Halloysite Nanotubes (HNTs) are nanomaterials composed of double layered aluminosilicate minerals with a predominantly hollow tubular structure in submicron range. HNTs are characterized by a wide range of applications in anticancer therapy, sustained agent delivery, being particularly interesting because of their tunable release rates and fast adsorption rates. However systematic investigations of their acoustic properties are still poorly documented. This paper shows a quantitative assessment of the effectiveness of HNTs as scatterers at conventional ultrasonic frequencies (5.7 -7 MHz) in low range of concentrations (1.5-5 mg/mL). Different samples of HNT (diameter: 40-50 nm; length: 0.5 to 2 microns, empty lumen diameter: 15-20 nm) containing agarose gel were imaged through a commercially available echographic system and acquired data were processed through a dedicated prototypal platform in order to extract the average ultrasonic signal amplitude associated to the considered sample. Relationships have been established among backscatter, HNT concentration and the employed echographic frequency. Our results demonstrated that improvement in image backscatter could be achieved incrementing HNT concentration, determining a non-linear signal enhancement due to the fact that they are poly-disperse in length. On the other hand the effect of different echographic frequencies used was almost constant at all concentrations, specifically using higher values of echographic frequency allows yielding a signal enhanced of a factor 1.75±0.26. © 2013 IEEE.

This review reports on the combined use of the atomic force microscopy (AFM) and several type of optical/fluorescence/laser scanning microscopy for investigating cells. It is shown that the hybrid systems of AFM with optical-derived microscopies enable to study in detail cell surface properties (such as topography), their mechanical properties (e.g., Young's modulus) mechanotransduction phenomena and allow to gain insight into biological-related pathways and mechanisms in the complex nanoworld of cells. (C) 2016 Wiley Periodicals, Inc.

Aim of this work was to investigate the automatic echographic detection of an experimentaldrug delivery agent, halloysite clay nanotubes (HNTs), by employing an innovative methodbased on advanced spectral analysis of the corresponding "raw" radiofrequency backscattersignals. Different HNT concentrations in a low range (5.5-66  1010 part/mL, equivalent to0.25-3.00 mg/mL) were dispersed in custom-designed tissue-mimicking phantoms and imagedthrough a clinically-available echographic device at a conventional ultrasound diagnostic frequency(10 MHz). The most effective response (sensitivity = 60%, specificity = 95%), was found ata concentration of 33  1010 part/mL (1.5 mg/mL), representing a kind of best compromisebetween the need of enough particles to introduce detectable spectral modifications in thebackscattered signal and the necessity to avoid the losses of spectral peculiarity associated tohigher HNT concentrations. Based on theoretical considerations and quantitative comparisonswith literature-available results, this concentration could also represent an optimal concentrationlevel for the automatic echographic detection of different solid nanoparticles when employinga similar ultrasound frequency. Future dedicated studies will assess the actual clinical usefulness ofthe proposed approach and the potential of HNTs for effective theranostic applications.

Halloysite clay Nanotubes (HNTs) are nanomaterials composed of double layered aluminosilicate minerals with a hollow tubular structure in the submicron range. They are characterized by a wide range of applications in anticancer therapy as agent delivery. In this work we aim to investigate the automatic detection features of HNTs through advanced quantitative ultrasound imaging employing different concentrations (3-5 mg/mL) at clinical conventional frequency, i.e. 7 MHz. Different tissue mimicking samples of HNT containing agarose gel were imaged through a commercially available echographic system, that was opportunely combined with ultrasound signal analysis research platform for extracting the raw ultrasound radiofrequency (RF) signals. Acquired data were stored and analyzed by means of an in-house developed algorithm based on wavelet decomposition, in order to identify the specific spectrum contribution of the HNTs and generate corresponding image mapping. Sensitivity and specificity of the HNT detection were quantified. Average specificity (94.36%) was very high with reduced dependency on HNT concentration, while sensitivity showed a proportional increase with concentration with an average of 46.78%. However, automatic detection performances are currently under investigation for further improvement taking into account image enhancement and biocompatibility issues.

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.

The natural properties of chitosan (CHI), such as biocompatibility and biodegradability, have stimulated its use as drug delivery carrier in several applications, including layer-by-layer assembly and polymer self assembly. In this work we have aimed at producing chitosan microtubes by using CaCO3 rods doped with poly allylamine hydrochloride (PAH) as templates. The shape of the CaCO3 particles could be controlled upon addition of PAH during synthesis. A CHI-PgA complex was formed upon electrostatic binding of polygalacturonic acid (PgA) to CHI, to produce capsules for bromopyruvic acid delivery. Morphological investigations of the size and shape of CaCO3 rods were performed by means of scanning and transmission electron microscopy techniques. Infrared spectroscopy was used to monitor the characteristic bands in PAH, CHI, PgA and CaCO3. Cellular uptake and cytotoxicity were investigated. Control of CaCO3 growth during synthesis towards elongated shapes by using PAH was confirmed. These results envisage the use of chitosan-polygalacturonic acid micro/nanotubes as efficient drug delivery system for encapsulation of bromopyruvic acid as blocker for glycolytic enzymes.

