Macroscopic Biaxial Order in Multilayer Films of Bent-Core Liquid Crystals Deposited by Combined Langmuir-Blodgett/Langmuir-Schaefer Technique.

Bent-core liquid crystals, a class of mesogenic compounds with non-linear molecular structures, are well known for their unconventional mesophases, characterized by complex molecular (and supramolecular) ordering and often featuring biaxial and polar properties. In the nematic phase, their unique behavior is manifested in the formation of nano-sized biaxial clusters of layered molecules (cybotactic groups). While this prompted their consideration in the quest for nematic biaxiality, experimental evidence indicates that the cybotactic order is only short-ranged and that the nematic phase is macroscopically uniaxial. By combining atomic force microscopy, neutron reflectivity and wide-angle grazing-incidence X-ray scattering, here, we demonstrate that multilayer films of a bent-core nematic, deposited on silicon by a combined Langmuir-Blodgett and Langmuir-Schaefer approach, exhibit macroscopic in-plane ordering, with the long molecular axis tilted with respect to the sample surface and the short molecular axis (i.e., the apex bisector) aligned along the film compression direction. We thus propose the use of Langmuir films as an effective way to study and control the complex anchoring properties of bent-core liquid crystals.

The influence of mannose-based esters on the mesophase behaviour of lyotropic liquid crystalline nanosystems as drug delivery vectors.

Lyotropic Liquid Crystalline (LLC) nanoparticles represent an emerging class of smart, biocompatible, and biodegradable systems for the delivery of drugs. Among these, structures with complex 3D architectures such as cubosomes are of particular interest. These are non- lamellar assemblies having hydrophobic and hydrophilic portions able to carry drugs of different nature. They can further be modulated including suitable additives to control the release of the active payload, and to promote an active targeting. Starting from monoolein (GMO) cubic phase, different concentrations of mannose-based esters were added, and the eventual structural modifications were monitored to ascertain the effects of the presence of glycolipids. Moreover, the structural properties of these nanosystems loaded with Dexamethasone (DEX), a very well-known anti-inflammatory steroid, were also studied. Experiments were carried out by synchrotron Small Angle X-ray Scattering (SAXS), Raman Microspectroscopy (RMS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) measurements. The drug delivery potential (i.e. entrapment efficiency and release properties) of the obtained nanoparticles was evaluated. Finally, in vitro cytocompatibility and anti-inflammatory activity studies of the prepared formulations were carried out. Inclusion of mannose-based surfactants up to 10 mol% influenced the structural parameters of Im3m cubic phase and swollen cubic phases were obtained with the different glycolipids with lattice parameters significantly higher than GMO. A complete cytocompatibility and an increased DEX activity were observed, thus suggesting the possibility to use GMO/glycolipids nanoparticles to formulate innovative drug delivery systems.

Characterisation of Bee Pollen from the Marche Region (Italy) According to the Botanical and Geographical Origin with Analysis of Antioxidant Activity and Colour, Using a Chemometric Approach.

Attempts have often been made to isolate and characterise monofloral pollens to correlate nutritional with botanical properties. Nevertheless, pollen harvested in a particular area that can have a high biodiversity could have healthier properties. In addition, the analysis of the pollen's botanical composition can be important for characterising the typical flora of a specific geographical area. On this basis, various pollens collected in different locations of the Marche region (Italy) and in different harvesting periods were analyzed for botanical composition and antioxidant (total phenolic content, ABTS, DPPH and ORAC tests), granulometry and colour (CIE L*a*b*) properties to evaluate the biodiversity of pollen sources within a particular geographical area and to correlate this to the nutraceutical characteristics. Antioxidant activity results showed values generally higher than those of monofloral pollens harvested in the same areas but manually separated according to colour, shape and size. This suggests that even the floral species present in low percentages may have an influence on the nutraceutical properties of these products. The multivariate statistical elaboration of the obtained results permitted the separation of samples containing a prevalent botanical species and the grouping of all the samples into separate clusters corresponding to different areas of Marche.

Nanoscale Structure of Langmuir-Blodgett Film of Bent-Core Molecules.

