Nanomedicine advancing from bench-to-bedside: the role of materials

Resume : This presentation will introduce new concept on iCRT and applications for medical implants and drug development. The sol-gel process for preparing silicate-based glasses and bioactive glasses is well-known and has been extensively researched over the last 20 years. In more recent times, the use of these sol-gel structures as controlled release drug delivery systems has also been well-studied to good effect. We are currently actively engaged in the design and appropriate synthesis of another important class of biomaterials by sol-gel methodologies – phosphate glasses, a new class of iCRT system. Phosphate glasses are becoming increasingly valuable for certain biomedical applications because of the high level of control over their dissolution rates that can be achieved. Primarily, this control over glass dissolution rate is accomplished through modification of the glass composition. However, the challenge is how to preparation of these materials by sol-gel routes with inherent porosity and increased surface area which provides enhanced dissolution behaviour and extra functionality as compared with the fusion derived analogues. More specifically, due to their controlled dissolution and nano porosity, phosphate glasses are potentially applicable for use as novel drug delivery systems and medical implant applications. Herein is reported our approach to the preparation of porous, amorphous phosphate glasses by a non-aqueous sol-gel route. The effects of a number of reaction process variables on the quality of the phosphate glass products will be discussed. The sol-gel precursor, order of addition, drying temperature, drying time and calcination temperature have all been found to be important parameters. This presentation will be discussing the mechanism and kinetics of glass formation and how the different reaction parameters involved are impacting on product formation and performance. With a deep understanding of this process, it will then be possible to amend the phosphate sol-gel method in order to introduce a relevant drug compound without risk of compromising drug integrity and using those physical properties of the glass which can be manipulated to modulate drug release. Case studies of the iCRT applications will be presented, including a novel approach to a drug-eluting bioresorbable cardiovascular stent and new development for controlled release of very soluble and poorly soluble drugs.

Resume : Tissue engineering is an emerging interdisciplinary field that applies principles of biology and engineering to the development of viable substitutes that restore or improve the function of human tissues. Scaffolds play a crucial role in nerve tissue engineering due to their encouragement towards cells’ proliferation, ability to allow nutrients to permeate and their resemblance with the Extracellular Matrix (ECM), thus forming a unique microenvironment for cells, allowing them to interact in vivo. To this end, via electrospinning, a versatile process, we fabricated conductive nanofiber-based scaffolds, consisting of the biodegradable polymer Polyvinyl alcohol (PVA) and the conductive polymer Poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), and we evaluated their surface properties, emphasizing on the way that manipulate cell growth and adhesion. A neural cell-line was deposited onto the fabricated scaffolds in order to evaluate their cytocompatibility. MTT cytotoxicity assay was used for the examination of cells’ proliferation and revealed excellent compatibility. In agreement with MTT findings, methylene blue staining and SEM imaging further reinforced scaffold’s cytocompatibility. Degradation and swelling studies were carried out in order to examine scaffold’s physicochemical behaviour. Results indicated that the conductive non-woven scaffolds are cytocompatible with promising cell proliferation properties, thus providing good potential for further utilization in nerve tissue engineering applications.