Halloysite is aluminosilicate clay With hollow tubular structure of 50 lint external diameter and 15 nm diameter)lumen. Halloysite biocompatibility study is important for its potential applications in polymer composites, boric 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 it good candidate for household materials zinc] medicine. A combination of transmission electron microscopy (TEM), scanning electron microscopy (SI-E-M), and scanning force microscopy (SFM) imaging techniques have been employed to elucidate the Structure of halloysite nanotubes.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that par- ticipates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic forcemicroscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of -cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Halloysite Clay nanotubes (HNTs) are naturally occurring nanotubes composed of double layered aluminosilicate minerals with a hollow tubular structure in the nanometer range. In recent years, HNTs have attracted specific research attention as a possible new material for various biological applications, including drug and gene delivery vehicles, ultrasound contrast agents, cancer and stem cells isolation. Therefore, assessment of HNT biocompatibility has gained importance to demonstrate its suitability for clinical purposes. In this study, HNTs were densely coated with poly(ethylene glycol) (PEG) and MTT measurements were performed on MCF-7 (breast cancer) and HeLa (cervical cancer) cells to quantify the biocompatibility of PEG-coated HNTs as a function of nanotube dosage and incubation time. PEG-coated HNTs resulted fully biocompatible for both cell lines at concentrations up to 0.1 mg/mL (>70% of cells survived after 72-h incubation), making them suitable candidates for nanomedicine applications at moderate levels of exposure.

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 drug's bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by-layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drug's 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. © 2011 Elsevier B.V.

Fluoro-magnetic nanoparticles play an important role in biomedical applications since their size and concentration in tumors allow a very high resolution and an accurate mapping of lesions. Fluorescein isothiocyanate (FITC) has been entrapped inside crystals of magnetic nanoparticles (MNPs) during crystallization. This causes changes of nanoparticle crystal architecture towards elongated rods. TEM and SEM-EDX show elongated crystals with high iron concentration. The intensity of fluoro-MNP fluorescence was detected by fluorescence spectrophotometry and confocal microscopy. The benzene ring structure of FITC and its carboxylic group were clearly detected in the fluoro-MNP spectrum by using FTIR, compared to MNPs prepared in the absence of FITC. Rods were functionalized by hydrogel cross-linking structure (PEG-CMC) onto the fluoro-MNPs surface by using alternate layer-by-layer (LbL) adsorption. These hydrogel properties are used as a preserver for protein delivery. ALK1fc as specific TGF?1 inhibitor, was encapsulated inside (PEG-CMC) layers during LbL assembly. Zeta potential measurement, X-ray diffraction and SDS PAGE-silver staining results confirmed the encapsulation of ALK1fc. The efficiency of encapsulated ALK1fc was quantified by immunofluorescence assay against localization of TGF?1. Stained TGF?1 appeared a purple color and is distributed in the cytoplasm of untreated HLF (a liver cancer invasive cell line), whereas it disappeared in a HLF sample treated with encapsulated ALK1fc.

Fluoro-magnetic nanoparticles play an important role in biomedical applications since their size and concentration in tumors allow a very high resolution and an accurate mapping of lesions. Fluorescein isothiocyanate (FITC) has been entrapped inside crystals of magnetic nanoparticles (MNPs) during crystallization. This causes changes of nanoparticle crystal architecture towards elongated rods. TEM and SEM-EDX show elongated crystals with high iron concentration. The intensity of fluoro-MNP fluorescence was detected by fluorescence spectrophotometry and confocal microscopy. The benzene ring structure of FITC and its carboxylic group were clearly detected in the fluoro-MNP spectrum by using FTIR, compared to MNPs prepared in the absence of FITC. Rods were functionalized by hydrogel cross-linking structure (PEG-CMC) onto the fluoro-MNPs surface by using alternate layer-by-layer (LbL) adsorption. These hydrogel properties are used as a preserver for protein delivery. ALK1fc as specific TGF beta 1 inhibitor, was encapsulated inside (PEG-CMC) layers during LbL assembly. Zeta potential measurement, X-ray diffraction and SDS PAGE-silver staining results confirmed the encapsulation of ALK1fc. The efficiency of encapsulated ALK1fc was quantified by immunofluorescence assay against localization of TGF beta 1. Stained TGF beta 1 appeared a purple color and is distributed in the cytoplasm of untreated HLF (a liver cancer invasive cell line), whereas it disappeared in a HLF sample treated with encapsulated ALK1fc.