Bent-core mesogens (BCMs) are a class of thermotropic liquid crystals featuring several unconventional properties. However, the interpretation and technological exploitation of their unique behavior have been hampered by the difficulty of controlling their anchoring at surfaces. To tackle this issue, we report the nanoscale structural characterization of BCM films prepared using the Langmuir-Blodgett technique. Even though BCMs are quite different from typical amphiphilic molecules, we demonstrate that stable molecular films form over water, which can then be transferred onto silicon substrates. The combination of Brewster angle microscopy, atomic force microscopy, and X-ray reflectivity measurements shows that the molecules, once transferred onto a solid substrate, form a bilayer structure with a bottom layer of flat molecules and an upper layer of upright molecules. These results suggest that Langmuir-Blodgett films of BCMs can provide a useful means to control the alignment of this class of liquid crystals.

A vibrational in vitro approach to evaluate the potential of monoolein nanoparticles as isofuranodiene carrier in MDA-MB 231 breast cancer cell line: New insights from Infrared and Raman microspectroscopies.

Isofuranodiene (IFD) is a sesquiterpene occurring in several plant species, which proved to have multiple anticancer activities. IFD has a lipophilic nature and, hence, a very low water solubility and a poor bioavailability; moreover, it is not stable, undergoing the "Cope rearrangement" to the less active curzerene. The use of appropriate delivery systems can thus be considered as a valid tool to enhance IFD bioavailability, solubility, stability and at the same time also to improve its intracellular uptake and pharmacological activity. Within this frame, monoolein (GMO) nanoparticles loaded with IFD were prepared and their enhanced anticancer activity, compared to pristine IFD, was assessed. In this study, for the first time, an in vitro Fourier Transform Infrared and Raman Microspectroscopy approaches were exploited to evaluate the effects of IFD, alone and loaded in GMO nanoparticles, on MDA-MB 231 breast cancer cell line. The anti-cancer effects of IFD were evidenced by both the spectroscopic techniques and discriminated from the GMO-induced changes in the culture environment; moreover, a synergistic effect of IFD and GMO administration can be envisaged by the experimental results.

Cubic and Hexagonal Mesophases for Protein Encapsulation: Structural Effects of Insulin Confinement.

Monoolein-based cubic and hexagonal mesophases were investigated as matrices for insulin loading, at low pH, as a function of temperature and in the presence of increasing amounts of oleic acid, as a structural stabilizer for the hexagonal phase. Synchrotron small angle X-ray diffraction, rheological measurements, and attenuated total reflection-Fourier transform infrared spectroscopy were used to study the effects of insulin loading on the lipid mesophases and of the effect of protein confinement in the 2D- and 3D-lipid matrix water channels on its stability and unfolding behavior. We found that insulin encapsulation has only little effects both on the mesophase structures and on the viscoelastic properties of lipid systems, whereas protein confinement affects the response of the secondary structure of insulin to thermal changes in a different manner according to the specific mesophase: in the cubic structure, the unfolding toward an unordered structure is favored, while the prevalence of parallel ß-sheets, and nuclei for fibril formation, is observed in hexagonal structures.

Antioxidant Activity Level, Bioactive Compounds, Colour and Spectroscopic Analysis (UV-Vis and FT-IR) of Flavoured Drinks Made with Wine and Sour Cherries (Prunuscerasus Var. austera).

In recent years, the increase in consumer interest towards simpler and authentic lifestyles has led to an explosive growth in the production and business of typical agri-food products and, among these, of wines and its derived beverages. With the aim of promoting a typical Italian beverage, the so-called "Vino di visciole" or "Visner", listed in the national table of traditional agri-food products, the antioxidant and colour properties of fifteen samples from different provinces of the Marche region and obtained with different recipes were analysed. The "in vitro" total antioxidant activity (TAA) determined using ABTS assays, total phenolic content (TPC), total flavonoid content (TFC), total anthocyanins content (TAC), and colour (Somers assay) were measured. In addition, a spectroscopic FT-IR and UV-Vis analysis was carried out to analyse samples with multivariate techniques. The results showed that the production area, the recipe, and the type of cherries used to make the alcoholic beverage do not influence the antioxidant properties and the phytochemical contents of the samples. The multivariate treatment of the spectroscopic features (mainly UV-Vis) rather allowed the differentiation of samples with high antioxidant activity using easy and low-cost instrumental techniques that require little time and can be employed in routine analysis.