Resume : In nano-pharmaceutical field the herbal extracts supported on nanometric carriers covers a large series of applications such as to heal variety of diseases with less toxic effects and better antioxidant and therapeutic effects. Nevertheless some limitations of herbal extracts like liver metabolism, instability in acidic environment lead to, drugs levels, below therapeutic concentration in blood, in consequence there is considerably decreased pharmaceutical effect. This study focuses on a new drug delivery system based on architectures of nano biopolymers for Silybum marianum L. extract. Silybum marianum L. species common name “milk thisle” have multiple pharmaceutical properties like hepatoprotective antihepatotoxic and antioxidant action due to complex chemical composition which contains besides silymarin, a number of compounds such as amino acids (glycin, cystein, leucine, tiamin, glutamic acid), taxifolin, betain, quercetin, dihydrokaempferol, apigenin, naringin, polyhydroxy chromone, lipids, linoleic acid, oleic acid, palmitic acid, sterols (cholesterol, stigmasterol, sitosterol). Incorporation into biopolymer-based nanoparticles seems to significantly help the oral delivery of herbal extracts, because these nanoparticles can protect the pharmaceutical active principles from degradation in the gastrointestinal tract and, by virtue of their unique absorption mechanism through the lymphatic system. The free radical scavenging effect was studied using the chemiluminescence technique and DPPH(2,2-diphenyl-1-picrylhydrazyl) method. The viability of cells were detected by MTS - assay that emphasize significant stimulation of the growth of mouse fibroblast 3T3 in a dose-dependent manner. Also evaluating the antiproliferative effect on human U87 glioma cell line showed promising results. In addition AFM and UV-Vis reveal a series of properties such as spectral characteristcs and specific topography and DLS was used to measure the hydrodynamic size and polydispersity index of drug delivery nano systems. Keywords: drug delivery nano systems, herbal extracts, antioxidant activity, antiproliferative effect

Resume : The high interest of graphene or graphene oxide due to special physico-chemical properties and versatility, has attracted significant attention in many areas of scientific research including nanomedicine and drug delivery fields. In this study, we investigated the self assembly effect of the several polyphenolic compounds with graphene/ graphene oxide related to the antioxidant activity and specific morphology. Polyphenolic compounds are well recognized having therapeutic properties like chemopreventive properties, antioxidant, anticarcinogenic, antimutagenic, antiproliferative and anti-inflammatory effects. Also polyphenolic compounds contribute to inducing apoptosis by arresting cell cycle, regulating carcinogen metabolism and ontogenesis expression, inhibiting DNA binding and cell adhesion, migration, proliferation or differentiation, and blocking signaling pathways. If graphene and graphene oxide could be a carrier for polyphenoloc compound the antioxidant properties need to be investigated in a first step.In this context, the study presents the evolution of antioxidant activity for graphene and graphene oxide before and after the process of assembling with various polyphenolic compounds. Chemiluminescence CL method (with chemiluminescence system generated by the luminol and hydrogen peroxide in an alkaline medium) and DHPP (2,2-diphenyl-1-picrylhydrazyl) technicqe was used to identify the oxidation potential and capacity of free radicals scavenging. In addition morphological characteristics was elucidation with atomic force microscopy AFM, Raman and FT-IR Keywords: graphene, graphene oxide, antioxidant activity, chemiluminescence, atomic force microscopy

Resume : Currently, photodynamic therapy (PDT) has emerged as an important treatment modality for a variety of cancers, cardiovascular and ophthalmic diseases. Upon irradiation, the activated photosensitizers (PSs) transfer their excess energy to the surrounding oxygen to form reactive oxygen species, which will cause irreversible damage of diseased cells and tissues. However, the clinical use of many PSs has been hampered by their significant side effects including nonspecific damage to normal tissues due to low selectivity to specific cells or tissues. In this study, we prepared spherical nanodrug, chlorin e6 (Ce6)-conjugated octaammonium polyhedral oligomeric silsequioxane (OA-POSS) for enhancing the PDT efficacy of PS. We first synthesized Ce6-conjugated OA-POSS (PC) and then oleic acid was introduced to PC (PCO). The resulting nanodrugs exhibited spherical shape with a size below 100 nm. ATR-FTIR and zeta-potential analyses confirmed the introduction of Ce6 to OA-POSS. The resulting nanodrug, PCO, exhibited significantly enhanced phototoxicity against human breast cancer cells ascribed to apoptotic cell death, while its dark-toxicity was negligible in the range of 0.1-2 Ug/mL. In addition, the PCO treatment caused the disruption of mitochondrial membrane potential and the increased cytochrome c release. Based on these results, the PCO nanodrug may contribute to the development of a new generation of PS for enhanced PDT treatment of cancers.