Frontispiece: Fe3 O4 nanoparticles (nano-Fe3 O4 ) LbL functionalized by coating biodegradable polyelectrolyte multilayers have been developed. Curcumin-loaded magnetic polymer coated nanoparticles are tested against an ovarian cancer cell line, SKOV-3 , as shown in confocal laser scanning image. Further details can be found in the article by S. Mancarella, V. Greco, F. Baldassarre, D. Vergara, M. Maffia, S. Leporatti on page 1365.

Halloysite nanotubes are cheap, abundant in their deposits, natural green clays with cylindrical structure having a chemical composition similar to that of kaolin. Because of their lumens, high aspect length-diameter ratio and low hydroxyl density on their surface they are readily suitable for a number of interesting applications. In this review we focus only on their use as 'nano-bazooka' drug carriers, able to shoot their cargo against major diseases. Their structure, controlled release and loading are described. We emphasize especially their possible use as novel drug delivery systems with applications in nanomedicine.

Halloysite is a nanostructured clay mineral withhollow tubular structure, which has recently found an importantrole as delivery system for drugs or other active molecules. Oneof these is curcumin, main constituent in the rhizome of the plantCurcuma Longa, with a series of useful pharmacologicalactivities, hindered by its poor bioavalaibility and solubility inwater. In this study, Halloysite Clay Nanotubes (HNTs) werecharacterized in terms of both structure and biocompatibilityand they were used for curcumin delivery to cancer cells. Theperformed 3 -(4,5 - dimethythiazol - 2- yl) - 2,5 - diphenyl -tetrazolium bromide (MTT) assay showed that HNTs have a highbiocompatibility, also when coated with polymers, whilecurcumin is highly toxic for cancer cells. The release kinetics ofcurcumin from HNTs was investigated by the dialysis bagmethod, showing a slow and constant release of the drug, whichcan be further controlled by adding layers of polyelectrolytes tothe external surface of the tubes. Successful polymer coating wasfollowed by Zeta potential. The Trypan Blue assay showed acytotoxic effect of loaded HNTs, proportional to theconcentration of tubes and the incubation time. Successful HNTsuptake by breast cancer cells was demonstrated by ConfocalLaser Scanning Microscopy images. All results indicate thatHalloysite Nanotubes are a promising carriers for polyphenoldelivery and release.

Halloysite nanotubes (HNTs) are efficient nano-containers capable of entrapping a range of active agents within the inner lumen, followed by their retention and slow release. Halloysite is a green environmentally friendly object available in commercial quantities. The lumen of the halloysite tube accomodates globular protein diameters, allowing their entrapment within the inner lumen of the halloysite while retaining their activity for use in biocatalysis. In this work a combination of high resolution imaging technique such as TEM, SEM and SFM have been employed to elucidate the structure. We have investigated their visco-elastic properties by force-indentation measurements and performed cytotoxicity tests utilizing neoplastic cell lines (breast and cervical cancer cells). Furthermore their uptake has been confirmed by Confocal Microscopy after their functionalisation with fluorescence molecules. The results indicate that halloysite nanotubes have been readly uptaken by neoplastic cells and exhibit a high level of biocompatibility.

There is an increasing amount of research on-going to produce functional nanometer-scale containers, and growing demand for their use in biomedical applications. Such containers would be inexpensive materials with a simple means of fabrication, thus calling for natural resources and nanotubes are good candidates for this. Halloysite clay is a two-layered aluminosilicate, chemically similar to kaolin, which has hollow tubular structure in the submicron range (1-3). As for most natural materials, the size of halloysite particles varies of 50-70 nm in external diameter, ca 15 nm diameter lumen and 1 ± 0.5 ?m length. Halloysite nanotubes are capable of entrapping a range of active agents within the inner lumen, followed by their retention and slow release (3-11). Different chemistry of the inner and outer surfaces in halloysite tubes would also allow for separate modification of inner and outer walls, e.g., for selective labelling. The lumen diameter of halloysite tube fits well to macromolecule and protein diameters, allowing their encasing in the tube. Biocompatibility is one of the main prerequisites for safe usage of halloysite in delivery of biologically active substances, in medical and household products. However, a comprehensive study of halloysite biocompatibility has not been done yet. In this work, we focused on studying halloysite nanotubes interaction (both untreated and fluorescently labelled) with cells. We analysed halloysite toxicity and visualized the process of cell uptake of fluorescently labelled clay nanotubes.