Synchrotron Characterization of Hexagonal and Cubic Lipidic Phases Loaded with Azolate/Phosphane Gold(I) Compounds: A New Approach to the Uploading of Gold(I)-Based Drugs.

Gold(I) phosphane compounds have recently attracted a renewed interest as potential new protagonists in cancer therapy. A class of phosphane gold(I) complexes containing azolate ligands has been successfully tested against several cancer cell lines and, in particular, against basal-like breast (BLB) cancer, a form characterized by strongly severe diagnosis and short life lapse after classic chemotherapy. Even though the anticancer activity of gold(I) phosphane compounds is thoroughly ascertained, no study has been devoted to the possibility of their delivery in nanovectors. Herein, nonlamellar lyotropic liquid crystalline lipid nanosystems, a promising class of smart materials, have been used to encapsulate gold(I) azolate/phosphane complexes. In particular, ((triphenylphosphine)-gold(I)-(4,5-dichloroimidazolyl-1H-1yl)) (C-I) and ((triphenylphosphine)-gold(I)-(4,5-dicyanoimidazolyl-1H-1yl)) (C-II) have been encapsulated in three different lipid matrices: monoolein (GMO), phytantriol (PHYT) and dioleoyl-phosphatidylethanolamine (DOPE). An integrated experimental approach involving X-ray diffraction and UV resonant Raman (UVRR) spectroscopy, based on synchrotron light and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, has been employed to establish the effects of drug encapsulation on the structure and phase behavior of the host mesophases. The results indicate that gold(I) complexes C-I and C-II are successfully encapsulated in the three lipid matrices as evidenced by the drug-induced phase transitions or by the changes in the mesophase lattice parameters observed in X-ray diffraction experiments and by the spectral changes occurring in UV resonant Raman spectra upon loading the lipid matrices with C-I and C-II.

Nanostructure of Unconventional Liquid Crystals Investigated by Synchrotron Radiation.

The macroscopic properties of novel liquid crystal (LC) systems-LCs with unconventional molecular structure as well as conventional LCs in unconventional geometries-directly descend from their mesoscopic structural organization. While X-ray diffraction (XRD) is an obvious choice to investigate their nanoscale structure, conventional diffractometry is often hampered by experimental difficulties: the low scattering power and short-range positional order of the materials, resulting in weak and diffuse diffraction features; the need to perform measurements in challenging conditions, e.g., under magnetic and/or electric fields, on thin films, or at high temperatures; and the necessity to probe micron-sized volumes to tell the local structural properties from their macroscopic average. Synchrotron XRD allows these problems to be circumvented thanks to the superior diffraction capabilities (brilliance, q-range, energy and space resolution) and advanced sample environment available at synchrotron beamlines. Here, we highlight the potentiality of synchrotron XRD in the field of LCs by reviewing a selection of experiments on three unconventional LC systems: the potentially biaxial and polar nematic phase of bent-core mesogens; the very high-temperature nematic phase of all-aromatic LCs; and polymer-dispersed liquid crystals. In all these cases, synchrotron XRD unveils subtle nanostructural features that are reflected into macroscopic properties of great interest from both fundamental and technological points of view.

Nanostructured liquid crystalline particles as delivery vectors for isofuranodiene: Characterization and in-vitro anticancer activity.