Resume : The meddling of nanotechnology and medicine comes to bear new approaches towards the treatment of various diseases, such as neurodegenerative ones. Electrospinning, a well established technique for nanofiber production is an easy and versatile technique that has recently been used to fabricate fibrous tissue-engineered scaffolds, which have great similarity to the Extracellular Matrix on fiber structure. In this study we proceed to the fabrication of a Polyvinyl Alcohol (PVA) /Poly (ε-caprolactone) (PCL) fibrous scaffold by dual system electrospinning and explored its application as nerve guide substrate in vitro. The dual physicochemical behaviour of the polymers revealed a unique performance of a combining polymer, with both hydrophobic and hydrophilic properties. The in vitro study indicated that electrospun PVA/PCL nano-fibrous scaffolds promoted neural cell adhesion, elongation and proliferation. Oxygen plasma treatment of the scaffolds, combined with the presence of Nerve Growth Factor further promoted cell adhesion with the presence of neuritis outgrowth. Due to its advantage of high surface area for cell attachment, it is believed that this electrospun nerve fiber-based scaffold could find further application in cell therapy for nerve regeneration in future, in order to improve functional regeneration outcome, especially for longer nerve defect restoration.

Resume : The body is unable to control massive blood loss without treatment. Hemostatic agents have been used in surgical operation for many years to control bleeding. Available hemostatic agents are often ineffective, expensive or raise safety concerns. In this study, we fabricated poly(vinyl pyrrolidone)(PVP) nanofibrous mats containing blood coagulation agents such as calcium chloride and thrombin to use as a novel non-woven hemostatic agent. PVP is inexpensive and safe material approved by FDA for human use. PVP nanofibrous sheets were fabricated by electrospinning of 10 wt% PVP aqueous ethanol solution containing thrombin and calcium chloride. Electrospun PVP nanofibrous mat structures revealed randomly aligned fibers with average diameter of 580 nm. Surface characteristic of PVP nanofibrous mats were confirmed to be soluble and hydrophilic surface determined by water contact angle measurement. Quantification of residual solvent on fabricated nanofibers determined by gas chromatography was a concentration range of 100-200 ppb. PVP nanofibers showed a relatively low cytotoxicity and the cellular survival rate was more than 90%. In vitro whole blood coagulation experiment showed expeditious blood coagulation within 3 minutes after contact with fabricated nanofibrous sheet, because PVP nanofibrous mats were soluble by contacting blood and helped facilitate clot formation by providing calcium ion and thrombin to the wound. In vivo studies using haemorrhaging liver rat model showed a great promise of PVP nanofibers to aid in rapid hemostasis. These results suggest that electrospun PVP nanofibrous mats containing blood clotting agents will be a promising hemostatic agent to reduce massive blood loss.

Resume : There is a strong interest in fabricating artificially structured platforms that allow reprogramming cell shape or enlarging cell culture harvest. In the particular context of neural engineering and regeneration, periodic topographical cues have already demonstrated ability to promote neural differentiation. Moreover, these types of patterns are attracting much attention to guide neurite outgrowth and re-establish neural connections, in combination or not with biological cues. Additionally, reduced graphene oxide (rGO) has a great interest for neural regeneration due to its extraordinary properties. In this work, we have used laser interference (LI) for producing fringed patterns with periods < 10 um on ultra-thin rGO films fabricated on glass coverslips by spin-coating of a GO aqueous suspension and subsequent thermal treatment. In contrast with laser or electron writing techniques, LI is easy-to-use, produces patterns in large areas using a few laser pulses and the period can easily be modified by changing the projection optics. As a proof of concept, we have cultured rat embryonic neural progenitor cells on these patterned rGO films and explored the influence of the patterns on neural adhesion, morphology, viability, alignment and differentiation. Our results evidence a clear induction of neural alignment along the lines defined by the patterns. The role of the period and pattern structure on neural cell behaviour will be finally discussed.