Halloysite Clay nanotubes (HNTs) are naturallyoccurring nanomaterials composed of double layeredaluminosilicate minerals with a hollow tubular structure. Due totheir interesting structural characteristics, chemically activeexternal and internal surfaces, cheap and abundant availability,HNTs have recently become the subject of research attention as anew type of material for various biological applications, includingdrug and gene delivery vehicles, cancer cells isolation, boneimplants, ultrasound contrast agents, cancer and stem cellsisolation and cosmetics. Therefore, assessment of HNTbiocompatibility has gained importance to demonstrate itssuitability for clinical purposes. In this study, HNTs were denselycoated with poly(ethylene glycol) (PEG) and MTT measurementswere carried out on two different human cancer cell lines, namelyHeLa (cervical cancer) cells and HepG2 (hepatocarcinoma) cells,to quantify the biocompatibility of PEG-coated HNTs as afunction of nanotube dosage and incubation time. While noncoatednanotubes exhibited significant concentration- and timedependenttoxicity, PEG-coated HNTs resulted fullybiocompatible for concentrations up to 0.5 mg/mL and forincubation time up to 72 h, making them suitable candidates fornanomedicine applications.

TGF?1 pathway antagonists have been considered promising therapies to attenuate TGF? downstream signals in cancer cells. Inhibiting peptides, as P-17 in this study, are bound to either TGF?1 or its receptors, blocking signal transduction. However, for efficient use of these TGF?1antagonist as target therapeutic tools, improvement in their delivery is required. Here, a plasmid carrying specific shDNA (SHT-DNA), small interfering RNA (siRNA), and the peptide (P-17) were loaded separately into folic acid (FA)-functionalized nano-carriers made of Bovine Serum Albumin (BSA). The two building blocks of the carrier, (BSA and FA) were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane of hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. Finally, cellular studies were performed to assess the targeting efficiency of the hybrid carriers. These vectors were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. Graphical Abstract: A novel fabrication of Hybrid Polymeric-Protein Nano-Carriers (HPPNC) for delivering TGF ?1 inhibitors to HCC cells has been developed. SHT-DNA, siRNA and P-17 have been successfully encapsulated. TGF ?1 inhibitors-loaded HPPNC were efficiently uptaken by HLF cells. [InlineMediaObject not available: see fulltext.].

m: The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. Materials & methods: Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO3) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. Results & discussion: Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 pm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. Conclusion: This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts.MATERIALS & METHODS:Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested.RESULTS & DISCUSSION:Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro.CONCLUSION:This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM-FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal-AFM-TIRF) microscopy.

Glucose consumption in many types of cancer cells, in particular hepatocellular carcinoma (HCC), was followed completely by over-expression of type II hexokinase (HKII). This evidence has been used in modern pharmacotherapy to discover therapeutic target against glycolysis in cancer cells. Bromopyruvate (BrPA) exhibits antagonist property against HKII and can be used to inhibit glycolysis. However, the clinical application of BrPA is mostly combined with inhibition effect for healthy cells particularly erythrocytes. Our strategy is to encapsulate BrPA in a selected vehicle, without any leakage of BrPA out of vehicle in blood stream. This structure has been constructed from chitosan embedded into oleic acid layer and then coated by dual combination of folic acid (FA) and bovine serum albumin (BSA). With FA as specific ligand for cancer folate receptor and BSA that can be an easy binding for hepatocytes, they can raise the potential selection of carrier system.

Micelles as colloidal suspension have attracted considerable attention due to their potential use for both cancer diagnosis and therapy. These structures have proven their ability to deliver poorly water-soluble anticancer drugs, improve drug stability, and have good penetration and site-specificity, leading to enhance therapeutic efficacy. Micelles are composed of hydrophobic and hydrophilic components assembled into nanosized spherical, ellipsoid, cylindrical, or unilamellar structures. For their simple formation, they are widely studied, either by using opposite polymers attachment consisting of two or more block copolymers, or by using fatty acid molecules that can modify themselves in a rounded shape. Recently, hybrid and responsive stimuli nanomicelles are formed either by integration with metal nanoparticles such as silver, gold, iron oxide nanoparticles inside micelles or by a combination of lipids and polymers into single composite. Herein, through this special issue, an updated overview of micelles development and their application for cancer therapy will be discussed.

The Epithelial to mesenchymal transition (EMT) is the process that drives epithelial tumor cells to acquire an invasive phenotype. The role of transforming growth factor-beta 1 (TGF-beta 1) in EMT is still debated. We used confocal laser scanning microscopy and scanning force spectroscopy to perform a morphomechanical analysis on epithelial breast cancer cells (MCF-7), comparing them before and after TGF-beta 1 exogenous stimulation (5 ng/mL for 48 h). After TGF-beta 1 treatment, loss of cell-cell adherence (mainly due to the reduction of E-cadherin expression of about 24%) and disaggregation of actin cortical fibers were observed in treated MCF-7. In addition, TGF-beta 1 induced an alteration of MCF-7 nuclei morphology as well as a decrease in the Young's modulus, owing to a rearrangement that involved the cytoskeletal networks and the nuclear region. These relevant variations in morphological features and mechanical properties, elicited by TGF-beta 1, suggested an increased capacity of MCF-7 to migrate, which was confirmed by a wound healing assay. By means of our biophysical approach, we highlighted the malignant progression of breast cancer cells induced by TGF-beta 1 exposure. We are confirming TGF-beta 1's role in EMT by means of morphomechanical evidence that could represent a turning point in understanding the molecular mechanisms involved in cancer progression.

