Stefano Leporatti

Cytocompatibility and Uptake of Halloysite Clay Nanotubes

Halloysite is aluminosilicate clay with hollow tubular structure of 50 nm external diameter and 15 nm diameter lumen. Halloysite biocompatibility study is important for its potential applications in polymer composites, bone implants, controlled drug delivery, and for protective coating (e.g., anticorrosion or antimolding). Halloysite nanotubes were added to different cell cultures for toxicity tests. Its fluorescence functionalization by aminopropyltriethosilane (APTES) and with fluorescently labeled polyelectrolyte layers allowed following halloysite uptake by the cells with confocal laser scanning microscopy (CLSM). Quantitative Trypan blue and MTT measurements performed with two neoplastic cell lines model systems as a function of the nanotubes concentration and incubation time indicate that halloysite exhibits a high level of biocompatibility and very low cytotoxicity, rendering it a good candidate for household materials and medicine. A combination of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning force microscopy (SFM) imaging techniques have been employed to elucidate the structure of halloysite nanotubes.

Polyelectrolyte multilayer capsule permeability control

Abstract The permeability properties of hollow polyelectrolyte multilayer capsules for different substances were investigated as a function of pH and salt concentration. Capsules were prepared by layer-by-layer (LBL) adsorption of oppositely charged polyelectrolytes onto the surface of melamine formaldehyde and CdCO3 particles followed by core removal. It was shown that the capsules are closed at a pH value of 8 and higher, but at a pH lower than 6 the macromolecules permeate into the capsule interior. For low molecular weight molecules capsules, templated on CdCO3 cores were found to be less permeable than MF-derived capsules. The open and closed states of the capsule wall are reversible. It provides thus an opportunity to encapsulate different materials into polyelectrolyte capsules.

Drug-loaded polyelectrolyte microcapsules for sustained targeting of cancer cells ☆

In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drugs bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by-layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drugs exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings.

Biological cells as templates for hollow microcapsules.

Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Halloysite clay nanotubes for resveratrol delivery to cancer cells.

Halloysite is natural aluminosilicate clay with hollow tubular structure which allows loading with low soluble drugs using their saturated solutions in organic solvents. Resveratrol, a polyphenol known for having antioxidant and antineoplastic properties, is loaded inside these clay nanotubes lumens. Release time of 48 h is demonstrated. Spectroscopic and ζ-potential measurements are used to study the drug loading/release and for monitoring the nanotube layer-by-layer (LbL) coating with polyelectrolytes for further release control. Resveratrol-loaded clay nanotubes are added to breast cell cultures for toxicity tests. Halloysite functionalization with LbL polyelectrolyte multilayers remarkably decrease nanotube self-toxicity. MTT measurements performed with a neoplastic cell lines model system (MCF-7) as function of the resveratrol-loaded nanotubes concentration and incubation time indicate that drug-loaded halloysite strongly increase of cytotoxicity leading to cell apoptosis.

Biological applications of LbL multilayer capsules: From drug delivery to sensing

Polyelectrolyte multilayer (PEM) capsules engineered with active elements for targeting, labeling, sensing and delivery hold great promise for the controlled delivery of drugs and the development of new sensing platforms. PEM capsules composed of biodegradable polyelectrolytes are fabricated for intracellular delivery of encapsulated cargo (for example peptides, enzymes, DNA, and drugs) through gradual biodegradation of the shell components. PEM capsules with shells responsive to environmental or physical stimuli are exploited to control drug release. In the presence of appropriate triggers (e.g., pH variation or light irradiation) the pores of the multilayer shell are unlocked, leading to the controlled release of encapsulated cargos. By loading sensing elements in the capsules interior, PEM capsules sensitive to biological analytes, such as ions and metabolites, are assembled and used to detect analyte concentration changes in the surrounding environment. This Review aims to evaluate the current state of PEM capsules for drug delivery and sensing applications.

Polyelectrolyte multilayer capsules templated on biological cells: core oxidation influences layer chemistry.