Resume : Electrospun nanofibers have architecture analogous to the natural environment of tissue in physical structure and biological function. Hence, electrospun nanofibers made from biodegradable polymers, such polycaprolactone (PCC) and polylactic acid (PLA) have great potential for biomedical devices, tissue engineering scaffolds and drug delivery carriers. However, the hydrophobic nature of PCL and PLA strongly decrease the biocompatibility of nanofibers made from these compounds. Therefore, the surface of the nanofibers must be modified in order to increase the surface wettability by introducing reactive groups (carboxyls, amines, anhydrides, etc.). The plasma deposition of functional coatings is the method of choice for the adjustment of surface composition and morphology of materials without affecting their bulk properties. In this work the deposition of the reactive carboxyl and anhydride groups is performed by co-polymerization of maleic anhydride and acetylene using atmospheric pressure dielectric barrier discharge (AP-DBD). The electrospun PCL nanofibers were prepared by electrospinning technology NanospiderTM. The low cost AP-DBD allowed the deposition of stable carboxyl and anhydride coatings onto the PCL nanofibers. It was found that by adjusting the PCL concentration, electrospinning voltage and substrate velocity the morphology and porosity of the nanofiber mesh can be optimized. By using the well known derivatization with trifluoroethanol we determined that up to 6 at.% of carboxyl groups can be deposited at the substrate surface. The homogeneity of the plasma coating was studied by Scanning Electron Microscopy, Energy Dispersive, IR and X-ray Photoelectron Spectroscopies.

Resume : Methicillin resistant Staphylococcus aureus (MRSA) represents one of the main causes of mortality and morbidity in hospitalized patients. The aim of this research was to develop a biocompatible nanomaterial based on magnetite and different natural, plant-derived compounds with antimicrobial and antibiofilm effect, efficient against MRSA strains. Functionalized magnetite nanoparticles (Fe3O4@) have been synthesized by co-precipitation, characterized by IR, SEM and HR TEM and functionalized with eugenol, eucalyptol, carvone, limonene and β-pinene. The minimum inhibitory concentrations (MICs) and biofilm development were established by microdilution method. Adherence of MRSA strains to the inert substrata was assessed by Cravioto modified method. The biocompatibility of the prepared nanoarhitectonics was assessed in vitro by fluorescence microscopy and MTT assay, using human cultured endothelial cells. Subinhibitory concentrations of Fe3O4@Eugenol and Fe3O4@Eucalyptol exhibited the most significant effects on S. aureus viability and attachment. All tested functionalized magnetite variants proved to inhibit S. aureus strains biofilm formation, in the following decreasing order: Fe3O4@Eug> Fe3O4@Euc> Fe3O4@Carv > Fe3O4@ β-pin> Fe3O4@Lim. Microscopy analysis and the MTT assay demonstrated that all used nanosystems (tested at MICs levels) exhibited a low citotoxicity in vitro, allowing the normal growth and metabolism of cultured endothelial cells.This study brings new insights in the development of modern therapies based on nanotechnology aiming to fight resistant infections by using natural products

Resume : The goal of our work was to test the possibility of attaching to carbon “nucleus” (ultra dispersed diamonds, UDDs), specific antibodies to the tumor receptors and metabolic drug (Doxorubicin, DOX). As carbon “nucleus” UDDs were used. The UDDs were obtained by detonation synthesis and purified by strong oxidative acid treatment in concentrated HNO3 at 750 C for 1 day. Then UDDs were incubated with N-(3-dimetylaminopropyl) - N - ethyl-carbodiimide hydrochloride (Sigma) and functionalized the surfaces with amino groups. After adding DOX-lactose monohydrate in dH2O and incubation at room temperature during 24 hours covalent binds between DOX and UDDs were formed. Thin films of UDD-DOX were placed on glass using Matrix-assisted Pulsed Laser Evaporation (MAPLE) deposition technology. Antibodies to Epidermal Growth Factor (# WH0001950M1 Sigma, for ELISA) or antibodies to Epidermal Growth Factor Receptor (# RMPD 20 clone SP9, Diagnostic Biosystems, for ICH) were deposited on the surface of the UDD-DOX films with MAPLE technology too. Biological properties of UDD-DOX- antibody constructs we determined by culturing it with tumor cells (MCF-7 cell line) and using microscopy, immune cytochemistry staining and ELISA. In result a stable compound of UDDs with Doxorubicin was obtained. The percentage of free Doxorubicin was 0.05%. Also we demonstrated that the UDD-DOX- antibody construct has dose-dependent cytotoxic effect on tumor cells (MTT-assay). Simultaneously, both antibodies after MAPLE deposition maintained from 75 to 83% of the functional activity and specificity. Thus, we can conclude about the prospects of selected methods and approaches for creating an antitumor agent with capabilities targeted delivery of drugs.