The unique physico-chemical properties of silver nanoparticles (AgNPs) make them a powerful tool in many fields, ranging from cosmetics, biomedicals, household products, and wound dressing. Several evidences suggest the strong toxicity of AgNPs both in vitro and in vivo, but few data are available to full understanding of their adverse effects on cellular components and cytoskeleton. In this work, we assessed the toxicity of citrate-capped AgNPs on cortical actin and organelles, namely mitochondria and lysosomes, on epithelial breast cancer cells (MCF-7). The impact of AgNPs on cells was firstly evaluated in term of viability, oxidative stress, mitochondria membrane potential alteration, and apoptosis activation. Afterwards, we carefully estimated the qualitative and quantitative morphological alterations of cortical F-actin and organelles by confocal microscopy and specific software tools, coupled with a biomechanical analysis by atomic force microscopy (AFM). This multidisciplinary approach, which combines the standard biological assays with systematic morphometric and biomechanical analysis on cells, permits to understand at different levels the intracellular response elicited by AgNPs in order to provide new scenarios in toxicity assessment.

The EMT phenomenon is based on tumour progression. The cells lose their physiologic phenotype and assumed a mesenchymal phenotype characterized by an increased migratory capacity, invasiveness and high resistance to apoptosis. In this process, RHO family regulates the activation or suppression of ROCK (Rho-associated coiled-coil containing protein kinase) which in turn regulates the cytoskeleton dynamics. However, while the biochemical mechanisms are widely investigated, a comprehensive and careful estimation of biomechanical changes has not been extensively addressed. In this work, we used a strong ROCK inhibitor, Y-27632, to evaluate the effects of inhibition on living breast cancer epithelial cells by a biomechanical approach. Atomic Force Microscopy (AFM) was used to estimate changes of cellular elasticity, quantified by Young's modulus parameter. The morphometric alterations were analyzed by AFM topographies and Confocal Laser Scanning Microscopy (CLSM). Our study revealed a significant modification in the Young's modulus after treatment, especially as regards cytoskeletal region. Our evidences suggest that the use of Y-27632 enhanced the cell rigidity, preventing cell migration and arrested the metastasization process representing a potential powerful factor for cancer treatment.

The internalisation efficiency and cytotoxicity of polymeric microparticles and capsules were studiedfor three different adherent cell lines: HeLa (human cervice cancer), MCF-7 (human breast cancer)and B104 (murine neuroblastoma). Microparticles of colloidal CaCO3 cores coated with either layerby-layer self-assembled oppositely charged biosynthetic (PSS\PAH) or biodegradable (DXS\PRM)polyelectrolytes and hollow capsules thereof, obtained upon removal of the CaCO3 cores by EDTA,were fabricated. Fluorescent-labeled polymer layers coated onto CaCO3 cores were employed forevaluating cell uptake efficiency. Confocal laser scanning microscopy was used to confirm dissolutionof preformed biodegradable multilayers. Cellular viability for all type of colloidal entities waspreserved, indicating safety and tolerability of the potential drug carriers.

Halloysite nanotubes (HNTs) are nanomaterials composed of double layered aluminosilicate minerals characterized by a wide range of medical applications. Nonetheless, systematic investigations of their imaging potential are still poorly documented. This paper shows a parametric assessment of the effectiveness of HNTs as scatterers for safe ultrasound (US)-based molecular imaging. Quantitative evaluation of average signal enhancement produced by HNTs with varying set up configuration was performed. The influence of different levels of power (20%, 50%, and 80%) of the signal emitted by clinical equipment was determined, to assess the efficacy of different HNT concentrations (1.5, 3, and 5 mg/mL) at conventional ultrasonic frequencies (5.7-7 MHz), even in case of specific limitation regarding US mechanical interaction with target tissues. Different samples of HNT containing agarose gel were imaged through a commercially available echographic system and acquired data were processed through a dedicated prototypal platform to extract the average ultrasonic signal amplitude. The rate of signal enhancement achieved by different concentration values was quantified and the contribution of frequency increment was separately evaluated. Despite influencing the level of mechanical excitation on HNTs and tissues, our results demonstrated how increasing the power of the emitted signal negatively affected the measured backscatter. Conversely, noticeable improvements in signal backscatter could be achieved incrementing HNT concentration and the echographic frequency employed; specifically the signal enhancement over the used concentration range could be improved by averagely 20%, corresponding to 4.86 ± 0.80 (a.u.), when employing the higher value of echographic frequency. © 2013 IEEE.