Abstract Polyelectrolyte multilayer capsules have been fabricated in aqueous media by step-wise assembling of sodium polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH) on human red blood cells and melamine formaldehyde resin particles as templates, followed by removal of these templates by two different procedures. The melamine formaldehyde core can be dissolved in pH 1.1. The removal of the cell cores was achieved by an oxidation with sodium hypochlorite solution (pH 12). The effect of these treatments on the chemical composition of the capsules and of a randomly formed polyelectrolyte complex was studied. The melamin resin templated capsules have basically the same composition as the untreated complex revealing no chemical changes induced by the acid. But about 20% of the total hollow capsules mass can be attributed to a rest of melamin resin. The treatment with NaOCl solution changed the chemical composition of the capsules drastically. The amino groups of polyallylamine were oxidized to nitriles, nitroso-, nitro-, azo- and carbonyl groups. Positive charges disappeared. Covalent bonds were formed which crosslink the polymer chains. Simultaneously, the amount of PSS is strongly reduced to 10% of the original value. The stability of the capsules can be understood as a result of crosslinking and hydrophobic interactions. The cell templated capsules are monodisperse, being replicas in size and shape of the template but their chemical composition is different compared with the initial polyelectrolyte multilayer film. The altered chemical properties are responsible for new physical and mechanical properties such as higher elasticity, high chemical stability, as well as selective adsorption and permeation for charged ions.

Lapatinib/Paclitaxel polyelectrolyte nanocapsules for overcoming multidrug resistance in ovarian cancer.

The sonication-assisted layer-by-layer (SLBL) technology was developed to combine necessary factors for an efficient drug-delivery system: (i) control of nanocolloid size within 100 - 300 nm, (ii) high drug content (70% wt), (iii) shell biocompatibility and biodegradability, (iv) sustained controlled release, and (v) multidrug-loaded system. Stable nanocolloids of Paclitaxel (PTX) and lapatinib were prepared by the SLBL method. In a multidrug-resistant (MDR) ovarian cancer cell line, OVCAR-3, lapatinib/PTX nanocolloids mediated an enhanced cell growth inhibition in comparison with the PTX-only treatment. A series of in vitro cell assays were used to test the efficacy of these formulations. The small size and functional versatility of these nanoparticles, combined with their ability to incorporate various drugs, indicates that lapatinib/PTX nanocolloids may have in vivo therapeutic applications.

Novel polyelectrolyte multilayer micro- and nanocapsules as magnetic carriers.

Polyelectrolyte multilayer (PEM) capsules are introduced as versatile magnetic carrier systems. Superparamagnetic magnetite is mounted to the multilayer shell itself or is a component of the capsule interior. The PEM is formed at different (decomposable) colloidal templates, e.g. melamine formaldehyde resin, glutaraldehyde fixed red blood cells, emulsion oil droplets. The results are illustrated by transmission electron microscopy and confocal laser scanning microscopy.

Resveratrol downregulates Akt/GSK and ERK signalling pathways in OVCAR-3 ovarian cancer cells

Phytochemicals constitute a heterogeneous group of substances with an evident role in human health. Their properties on cancer initiation, promotion and progression are well documented. Particular attention is now devoted to better understand the molecular basis of their anticancer action. In the present work, we studied the effect of resveratrol on the ovarian cancer cell line OVCAR-3 by a proteomic approach. Our findings demonstrate that resveratrol down-regulates the protein cyclin D1 and, in a concentration dependent manner, the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). The dephosphorylation of these kinases could be responsible for the decreased cyclin D1 levels observed after treatment. We also showed that resveratrol reduces phosphorylation levels of the extracellular signal-regulated kinase (ERK) 1/2. Chemical inhibitors of phosphatidylinositol 3-kinase (PI3K) and ERK both increased the in vitro therapeutic efficacy of resveratrol. Moreover, resveratrol had an inhibitory effect on the AKT phosphorylation in cultured cells derived from the ascites of ovarian cancer patients and in a panel of human cancer cell lines. Thus, resveratrol shows antitumor activity in human ovarian cancer cell lines targeting signalling pathway involved in cell proliferation and drug-resistance.