Resume : During the last decade, drug delivery applications and especially targeted delivery of anti-cancer drugs i.e. doxorubicin, cyclodextrin etc., has been under intense study, due to the high potential health impact that they present. In this context, a magnetically-navigated drug delivery system is developed, based on a nanocapsule which will consist of multi-wall carbon nanotubes as the main carrier component, magnetite nanoparticles for the navigation of the nanocapsule, an anti- cancer drug and fluorescent molecules for the visualization of the nanocapsule movement in a microfluidic channel. The magnetic navigation of the nanocapsule in a microfluidic channel will be also presented, while all the individual components of the drug delivery system are characterized both structurally and morphologically. In addition, a study based on the interaction of cancerous mouse lesions with the drug loaded nanocapsule will be presented based on micro-Raman spectroscopic analysis. Tissues give rise to distinct spectral peaks when illuminated with light, through the variation or vanishing of the lipid and protein-assigned vibrational modes that are usually observed in healthy and cancerous tissue areas. Vibrational spectroscopy and Raman spectroscopy in particular, offers unique advantages as a label-free technique, in terms of spectral resolution, being able to distinct narrow spectral alterations thus providing insight into the structural condition of specific tissue biomarkers. This work aims to highlight the advantages of magnetically-controlled drug delivery systems for cancer treatment as well as the potential use of Raman spectroscopy as an in vitro/in vivo medical diagnostics tool.

Resume : Eu3+ doped CaF2 and SrF2 nanoparticles (NPs) were synthesized through a hydrothermal reaction [1] with different reaction times. Sodium citrate or potassium citrate were chosen as capping agent to obtain water dispersible NPs. X-ray Powder Diffraction shows that the NPs are single phase with fluorite structure. TEM images show that the size of the NPs changes with the reaction time. The analysis of the EXAFS spectra shows that the lanthanide ions enter the fluorite structure as substitutional defects on the metal site, as also confirmed by laser spectroscopy. Eu3+ spectroscopy allowed finding the site symmetry of Eu3+ in-side the structure, by studying the emission bands. All the samples present two different Eu3+ sites, one with higher symmetry and the other one with lower symmetry. For NPs synthesized at longer reaction time, the emission of Eu3+ in sites with higher symmetry is predominant with respect to the emission from Eu3+ sites with lower symmetry. Emission decay measurements revealed that lifetimes of the 5D0 state of Eu3+ doped NPs increases for NPs grown with longer reaction times. Lifetimes for Eu3+ emission around 20 ms were found, indicating that the present NPs can be good candidates as markers for time resolved fluorescence techniques in biomedical imaging for which fast autofluorescence is a relevant drawback to avoid. [1] M. Pedroni, F. Piccinelli, T. Passuello, M. Giarola, G. Mariotto, S. Polizzi, M. Bettinelli, and A. Speghini, Nanoscale, 2011, 3, 1456?60.