The requested features of pharmaceutical drug delivery (such as biodegradability or targeting) for intravenous administration are reasonably well met by liposomes, microcapsules, and nanoparticles for water-soluble drugs. The development of nanoparticulate drugs displaying all of these properties for poorly soluble pharmaceuticals still represents a challenge. Low solubility in water, however, tends to be an intrinsic property of many drugs, including some powerful anti-cancer agents. Intravenous administration of relatively large aggregates of an insoluble drug may result in embolisation of these particles into small blood capillaries and may cause unwanted effects like tissue ischemia. Hence, it does not allow for achieving therapeutically significant concentrations. Many promising drug candidates never enter further development processes because of solubility problems. On multiple occasions micelles can serve as drug delivery systems for poorly soluble pharmaceuticals. However, there are problems, which include low loading efficacy of the drug into the micelles; problems with controlling the release rate of the drug, and with micelle stability.

Theranostic approaches using nanotechnology have been a hot research area for the past decade. All nano drug delivery techniques and architectures have some limitations, as do diagnostic nano-approaches. Thus, combining nano drug delivery strategies with diagnostic techniques using nanoparticles for improving imaging modalities has been the key to fill up those gaps. In the past decade, lots of approaches have been made with different combinations of biomaterials fabricated/synthesized to nanostructures with modified surface functionalization to improve their overall theranostic properties. This article summarizes recent research works based on the biomaterials used for fabricating these nanostructures. Their combinations with other biomaterials have been demonstrated with their overall advantages and limitations.

Oil bodies (OBs) are specialised organelles ubiquitously detected in plant oil seeds, which serve as lipid storage compartments. OBs consist of a hydrophobic core of triacylglycerol (TAGs), surrounded by a monolayer of phospholipids (PLs) embedded with some specific proteins with a size ranging from 0.5 to 2 mu m. In this work, we report an easy method to reconstitute OBs starting from their constituents and to encapsulate lipophilic molecules, i.e. the fluorescent fluorescein isothiocyanate (FITC) and carboxyfluorescein (CF), into reconstituted OBs. This methods allowed us to produce OBs 4- to 10-fold smaller (50-200 nm) than the native one and to obtain a good recovery (about 40%) of both the fluorescent compounds used in the present work. The properties of reconstituted OBs were investigated by a combination of Brewster angle microscopy, scanning force microscopy, zeta-potential techniques. OBs were stable and formed ordered monolayers when patterned on hydrophobic substrates whereas they showed a higher tendency to aggregate into larger, coalescing OBs when were deposited onto hydrophilic substrates or at the air/water interface. Furthermore, we verified the uptake of FITC-loaded OBs by the MCF-7 breast cancer cell line. Our results indicated that OBs could be envisaged as novel carriers to deliver hydrophobic bioactive compounds

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFbeta. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

In this work, tunable nonwoven mats based on poly(3-hydroxybutyrate) (PHB) and type I collagen (Coll) were successfully produced by electrospinning. The PHB/Coll weight ratio (fixed at 100/0, 70/30, and 50/50, resp.) was found to control the morphological, thermal, mechanical, and degradation properties of the mats. Increasing collagen amounts led to larger diameters of the fibers (in the approximate range 600-900 nm), while delaying their thermal decomposition (from 245 degrees C to 262 degrees C). Collagen also accelerated the hydrolytic degradation of the mats upon incubation in aqueous medium at 37 degrees C for 23 days (with final weight losses of 1%, 15%, and 23% for 100/0, 70/30, and 50/50 samples, resp.), as a result of increased mat wettability and reduced PHB crystallinity. Interestingly, 70/30 meshes were the ones displaying the lowest stiffness (similar to 116 MPa; p < 0 05 versus 100/0 and 50/50 meshes), while 50/50 samples had an elastic modulus comparable to that of 100/0 ones (similar to 250 MPa), likely due to enhanced physical crosslinking of the collagen chains, at least at high protein amounts. All substrates were also found to allow for good viability and proliferation of murine fibroblasts, up to 6 days of culture. Collectively, the results evidenced the potential of as-spun PHB/Coll meshes for tissue engineering applications.

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, ?- and ?-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.

Novel synthetic peptides represent smart molecules for antigen antibody interactions in several bioanalytics applications, from purification to serum screening. Their immobilization onto a solid phase is considered a key point for sensitivity increasing. In this view, we exploited Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) with a double aim, specifically, as investigative tool for spacers monolayer assembling and its functional evaluation, as well as high sensitive method for specific immunosorbent assays. The method was applied to pancreatic ductal adenocarcinoma (PDAC) detection by studying the interactions between synthetic phosphorylated and un-phosphorylated alpha-enolase peptides with sera of healthy and PDAC patients. The synthetic peptides were immobilized on the gold surface of the QCM-D sensor via a self-assembled alkanethiol monolayer. The presented experimental results can be applied to the development of surfaces less sensitive to non-specific interactions with the final target to suggest specific protocols for detecting PDAC markers with un-labeled biosensors.

