-25%), while the inner one by plain POPC. To deeply investigate the interaction of sCT with this model and the role-played by GM1, we prepared the outer leaflet adding sCT at a concentration such that the number of proteins equals that of GM1. Atomic Force Microscopy revealed the occurrence of two distinct kinds of flat surfaces, with globular aggregates localized exclusively on top of the highest one. To unravel the nature of the interaction, we studied by ζ-potential technique liposomes composed as the outer leaflet of the model. Results demonstrated that an electrostatic interaction sCT-GM1 occurred. Finally, to investigate the interaction thermodynamics between sCT and the outer leaflet, Langmuir films as the outer monolayer and containing increasing content of sCT were studied by compression isotherms and Brewster Angle Microscopy experiments. Based on the all body of results we propose an interaction model where GM1 plays a pivotal role.

Remote loading of aloe emodin in gemini-based cationic liposomes.

Anthraquinone compound aloe-emodin (AE) has shown antineoplastic, antibacterial, antiviral, and anti-inflammatory properties and scavenging activity on free radicals. Because of these therapeutic features, AE has been attracting increasing interest and could be applied in the curing of many diseases. However, until now the physicochemical features of this compound have not been fully investigated; furthermore, its wide application might be hindered by its scarce solubility in aqueous media (∼19 µM). The inclusion of AE in nanocarriers, such as cationic liposomes, could allow its delivery effectively and selectively to target sites, reducing side effects in the remaining tissues. In this work, the weak acid nature of AE, because of its two phenolic functions, was exploited to load it remotely in the internal aqueous phase of liposomes in response to a difference in pH between the inside and outside of the liposomes, pHin > pHout. The inclusion of AE in gemini-based cationic liposomes by the acetate gradient method was obtained at high AE/lipid ratios (up to 1:30).

Fusion of gemini based cationic liposomes with cell membrane models: implications for their biological activity.

The interaction of neutral and anionic phospholipid liposomes, used as cell models, with cationic liposomes formulated with 1,2-dimyristoyl-sn-glicero-3-phosphocholine and stereomeric cationic gemini surfactants was investigated by differential scanning calorimetry, fluorescence experiments and dynamic laser light scattering. This study was aimed at rationalizing the different biological features shown by liposomes based on different gemini stereoisomers observed in previous investigations. In fact, to correlate the observed biological activity of liposomes with the molecular structure of their components is critical for a rational and systematic approach to the design of new carriers for drug delivery. The obtained results show that the different stereochemistry of the gemini surfactant controls the interaction and the extent of fusion with different cell models.

Synthesis and physicochemical characterization of pyrrolidinium based surfactants.

Three new pyrrolidinium based surfactants were synthesized and characterized as pure aggregate components and in mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine to understand how the molecular structure of the cationic amphiphile and its mole percentage might affect the physicochemical properties of the resulting aggregates. The thermotropic behavior of the mixed liposomes was investigated by differential scanning calorimetry on multilamellar vesicles, whereas their size and surface potential were investigated on large unilamellar vesicles by dynamic laser light scattering and fluorescence experiments, respectively. The presence of either methoxy or hydroxy groups in the positions 3 and 4 of the pyrrolidinium ring as well as the presence of a second alkyl chain on pyrrolidinium nitrogen, controls the aggregates features.

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes.

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.

Extended cardiolipin anchorage to cytochrome c: a model for protein-mitochondrial membrane binding.

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c-CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c-CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c-CL complex.

Efficiency of liposomes in the delivery of a photosensitizer controlled by the stereochemistry of a gemini surfactant component.

Liposomes formulated with dimyristoyl-sn-glycero-phosphatidylcholine, DMPC, and either one of the cationic gemini surfactants (S,S)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane bromide, 1a, and (S,R)-2,3-dimethoxy-1,4-bis(N-hexadecyl-N,N-dimethylammonio)butane bromide, 1b, were investigated as vehicles of the photosensitizer m-tetrahydroxyphenylchlorin, m-THPC, to cell models of malignant glioma. The delivery efficiency of DMPC/1a and DMPC/1b liposome formulations were evaluated on the murine glioblastoma cell line C6 and on the human glioblastoma cell line LN229 by flow cytometry and laser scanning confocal microscopy. The stereochemistry of the spacer of the gemini was found to strongly influence the delivery efficiency of m-THPC to cells, the mode of interaction with the cell membrane, and the intracellular distribution of m-THPC. The physicochemical features of liposomes were investigated with the aim of explaining the parameters that control their biological features. Differences that could account for the different biological activity of the formulations concern the values of surface potential and the environment of m-THPC at the water/liposome interface.

New cationic liposomes as vehicles of m-tetrahydroxyphenylchlorin in photodynamic therapy of infectious diseases.

Antimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. For optimizing the antibacterial activity of the photosensitizer m-tetrahydroxyphenylchlorin, it has been encapsulated in mixed cationic liposomes composed of different ratios of dimyristoyl- sn-glycero-phosphatidylcholine and any of four cationic surfactants derived from l-prolinol. The delivery efficiency of the different liposomes formulations has been evaluated on a methicillin-resistant Staphylococcus aureus bacterial strain (MRSA), and one of the tested formulations shows a biological activity comparable to that of the free chlorin. In order to rationalize the physicochemical parameters of the carriers that control the biological activity, the new liposome formulations have been characterized by measuring (a) the zeta potential, (b) their capability of chlorin entrapping efficiency, i.e. entrapment efficacy, (c) the effect of storage on chlorin entrapment and (d) the localization of chlorin in the bilayer. The correlation of the physicochemical and biological features of formulations has allowed us to rationalize, to some extent, some of the parameters that may control the interactions with the biological environment.

Concentration as the switch for chiral recognition in biomembrane models.

Chiral recognition was observed in a biomembrane model. Micellar aggregates formed by enantiopure N-alkyl-N,N-dimethyl-N-(1-phenyl)ethylammonium bromide were in fact able to convert the racemic mixture of bilirubin-IXalpha into an enantiomerically enriched mixture. The stereochemical preference and the extent of enantiomeric enrichment depend on the length of the hydrophobic portion of the surfactant and on the concentration conditions, and changes in the stereochemical bias are reversible.