Resume : A disposable magnetic particle-based GMR sensor sytem for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed. The immunochemical system makes use of the competition assay principle, and employs an erythromycin (or tylosin)–BSA conjugate as coating molecule. After competition between free and coated analyte for the antibodies, the activity of the magnetic particle-labelled antiglobulins was measured by high-sensitive magnetic sensor. Using standard solutions of erythromycin and tylosin, the detection limit of the assay was 0.5 ng/mL for erythromycin and 3.0 ng/mL for tylosin, while the sensitivity (25% inhibition concentration) was 2.0 ng/mL for erythromycin and 4.0 ng/mL for tylosin. The suitability of the assay for quantification of erythromycin and tylosin in bovine muscle was also studied. Results obtained on real samples were confirmed by micro-liquid chromatography coupled on line with tandem mass spectrometry (micro-LC-MS-MS), using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest.

Resume : Pharmaceutical and biomedical companies are looking for innovative biomaterial-based solutions to improve their drug development strategies, by using novel drug delivery systems able to maximize efficacy and minimize side effects. Controlled release of active pharmaceutical ingredients (API) is a major challenge for the development of novel and efficient drug delivery systems as it holds a number of advantages over traditional drug delivery. The present contribution describes the application of injectable, thermo-sensitive polymer excipients able to localize and sustain the release of different APIs. These systems are biocompatible, biodegradable and easy to inject. They are liquid at room temperature and can easily encapsulate both, hydrophilic and hydrophobic drugs. Upon injection in the human body, they undergo a phase transition forming a 3D hydrogel structure from which the API is release in a controlled and localized fashion. These formulations possess a tunable composition, thus allowing modulation of degradation rate and drug release kinetics. Preliminary results on API release from materials and their in vivo behavior will be presented.

Resume : Tissue engineering is an approach in generating a renewable source of transplantable tissues by using the principles of engineering, physical sciences and medicine. In most cases, cells are seeded on biodegradable materials that have been fabricated in the form of porous scaffolds. In particular, the research for a suitable scaffold which serves as a structural support and substrate for cell adhesion, differentiation and neo tissue genesis is now a fundamental topic. The current study is a comparative study about design and fabrication of TiO2 nanostructured-scaffold. TiO2 nanospheres, nanowires and nanotubes were synthesized and characterized by Scanning Electron Microscopy (SEM), X- ray diffraction (XRD) methods. In order to investigate the effect of morphology of TiO2 nanostructured-scaffold on the regeneration of urothelial cells, normal urothelial cells were cultured on TiO2 nanostructured-scaffold, with different surface morphology (such as TiO2 nanospheres, nanowires and nanotubes). After 2 weeks culture, morphological alteration and ultra-structures of cells grown on the surface of TiO2 nanostructured-scaffold were investigated by SEM. Result showed, for all TiO2 nanostructured-scaffold with different morphology, urothelial cells culture was confluent and the surface of TiO2 nanostructured-scaffold totally covered with the cells without any morphological alteration. On the other hand ultra-structures analysis revealed more details and difference about cells grown on the TiO2 nanostructured-scaffold with different morphology.

Resume : Cell and material interact through biochemical and biophysical signals, including matricellular cues, topography, mechanical and hydrophobicity. Details of the mechanisms by which cells recognize these signals at the interface may help in engineering novel materials able to control and direct specific cellular functions. Although a plethora of biochemical and biophysical signaling acting at the cell-material interface have been reported in the recent literature, the way to properly present them to elicit a given cell response still remains largely unknown. In this lecture the basic signals that control the dynamics of cell-material interface will be discussed along with the strategies to correctly display and deliver them to the cell. Example of cell-material communication and miscommunication will also be presented.

Resume : Recently, we demonstrated the possibility to obtain ultra-pure inorganic silicon nanoparticles (Si-NPs) by using ultrashort laser assisted method, making them promising biocompatible and biodegradable candidates for potential applications in the emerging nanomedicine field. However efforts still have to be employed in order to answer their fate once in contact with the biological matrix. To achieve this goal, Si-NPs were generated from raw silicon micropowder dispersed in milli-Q water, by using “green” laser femtosecond (fs) method. The obtained Si-NPs were separated from unreacted raw materials and recovered by successive centrifugation/filtration/ultra-centrifugation steps, and then characterized by TEM-XRD, XPS and DLS/Zeta potential methods. The results showed that Si-NPs appeared as crystallized Si with spherical shape ranging from 20 nm to 80 nm with a peak around 50 nm and surface charge close to -35 mV. The stability was followed by ICP-MS method and tested in water and classical RPMI cell culture medium. Safety/cytotoxicity properties were assessed on human microvascular endothelial cells (HMEC) by cell viability assay (MTT test). The first results showed that the material was stable up to 3 days under continuous stirring at 37 °C. Moreover, only 20 % decrease in cell survival was observed after 72 h of incubation up to 50 µg/mL. This encouraging work provides chances to adopt potentially widespread applications of these materials in nanomedicine.