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-3? (GSK-3?). 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. © 2012 The Royal Society of Chemistry.

Nowadays, there is strong interest in the development of smart inorganic nanostructured materials as tools for targeted delivery in cancer cells. We proposed a novel synthetic procedure of calcium carbonate nanocrystals (NCs) and their use as drug delivery systems, studying the physical chemical properties and the in vitro interaction with two model cancer cells.Pure and thermodynamically stable CaCO3 NCs in calcite phase were synthesized by a readily and feasible method, easily scalable, that allows the control of NCs shape and size without any surfactant use. CaCO3 NCs were extensively investigated by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), X-ray Diffraction (XRD), Raman spectroscopy and Brunauer-Emmett-Teller analysis (BET). To deeper investigate their possible use as nanovectors for drug cancer therapies, CaCO3 NCs biocompatibility (by MTT assay), cell interaction and internalization were studied in in vitro experiments on HeLa and MCF7 cell lines. Confocal and transmission electron microscopies were used to monitor and evaluate NCs-cell interaction and cellular uptake.Data here reported demonstrated that synthesized NCs readily penetrate HeLa and MCF7 cells. NCs preferentially localize inside the cytoplasm, but were also found into mitochondria, nucleus and lysosomes. This study suggests that synthesized CaCO3 NCs are good candidates as effective intracellular therapeutic delivery system.

The large use of nanomaterials in many fields of application and commercial products highlights their potential toxicity on living organisms and the environment, despite their physico-chemical properties. Among these, silver nanoparticles (Ag NPs) are involved in biomedical applications such as antibacterial agents, drug delivery vectors and theranostics agents. In this review, we explain the common synthesis routes of Ag NPs using physical, chemical, and biological methods, following their toxicity mechanism in cells. In particular, we analyzed the physiological cellular pathway perturbations in terms of oxidative stress induction, mitochondrial membrane potential alteration, cell death, apoptosis, DNA damage and cytokines secretion after Ag NPs exposure. In addition, their potential anti-cancer activity and theranostic applications are discussed.

Halloysite clay nanotubes (HNTs) are natural materials with a characteristic hollow tubular structure in the nanometer range. Owing to this feature, they were found to be a suitable nanosized container for the loading of biologically active molecules like biocides and drugs. Also, HNTs have been reported to be of potential interest for other biological applications, such as gene delivery carriers, ultrasound contrast agents, cancer therapy and stem cells isolation. Therefore, biocompatibility of halloysite represents one the main requisites for the employment of HNTs for clinical purposes. Here we present a study aimed at assessing HNTs biocompatibility before and after their surface coating with poly(ethylene glycol) (PEG), a polymer which has been reported to increase biocompatibility, to prolong circulation time and to prevent protein adsorption and aggregation in biological environments. The dose- and time-dependent cytotoxicity of noncoated and PEG-coated HNTs obtained was evaluated in vitro by MTT cell viability assay carried out on both HeLa and HepG2 cells, two different human cancer cell lines. Binding and uptake of nanotubes were also analyzed at ultrastructural level by transmission electron microscopy (TEM). Interestingly, the results obtained showed that both the HNTs tested were actively taken up by the cells but, while noncoated nanotubes exhibited significant concentration- and time-dependent toxicity, PEG-coated HNTs were found to be highly biocompatible, being then suitable candidates for biomedical applications.

A smart nanocarrier system for cancer therapy, based on a recently developed technique for preparing pure nanometric calcium carbonate (CaCO3), was studied. Different approaches were used to obtain sustained release of cisplatin: at first, pure CaCO3 nanoparticles were evaluated as carriers, then the nanoparticles were functionalized with polymer or silanes, and finally they were employed as a substrate to build layer by layer (LbL) self-assembled polyelectrolyte nanocapsules. Loading efficiency and release kinetics were measured. The best loadings were obtained with the LbL nanocapsules, allowing for high loading efficiency and the possibility of controlling the release rate of the drug. The behavior of all the carriers was evaluated on four neoplastic cell lines, representative of different types of neoplastic disease, namely MCF-7 (breast cancer), SKOV-3 (ovarian cancer), HeLa (cervical cancer) and CACO-2 (human epithelial colorectal adenocarcinoma). Negligible cytotoxicity of the nanoparticles, functionalized nanoparticles, and nanocapsules was observed in experiments with all cell lines. Nanocapsules were functionalized with fluorescein isothiocyanate (FTTC) in order to track their kinetic of internalization and localization in the cell line by confocal laser scanning microscopy (CLSM). The cytotoxicity of the loaded capsules was evaluated, showing cell survival rates close to those expected for non-encapsulated cisplatin at the same nominal concentration. (C) 2015 Elsevier Inc. All rights reserved.

