Sandra Ristori

Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action

Background A promising strategy to create stimuli-responsive gene delivery systems is to exploit the redox gradient between the oxidizing extracellular milieu and the reducing cytoplasm in order to disassemble DNA/cationic lipid complexes (lipoplexes). On these premises, we previously described the synthesis of SS14 redox-sensitive gemini surfactant for gene delivery. Although others have attributed the beneficial effects of intracellular reducing environment to reduced glutathione (GSH), these observations cannot rule out the possible implication of the redox milieu in its whole on transfection efficiency of bioreducible transfectants leaving the determinants of DNA release largely undefined. Methodology/Principal Findings With the aim of addressing this issue, SS14 was here formulated into binary and ternary 100 nm-extruded liposomes and the effects of the helper lipid composition and of the SS14/helper lipids molar ratio on chemical-physical and structural parameters defining transfection effectiveness were investigated. Among all formulations tested, DOPC/DOPE/SS14 at 25∶50∶25 molar ratio was the most effective in transfection studies owing to the presence of dioleoyl chains and phosphatidylethanolamine head groups in co-lipids. The increase in SS14 content up to 50% along DOPC/DOPE/SS14 liposome series yielded enhanced transfection, up to 2.7-fold higher than that of the benchmark Lipofectamine 2000, without altering cytotoxicity of the corresponding lipoplexes at charge ratio 5. Secondly, we specifically investigated the redox-dependent mechanisms of gene delivery into cells through tailored protocols of transfection in GSH-depleted and repleted vs. increased oxidative stress conditions. Importantly, GSH specifically induced DNA release in batch and in vitro. Conclusions/Significance The presence of helper lipids carrying unsaturated dioleoyl chains and phosphatidylethanolamine head groups significantly improved transfection efficiencies of DOPC/DOPE/SS14 lipoplexes. Most importantly, this study shows that intracellular GSH levels linearly correlated with transfection efficiency while oxidative stress levels did not, highlighting for the first time the pivotal role of GSH rather than oxidative stress in its whole in transfection of bioreducible vectors.

Carborane derivatives loaded into liposomes as efficient delivery systems for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is an anticancer therapy based on the incorporation of (10)B in tumors, followed by neutron irradiation. Recently, the synthesis and delivery of new boronated compounds have been recognized as some of the main challenges in BNCT application. Here, we report on the use of liposomes as carriers for BNCT active compounds. Two carborane derivatives, i.e., o-closocarboranyl beta-lactoside (LCOB) and 1-methyl-o-closocarboranyl-2-hexylthioporphyrazine (H(2)PzCOB), were loaded into liposomes bearing different surface charges. The efficacy of these formulations was tested on model cell cultures, that is, DHD/K12/TRb rat colon carcinoma and B16-F10 murine melanoma. These induce liver and lung metastases, respectively, and are used to study the uptake of standard BNCT drugs, including borophenylalanine (BPA). Boron concentration in treated cells was measured by alpha spectrometry at the TRIGA mark II reactor (University of Pavia). Results showed high performance of the proposed formulations. In particular, the use of cationic liposomes increased the cellular concentration of (10)B by at least 30 times more than that achieved by BPA.

Using Liposomes as Carriers for Polyphenolic Compounds: The Case of Trans-Resveratrol

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol found in various plants, especially in the skin of red grapes. The effect of resveratrol on human health is the topic of numerous studies. In fact this molecule has shown anti-cancer, anti-inflammatory, blood-sugar-lowering ability and beneficial cardiovascular effects. However, for many polyphenol compounds of natural origin bioavailability is limited by low solubility in biological fluids, as well as by rapid metabolization in vivo. Therefore, appropriate carriers are required to obtain efficient therapeutics along with low administration doses. Liposomes are excellent candidates for drug delivery purposes, due to their biocompatibility, wide choice of physico-chemical properties and easy preparation. In this paper liposome formulations made by a saturated phosphatidyl-choline (DPPC) and cholesterol (or its positively charged derivative DC-CHOL) were chosen to optimize the loading of a rigid hydrophobic molecule such as resveratrol. Plain and resveratrol loaded liposomes were characterized for size, surface charge and structural details by complementary techniques, i.e. Dynamic Light Scattering (DLS), Zeta potential and Small Angle X-ray Scattering (SAXS). Nuclear and Electron Spin magnetic resonances (NMR and ESR, respectively) were also used to gain information at the molecular scale. The obtained results allowed to give an account of loaded liposomes in which resveratrol interacted with the bilayer, being more deeply inserted in cationic liposomes than in zwitterionic liposomes. Relevant properties such as the mean size and the presence of oligolamellar structures were influenced by the loading of RESV guest molecules. The toxicity of all these systems was tested on stabilized cell lines (mouse fibroblast NIH-3T3 and human astrocytes U373-MG), showing that cell viability was not affected by the administration of liposomial resveratrol.