Resume : The peptide fragments NGF(1-14) and BDNF(1-12) are able to mimic the activity respectively of nerve growth factor (NGF) and brain-derived growth factor (BDNF), neurotrophins with high therapeutic potentialities but with serious side effects and significant contra-indications, such as low serum stability and limited CNS penetration. We studied the immobilization of NGF(1-14) and BDNF(1-12) on gold nanoparticles through i) direct physisorption driven by environmental conditions (peptide concentration, pH, ionic strength) (pept/Au), and ii) adsorption mediated by a supported lipid bilayer (pept-SLB/Au). Physicochemical characterization was performed by studying plasmonic properties of the gold nanoparticles (UV-vis), hydrodynamic size (dynamic light scattering), surface charge (zeta potential), and topography changes (atomic force microscopy), both in adsorption as well as in desorption driven by copper ions addition and pH. In vitro live cell imaging experiments by confocal microscopy for the uptake into neuroblastoma and PC12 cells of peptides, pept/Au and pept-SLB/Au, in the presence or not of Cu2 , demonstrate the ionophore capability of the free peptides and the good premises of pept-SLB/Au as tunable nanoplatform in drug delivery application. Measurements are in progress to optimize the parameter of fabrication of the hybrid pept-SLB/Au system (lipid charge and composition) to efficiently control the cell response.

Resume : In recent years, nanotechnology has increasingly been demonstrating its great potential in overcoming the limitations of traditional strategies in cancer therapy. Latest advancements in material synthesis and characterization, as well as in targeting approaches, are indeed paving the way to the development of advanced nanovectors that can be accumulated and can selectively deliver therapeutic agents at a desired site of action. In this context, the exploitation of smart materials is promoting the development of actual theranostic and multifunctional nanosystems in an unprecedented way. In this talk, the ongoing research on cancer nanotechnology in our laboratory will be reviewed, with focus on lipid nanocarriers for strongly hydrophobic drugs and artificial anti-oxidants. We will introduce a nanoplatform for the targeting and release of sorafenib (a chemotherapeutic drug approved by FDA for treatment of liver and advanced renal carcinoma) based on magnetic solid lipid nanoparticles, and the use of cerium oxide nanoparticles as powerful inorganic anti-oxidants, exploitable for the treatment of a wide range of pathologies, including cancer. Our final goal is the development of a multi-functional active nanocarrier, able to attack tumour cells selectively and in a spatially controlled way by a combination of pharmacological and nanotechnology-based approaches.

Resume : Multi-shell fullerenes, also known as carbon nano-onions (CNOs) are structured by concentric shells of carbon atoms and display several unique properties, such as a large surface area to volume ratio, a low density and a graphitic multilayer morphology.1 In my research group we have developed a versatile and robust approach for the functionalisation of CNOs, involving the facile introduction of a number of simple functionalities onto their surface by treatment with in-situ generated diazonium compounds.2 Our results have shown that chemical functionalization of the CNOs dramatically enhance their solubility and attenuate their inflammatory properties. 3 Recently, we have developed novel intracellular imaging systems based on CNOs functionalised with BODIPY derivatives,4 with special attention to the biologically important near-infra red (NIR) region. CNOs functionalised with NIR emitting aza-BODIPY derivative showed good cellular uptake, and a pH dependent fluorescence on-off switching.5 We have also developed a synthetic multi-functionalisation strategy for the introduction of different functionalities (receptor targeting unit and imaging unit) onto the surface of the CNOs and we are now investigating the ability of these nanoconstructs to act as drug-delivery systems. [1] Beilstein J. Nanotechnol., 2014, 5, 1980. [2] Org. Lett. 2010, 12, 840. [3] Small, 2013, 9, 4194. [4] Nanoscale 2014, 66, 13761. [5] Mat. Chem. B, 2014, 2, 7459.