In this review we will report on recent advanced in polyelectrolyte capsules for targeted drug delivery (eg of growth factor inhibitor) against epatocarcinoma. Degradable polyelectrolyte multilayers capsules (PMCs) are of particular interest for cancer therapy since under physiological conditions they can be enzymatically degraded upon cell interaction. Small bioactive molecules such as TGF-Beta inhibitors can be incorporated inside them. Nano-to-microscale delivery systems can enhance efficacy at single cell level for targeted therapy. Layer-by-layer (LbL) self-assembled capsules are novel carriers maximizing drug administration and improving antimetastatic activity of TGF-Beta inhibitors in Hepatocellular Carcinoma (HCC).

Transforming growth factor (TGF)- is a pluripotent cytokine that displays several tissue-specific biological activities. In the liver, TGF- is considered a fundamental molecule, controlling organ size and growth by limiting hepatocyte proliferation. It is involved in fibrogenesis and, therefore, in worsening liver damage, as well as in triggering the development of hepatocellular carcinoma (HCC). TGF- is known to act as an oncosuppressor and also as a tumour promoter in HCC, but its role is still unclear.

Selective imaging of lysosomes by fluorescence microscopy using specific fluorescent probes allows the study of biological processes and it is potentially useful also for diagnosis. Lysosomes are involved in numerous physiological processes, such as bone and tissue remodeling, plasma membrane repair, and cholesterol homeostasis, along with cell death and cell signaling. Despite the great number of dyes available today on the market, the search for new fluorescent dyes easily up-taken by cells, biocompatible and bearing bright and long-lasting fluorescence is still a priority. Methods: Two thiophene-based fluorescent dyes, TC1 and TC2, were synthetized as lysosome-specific probes. Results: The new dyes showed high selectivity for fluorescent staining and imaging of lysosomes and disclosed high photostability, low toxicity and pH insensitivity in the range 2-10. Conclusions: The TC dyes exhibited high co-localization coefficients (>95%) and moderate quantum yields. They showed high biocompatibility and long-term retention, important features for biological applications. General significance: The results of the present work disclose a new class of organic dyes with potential wide applications as specific and efficient lysosome probes in the study of various biological processes.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers in Europe and the United States. It has a very low 5 years-survival rate and its diagnosis is often late and imprecise due to the lack of specificity of currently used markers for PDAC. As previously demonstrated PDAC patients' sera may contain autoantibodies towards phosphorylated a-enolase (ENOA), which in combination with other standard markers can increase specificity in diagnosis of PDAC. In this context we realized a microfluidic platform with integrated EIS biosensors. We achieved a specific antibodies detection by immobilizing onto electrodes peptides corresponding to a portion of ENOA. Phosphorylation of peptides was found to influence the recognition of antibodies in PDAC patients' sera detected by the developed biochip thus validating the EIS technique as a strong tool for quick, cost-saving and label-free analysis of serum samples. Biochip results are in agreement with those from traditional techniques, such as ELISA and western blot, but measurements are much more sensitive and specific, increasing the possibility of PDAC diagnosis. In addition this approach is faster and more reproducible compared to traditional techniques making the developed biochips ideal for a quick, cost-saving and label-free analysis of serum samples.

Aim of this work was to investigate the effect ofultrasound incident frequency on the echographic contrastenhancement power of an experimental drug delivery agent,halloysite clay nanotubes (HNTs), and to determine a suitableconfiguration in terms of both insonification frequency andparticle concentration for an effective employment as targetedcontrast agent. Various HNT concentrations (range 0.25-3.00mg/mL) were dispersed in custom-designed tissue-mimickingphantoms and exposed to different ultrasound frequencies (7-11MHz) through a conventional clinically-available echographicdevice. Off-line analysis included the evaluation of bothamplitude of backscattered ultrasound signals and imagebrightness. Amplitude of HNT-backscattered signals showed alinear increase with particle concentration, while imagebrightness enhancement was limited by logarithmic compressioneffects. On the other hand, backscatter amplitude showedsignificant increments with increasing ultrasound frequency upto 10 MHz, then showing a concentration-dependent behaviorwithout further enhancements. Overall, the most effectiveresponse was found when a 10-MHz ultrasound frequency wasemployed to insonify HNTs at a concentration of 1.5 mg/mL. Inconclusion, the present study optimized the combination ofincident ultrasound frequency and HNT concentration, in orderto obtain an echographic image enhancement suitable for medicalapplications. Future dedicated studies will assess the feasibility ofautomatic detection of HNTs within echographic images andtheir possible employment as theranostic agents.