Isofuranodiene is an oxygenated sesquiterpene containing a furan ring isolated from the essential oil of Smyrnium olusatrum L. (Apiaceae) owning notable anticancer activity. Despite its biological potential, the high lipophilicity along with a relatively low stability due to Cope rearrangement giving rise to a less active compound, make the perspective of its therapeutical use unlikely. On this basis, in the present work we evaluated bulk and dispersed non lamellar liquid crystalline phases as effective delivery vectors for isofuranodiene, and capable of preserving its structure and enhancing the biological activity. Small-angle X-ray scattering, dynamic light scattering, and UV resonance Raman spectroscopy were used to characterize the nanosystems in an integrated experimental approach. Encapsulation of isofuranodiene in the lipid matrix resulted in a transition from a cubic Im3m to a reversed hexagonal phase because of the highly lipophilic character of the drug, as obtained in SAXS measurements, and in significant shifts in the components of the Raman spectrum of isofuranodiene. The anticancer activity of isofuranodiene-loaded lipidic nanoparticles was assessed on MDA-MB 231 cell line by MTT assay and was found to be higher than that of pristine isofuranodiene.

Investigation of human pancreatic cancer tissues by Fourier Transform Infrared Hyperspectral Imaging.

Fourier-transform infrared hyperspectral imaging (FTIR-HSI) provides hyperspectral images containing both morphological and chemical information. It is widely applied in the biomedical field to detect tumor lesions, even at the early stage, by identifying specific spectral biomarkers. Pancreatic neoplasms present different prognoses and are not always easily classified by conventional analyses. In this study, tissue samples with diagnosis of pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumor were analyzed by FTIR-HSI and the spectral data compared with those from healthy and dysplastic samples. Multivariate/univariate approaches were complemented to hyperspectral images, and definite spectral markers of the different lesions identified. The malignant lesions were recognizable both from healthy/dysplastic pancreatic tissues (high values of phospholipids and triglycerides with shorter, more branched and less unsaturated alkyl chains) and between each other (different amounts of total lipids, phosphates and carbohydrates). These findings highlight different metabolic pathways characterizing the different samples, well detectable by FTIR-HSI.

Encapsulation of vitamin B12 into nanoengineered capsules and soft matter nanosystems for targeted delivery.

Targeted delivery of vitamins to a desirable area is an active branch in a modern pharmacology. The most important and difficult delivery of vitamin B12 is that to bone marrow and nerve cells. Herein we present a first step towards the development of two types of smart carriers, polymer capsules and lyotropic liquid-crystalline nanosystems, for vitamin B12 targeted delivery and induced release. A vitamin B12 encapsulation technique into nanoengineered polymeric capsules produced by layer-by-layer assembling of polymeric shells on CaCO3 templates has been developed. The effectiveness of the process was demonstrated by optical absorption spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM) and small-angle X-ray diffraction. TEM and AFM analyses performed on capsules after their drying, confirmed the presence of the vitamin B12 inside the capsules in the form of crystalline nanoaggregates, 50-300 nm in diameter. Soft lipid nanovectors consisting of amphiphilic phytantriol molecules, which in water excess spontaneously self-assembly in 3D well-ordered inverse bicontinuous cubic bulk phase, were used as alternative carriers for vitamin B12. It was shown that about 30% of the vitamin added in the preparation of the soft lipid system was actually encapsulated in cubosomes and that no structural changes occurred upon loading. The Vitamin stabilizes the lipid system playing the role of its structure-forming element. The biocompatible nature, the stability and the feasibility of these systems make them good candidates as carriers for hydrophilic vitamins.

Selective induction of apoptosis in MCF7 cancer-cell by targeted liposomes functionalised with mannose-6-phosphate.