Resume : Recent studies have identified a new class of circulating biomarkers such as miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolar- specificity, and multiplexing. Furthermore, cost effectiveness, simplicity of procedures for handling, workflow and reading must be carefully considered in a context of point-of-care testing for large screening. Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in serum. Polyethyleneglycol-based microgels have a core-shell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10^-15 M and a dynamic range from 10^-9 to 10^-15 M, with higher accuracy than qRT-PCR. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement in serum of such microgels will translate into diagnostic benefits. Reference Causa F, Aliberti A, Cusano A, Battista E, Netti P. JAm ChemSoc 2015

Resume : Alkaline-earth fluoride nanoparticles (NPs) doped with lanthanide ions are interesting materials that can find use in various technological fields, in particular in biomedical diagnostics [1, 2]. In this contribution, we report on the preparation of water dispersible core-shell alkaline-earth fluoride (CaF2 and SrF2) NPs activated with the Gd3+ and Nd3+ ions and their possible use as multimodal MRI and luminescent markers for bioimaging in the biological windows. A simple and environmental friendly hydrothermal procedure has been developed to prepare the core-shell structure, which increases the luminescence properties by shielding the lanthanide ions from deactivation processes due to water. The NPs are capped with hydrophilic moieties to ensure good colloidal stability in water. Preliminary imaging results as well as heating experiments using near infrared radiation in the biological window let foresee the use of these NPs as both optical probes and nanothermometers. [1] M. Pedroni, F. Piccinelli, T. Passuello, S. Polizzi, J. Ueda, P. Haro-Gonzalez, L. M. Maestro, D. Jaque, J. Garcia-Sole, M. Bettinelli and A. Speghini, Cryst. Growth Des., 2013, 13, 4906. [2] N. N. Dong, M. Pedroni, F. Piccinelli, G. Conti, A. Sbarbati, J. E. Ramirez-Hernandez, L. M. Maestro, M. C. Iglesias-de la Cruz, F. Sanz-Rodriguez, A. Juarranz, F. Chen, F. Vetrone, J. A. Capobianco, J. G. Sole, M. Bettinelli, D. Jaque and A. Speghini, ACS Nano, 2011, 5, 8665.

Resume : During the last decades the frequency of tumour diseases in both animals and humans registered a continuous increase. A tremendous intensification of the research activity in the field has evolved to identify the aetiology factors and seek for new therapies aimed at reducing mortality and increasing chances to healing. One very common method to counteract extensive development of cancer tumours is surgery, by means of which malign tissues are either completely removed from the body or have their size reduced to prevent metastases. Achieving a clean surgical margin represents a technical challenge with important clinical implications. Raman spectroscopy that extracts chemical information, was reported to have a sensitivity of 100%, a specificity of 100%, and overall accuracy of 93% in identifying carcinomas. We have investigated ex vivo samples from 10 patients (female cats and female dogs) using a LABRAM HR 800 Horiba Jobin Yvon micro-Raman spectrometer. The excitation source was a 632 nm HeNe laser and the spectra were collected from 130 cm-1 to 4000 cm-1. To date were reported Raman experiments on human subjects using 830 nm and 785 nm excitation wavelengths that limit the range to 1800 cm-1. The spectra were associated to histopathology and anatomopathology analyses and also compared with spectra taken from healthy tissue coming from spaying surgery. Excellent resolution and no fluorescence background were obtained from measurements carried out on scalpel blades. Acknowledgment This work has been supported from PCCA 2013-Contract 20/2014