Laura Ciani

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.

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.

Rational design of gold nanoparticles functionalized with carboranes for application in Boron Neutron Capture Therapy

In this paper we propose a bottom-up approach to obtain new boron carriers built with ortho-carborane functionalized gold nanoparticles (GNPs) for applications in Boron Neutron Capture Therapy. The interaction between carboranes and the gold surface was assured by one or two SH-groups directly linked to the boron atoms of the B10C2 cage. This allowed obtaining stable, nontoxic systems, though optimal biological performance was hampered by low solubility in aqueous media. To improve cell uptake, the hydrophilic character of carborane functionalized GNPs was enhanced by further coverage with an appropriately tailored diblock copolymer (PEO-b-PCL). This polymer also contained pendant carboranes to provide anchoring to the pre-functionalized GNPs. In vitro tests, carried out on osteosarcoma cells, showed that the final vectors possessed excellent biocompatibility joint to the capacity of concentrating boron atoms in the target, which is encouraging evidenced to pursue applications in vivo.

Advances in Lipid-Based Platforms for RNAi Therapeutics

Sequence-specific gene silencing, known as RNA interference (RNAi), is a natural process that can be exploited for knocking-down specific genes involved in the insurgence/development of pathological processes. In 2001 the discovery that small interfering RNA (siRNA) can induce gene silencing without immunoresponse turned RNAi into a promising technique for the control of post-transcriptional gene expression. Nowadays, the major challenge remains infusion in vivo. Therefore, vehicles providing protection and selective transport are to be developed for efficient systemic delivery. The most used vectors are lipid-based, offering a wide range of biocompatible formulations. Here their application in molecular medicine is discussed, especially with regard to recent clinical trials where conventional therapies have failed. The role played by extended physicochemical characterization for the success of RNAi therapeutics is also evidenced.

Carbonic anhydrase inhibitors: Design of spin-labeled sulfonamides incorporating TEMPO moieties as probes for cytosolic or transmembrane isozymes

A series of spin-labeled sulfonamides incorporating TEMPO moieties were synthesized by a procedure involving the formation of a thiourea functionality between the benzenesulfonamide and free radical fragment of the molecules. The new compounds were tested as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and showed efficient inhibition of the physiologically relevant isozymes hCA II and hCA IX (hCA IX being predominantly found in tumors) and moderate to weak inhibitory activity against hCA I. Some derivatives were also selective for inhibiting the tumor-associated isoform over the cytosolic one CA II, and presented significant changes in their ESR signals when complexed to the enzyme active site, being interesting candidates for the investigation of hypoxic tumors overexpressing CA IX by ESR techniques, as well as for imaging/treatment purposes.

DNA induced dimerization of a sulfhydryl surfactant in transfection agents studied by ESR spectroscopy

Synthetic vectors for gene delivery offer a wide variety of functional derivatization, which can be exploited to increase targeting and transfection efficacy. In this field, redox-sensitive agents based on the thiol/disulfide (-SH/-SS-) equilibrium are a class of promising transfectants. Here the thiol group content in lipoplexes formed by a triazine-based sulfhydryl surfactant (SH14) and a plasmid (pGEFP-N1) was probed by Electron Spin Resonance (ESR) of appropriately tailored nitroxides. By modelling the time decay of ESR intensity, details on the process of lipoplex formation were obtained. It was found that the concentration of available -SH groups depended on the contact time between SH14 and DNA, suggesting that lipoplex formation entailed disulfide bridge formation among SH14 molecules. This finding represents the first experimental evidence that the -SH/-SS- equilibrium plays a role in lipoplex formation when DNA is complexed by sulfhydryl-based transfecting agents, which may have profound influence on their mechanism of action.

Dissecting the Inhibition Mechanism of Cytosolic versus Transmembrane Carbonic Anhydrases by ESR

Spin-labeled sulfonamides incorporating TEMPO moieties showed efficient activity as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and, in particular, of the physiologically relevant isoenzymes hCA II, hCA IX, and hCA XIV. Here we report a detailed analysis of this class of inhibitors by means of ESR and X-ray crystallography, in comparison with inhibition tests against all mammalian CA isoforms, CA I-XIV. Local dynamics and structure were manifested in the ESR signal through modulation of internal magnetic anisotropies. Analysis and fitting of the ESR spectra of several spin-labeled sulfonamides with isoforms CA II (cytosolic), CA IX (catalytic domain and full length transmembrane, tumor-associated isoform) and CA XIV (transmembrane isozyme) provided information about polarity and dynamics of specific microenvironments sensed by the nitroxyl group within the active site cavity of these isozymes. The comparison of ESR and crystallographic data of hCA II complexed with one of these inhibitors constitutes a useful tool for the understanding of molecular hindrance and ordering within the enzyme active site, and provides theoretical bases to use these inhibitors for imaging purposes of hypoxic tumors overexpressing the transmembrane isozyme CA IX. Combining the sulfonamide zinc-binding group with the TEMPO moiety thus allowed to dissect the selective inhibition mechanism of different cytosolic and transmembrane carbonic anhydrases.