Liposomes are versatile platforms to carry anticancer drugs in targeted drug delivery; they can be surface modified by different strategies and, when coupled with targeting ligands, are able to increase cellular internalisation and organelle-specific drug delivery. An interesting strategy of antitumoral therapy could involve the use of lysosomotropic ligand-targeted liposomes loaded with molecules, which can induce lysosomal membrane permeabilization (LMP), leakage of cathepsins into the cytoplasm and subsequent apoptosis. We have previously demonstrated the ability of liposomes functionalised with a mannose-6-phosphate to reach lysosomes; in this research we compare the behaviour of M6P-modified and non-functionalised liposomes in MCF7 tumour cell and in HDF normal cells. With this aim, we first demonstrated by Western blotting the overexpression of mannose-6-phosphate/insulin-like growth factor (M6P/IGF-II) receptor in MCF7. Then, we prepared calcein-loaded liposomes and we revealed the increased uptake of M6P-functionalised liposomes in MCF7 cells respect to HDF cells by flow cytometry analysis. Finally, we loaded functionalised and not functionalised liposomes with N-hexanoyl-d-erythro-sphingosine (C6Cer), able to initiate LMP-induced apoptosis; after having studied the stability of both vesicles in the presence of serum by Dynamic Light Scattering and Spectrophotometric turbidity measurements, we showed that ceramide-loaded M6P-liposomes significantly increased apoptosis in MCF7 with respect to HDF cells.

Lyotropic Liquid-Crystalline Nanosystems as Drug Delivery Agents for 5-Fluorouracil: Structure and Cytotoxicity.

Lyotropic cubic liquid-crystalline systems have received increasing attention due to their unique microstructural and physicochemical properties as efficient nanocarriers for drug delivery. We report the preparation and characterization of bulk phases and cubosome dispersions of phytantriol loaded with the anticancer drug 5-fluorouracil, in neutral and anionic forms. In both cases, a Pn3m cubic phase was observed. The phytantriol phase behavior can be influenced by the addition of ionic agents, and, to this purpose, a positively charged lipid, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride salt (DOTAP), was included in the studied formulations. It was found to induce a variation of the spontaneous membrane curvature of the phytantriol lipid bilayer, generating a transition from the Pn3m to the Im3m cubic phase. When 5-fluorouracil, in its anionic form (5-FUs), was encapsulated in these latter systems, a further transition to the HII hexagonal phase was observed as a consequence of the formation of a complex phytantriol/DOTAP/5-FUs. The physicochemical characterization was performed with various complementary techniques including synchrotron small-angle X-ray scattering, dynamic light scattering, and attenuated total reflection Fourier transform infrared and UV resonance Raman spectroscopies. Encapsulation of 5-fluorouracil in the corresponding nanodispersions was evaluated, and their in vitro cytotoxicity was assessed in MDA-MB-231 cell line. Phytantriol cubosomes containing 5-fluorouracil showed a higher toxicity compared with the bare drug solution, and hence they represent potential nanocarriers in the delivery of 5-fluorouracil for cancer therapy.

Retroperitoneal liposarcomas: a representative literature review occasioned by a rare case of laterelapse abdominal liposarcoma.

Soft tissue sarcomas (STS) are rare and heterogeneous tumours representing approximately 0.7%-1% of all adult tumours. In the adults and among the retroperitoneal sarcomas (RPS), Liposarcoma (LS) is the most common variant accounting for 12% -20% of all sarcomas and up to 45% of sarcomas at retroperitoneal localization. A rare case of LS relapsed after 15 years is giving the occasion to review the published literature and emphasise the followings concepts: 1) Despite extensive surgery remains the mainstay of treatment for localized STS at present, anatomical complexity and occult localization result in local recurrence in the majority of patients; 2) The role of imaging and tumour markers is still limited; 3) Indefinite prolonged surveillance is a key point of treatment; 4) Referral to tertiary centres with dedicated Retroperitonal Surgeons and Oncology expertise is mandatory.

Neutral liposomes containing crown ether-lipids as potential DNA vectors.

Three crown ether derivatives, 1,2-O-dioleoyl-3-O-{2-[(12-crown-4)methoxy]ethyl}-sn-glycerol (12C4L), 1,2-O-dioleoyl-3-O-{2-[(15-crown-5)methoxy]ethyl}-sn-glycerol (15C5L) and 2,3-naphtho-15-crown-5 (NAP5), have been incorporated into 1-palmitoyl-2-oleoyl-phosphatydilcholine (POPC) liposomes. The size of the crown ether and the lipophilic moiety of 12C4L, 15C5L and NAP5 influence the stability and the properties of the extruded POPC liposomes determined at 25°C in buffered aqueous solution at pH7.4. The investigated liposomes are zwitterionic for POPC headgroups but can be turned into cationic aggregates in the presence of divalent cations. The capability of these systems to complex DNA has been demonstrated by SAXS experiments.