Dynamics of pattern formation in biomimetic systems

This paper is an attempt to conceptualize pattern formation in self-organizing systems and, in particular, to understand how structures, oscillations or waves arise in a steady and homogenous environment, a phenomenon called symmetry breaking. The route followed to develop these ideas was to couple chemical oscillations produced by Belousov-Zhabotinsky reaction with confined reaction environments, the latter being an essential requirement for any process of Life. Special focus was placed on systems showing organic or lipidic compartments, which represent more reliable biomimetic matrices.

Complexing a small interfering RNA with divalent cationic surfactants

Small interfering RNAs (siRNAs) are double strand RNA fragments of short sequence (∼20 bp). RNA interference came into focus only 13 years ago as a major biological breakthrough and, since then, many studies have described the involvement of siRNA in gene silencing. Application to gene therapy is extremely promising, provided that appropriate vectors are used. Optimising transfection efficacy strongly relies on the knowledge and tuning of physicochemical properties of transfection complexes, such as size, surface charge and internal interactions, which govern in vitro and in vivo stability. Here we report a study on siRNA complexation with micelles of two types of divalent cationic surfactants, i.e. three Gemini bis(quaternary ammonium) bromide with variable spacer length (12-3-12, 12-6-12, 12-12-12) and one weak electrolyte surfactant with a triazine polar head. The process of complex formation was followed by SANS, DLS and zeta potential. Charge density on micelles and counterion exchange were key factors in determining the extent of complexation, as it happens to polymer electrolytes interacting with micelles. A description of complex formation was given in terms of liquid–liquid micro-phase separation, due to internally structured coacervates progressively nucleating from the micelle solution upon siRNA addition. An affinity order between surfactants and siRNA could be established on the basis of the obtained results and their comparison.

Boron as a platform for new drug design

Introduction: Boron lies on the borderline between metals and non-metals in the periodic table. As such, it possesses peculiarities which render it suitable for a variety of applications in chemistry, technology and medicine. However, borons peculiarities have been exploited only partially so far. Areas covered: In this review, the authors highlight selected areas of research which have witnessed new uses of boron compounds in recent times. The examples reported illustrate how difficulties in the synthesis and physicochemical characterization of boronated molecules, encountered in past years, can be overcome with positive effects in different fields. Expert opinion: Many potentialities of boron-based systems reside in the peculiar properties of both boron atoms (the ability to replace carbon atoms, electron deficiency) and of boronated compounds (hydrophobicity, lipophilicity, versatile stereochemistry). Taken in conjunction, these properties can provide innovative drugs. The authors highlight the need to further investigate the assembly of boronated compounds, in terms of drug design, since the mechanisms required to obtain supramolecular structures may be unconventional compared with the more standard molecules used. Furthermore, the authors propose that computational methods are a valuable tool for assessing the role of multicenter, quasi-aromatic bonds and its peculiar geometries.

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin

A dimerizable cationic lipid with potential for gene delivery

Despite the use of currently optimized lipofection conditions, including transfection in serum‐depleted media, the efficiency of gene transfer is low and high transfection rates often induce cytotoxicity. A lipid formulation with transfection efficiency not inhibited by serum would provide an advance towards in vivo applications.

Interaction of the Belousov–Zhabotinsky Reaction with Phospholipid Engineered Membranes

Compartmentalized in liposome arrays, the Belousov-Zhabotinsky (BZ) oscillatory reaction might represent a good model for biochemical networks. In order to engineer such liposomes, we used small-angle X-ray scattering (SAXS) to study the effect of individual BZ reactant as well as of the entire BZ mixture on the structural properties of lipid layer(s) formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipids in aqueous media. These properties were compared with those of lipid layers doped with myristic acid (Myr-A), sodium tetradecyl sulfate (STS), and cholesterol (CHOL). In parallel, the effect on the BZ reaction exerted by doped DMPC liposomes was investigated by UV-vis spectroscopy, followed by image analysis of the recorded time series. SAXS experiments showed that chemical species involved in the BZ reaction bring small changes to the internal structure of DMPC bilayers. However, ferroin can reduce the liposome lamellarity by being adsorbed on the surface of lipid layers. Also, the presence of charged dopants such as STS and TA tends to reduce the lamellarity of liposomes, while CHOL brings marked changes in the BZ system due to chemical reaction with oxidant species. In particular, an increase of the oscillation frequency is observed when the BZ reaction is carried out in the presence of CHOL-DMPC liposomes. For this behavior, a possible explanation supported by numerical simulations is bromination of CHOL double bonds by BZ intermediates.

Synthesis, Spectroscopy and Electrochemistry of Lanthanide Bis-(ethylsulfanyl)tetraazaporphyrins

Reaction of the oespz2− ligand (oespz = 2,3,7,8,12,13,17,18-octakis (ethylsulfanyl)-5,10,15,20-porphyrazinato) with trivalent lanthanide ions, such as Nd3+, Eu3+, Dy3+, Yb3+ and Lu3+, leads to the direct synthesis of the corresponding sandwich complexes, in the radical, ligand-oxidized green form, whereas the reaction with La3+ affords the sandwich complex in the protonated, blue form. It is found that the ligand-oxidized complexes show rather weak electronic absorptions in the range 850–1150 nm and are, compared to lanthanide diporphyrins and diphthalocyanines, very stable both to oxidation and to reduction.