Biophysical characterization of complexes of DNA with mixtures of the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-hexanoylamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoylamine and 1,2-dioleoyl-sn-glycero-3-phosphocholine in the presence of bivalent metal cations for DNA transfection.

Neutral lipids have received up to now a little attention as genetic material carriers, despite some valuable features, such as the absence of toxicity and the high stability in serum of their complexes with DNA. We have prepared two quaternary complexes of DNA and mixtures of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-hexanoylamine (6PE) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoylamine (12PE) with DOPC in aqueous dispersions of bivalent metal cations (PE/DOPC-DNA-M(2+)). The aim was to evaluate the effect of the amide moiety on the transfection efficiency. These complexes form in a self-assembled manner, the DNA condensation being promoted by the metal cations. Synchrotron X-ray diffraction analysis was used to determine the structure of the complexes, which exhibit the lamellar symmetry of the L(α)(c) phase. The size and surface charge of the complexes have also been measured, and promising results of DNA transfections in vitro have been reported.

Metal cation induced cubic phase in poly(ethylene glycol)-functionalized dioleoylphosphatidylethanolamine aqueous dispersions.

Metal cations (Mn(2+) or Ca(2+)) in aqueous dispersions of mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-functionalized DOPE (DOPE-PEG(350)) induce, above a certain amount of the PEG lipid component, a phase transition from the inverted hexagonal phase H(II) to the bicontinuous inverted cubic phase Q(224) with space group Pn3m. The process is driven by the decrease of free elastic energy due to the Gaussian curvature of the cubic phase. The structural characterization of the phase behavior over the whole explored range of DOPE-PEG/DOPE weight ratio (3-25%) is reported, focusing on the role of the metal cation in the formation of the 3D cubic lattice. This result may represent a significant progress toward a design-based approach to drug delivery.

Nanoaggregates of copper porphyrazine in floating layers and Langmuir-Schaefer films.

Aggregation behavior of unsubstituted copper porphyrazine (CuPaz) on the water surface was studied by analysis of compression curves, Brewster angle microscopy (BAM), and optical spectroscopy. The structure and stability of the CuPaz aqua aggregates in the floating layers are determined by hydration degree that depends on initial surface concentration and surface pressure. Langmuir-Schaefer (LS) films of CuPaz were prepared by deposition of the variously structured floating layers and studied by X-ray scattering technique and optical spectroscopy. Stable and labile structures were detected and compared with the floating CuPaz aqua aggregates. Conditions of formation of the stable four-stacked nanoaggregates in LS films were determined. A model comprising both nucleation of CuPaz on the water surface and structural transformations in the solid films is proposed.

Pressure effects on lipidic direct phases: the dodecyl trimethyl ammonium chloride-water system.

The direct lyotropic polymorphism of dodecyltrimethylammonium chloride (DTAC) was investigated by synchrotron X-ray diffraction at different water concentrations under compression up to 2 kbar, i.e., in the pressure intermediate range where interesting biophysical transformations occur and the functional characteristics of cell membranes are altered. The results show that pressure induces the transition from the hexagonal phase to the micellar Pm3n cubic phase in hydrated samples (c between 0.5 and 0.6, c being the weight concentration of lipid in the mixture) and the transition from the bicontinuous Ia3d cubic phase to the hexagonal phase in drier samples (c = 0.8). By increasing the pressure on very dry samples, a lamellar L(alpha) phase was observed to form transitorily at the Ia3d cubic-hexagonal phase transition. Phase compressibility and then the lipid and water partial molecular compressibilities were derived as a function of pressure and concentration. As a result, we assessed the very low compressibility of the hydration water within the lipid phases, and we demonstrated that the compressibility of DTAC is very dependent on pressure. Moreover, the molecular parameters of DTAC calculated in the different phases during compression confirmed that pressure induces small but continuous conformational changes, definitely different from the large changes observed in lipid molecules forming type II structures.