Nanotechnology for targeted personalized medicines and theranostics

Resume : The administration of nanoparticles to tumors followed by alternating magnetic field application was shown to efficiently destroy tumors both preclinically and clinically, especially glioma. However, antitumor efficacy remains suboptimal and requires further improvements. We therefore developed a new type of nanoparticles synthesized by magnetotactic bacteria called magnetosomes. Due to their chain arrangement that leads to uniform distribution, ferrimagnetic properties that enhance their heating power and to a controlled release of endotoxins that attract polynuclearneutrophiles, we show that chains of magnetosomes achieve full destruction of intracranial U87-Luc glioma tumors under AMF application in 40% of treated mice using a rather low quantity of magnetosomes administered of 13 µg of magnetosomes per mm3 of tumor. By contrast, under the same treatment conditions, signs of antitumor activity are not observed with chemically synthesized nanoparticles currently used in the magnetic hyperthermia treatment of tumors. It also appears that full glioma destruction is achieved when magnetosomes occupy only 10% of the whole tumor volume, which suggests the involvement of an indirect mechanism of tumor destruction, which is desired for the treatment of infiltrating tumors, such as glioma, for which whole tumor coverage by nanoparticles can hardly be achieved.

Resume : Nanotechnology offers the possibility to exploit and modulate materials’ properties at the nanoscale providing sustainable and effective cancer therapies.[1] Nowadays, combinatorial therapies are preferred to single therapies in the clinical treatments against cancer.[2] However, the development of nanotools gathering different therapeutic properties is still challenging. Here, we propose iron oxide nanocubes (NCs) as a possible platform for developing a multimodal strategy able to treat cancer.[3] The heating performances of the NCs (evaluated in term of SAR) allow their efficient application in hyperthermia treatment.[4] Here, the hyperthermia effect was enhanced by combining drug delivery and tumor targeting units.[5] The bioconjugation strategy chosen provided the efficient binding of a platinum-based drug and an antibody fragment. The biomolecules bound on the nanocubes were active against folate receptor α (FRα) and provided specificity for the desired cell lines. While the nanocubes were biocompatible, they expressed severe toxicity when functionalized with the platinum-derived PEG compound and when exposed to magnetic hyperthermia treatment. The nanocubes were able to kill the cancer cells in an in vitro model, inducing toxicity mediated by platinum release. The targeting moieties directed the toxicity specifically against the desired cell line. Moreover, hyperthermia further increased the nanocubes toxicity, inducing cell membrane damages. Thus, combining drug delivery, targeting and hyperthermia we developed a platform suitable for multi-therapeutic purposes against cancer cells. 1. Pelaz, B., et al., Diverse Applications of Nanomedicine. ACS Nano, 2017. 11(3): p. 2313-2381. 2. Mokhtari, R.B., et al., Combination therapy in combating cancer. Oncotarget, 2017. 8(23): p. 38022-38043. 3. Guardia, P., et al., Water-Soluble Iron Oxide Nanocubes with High Values of Specific Absorption Rate for Cancer Cell Hyperthermia Treatment. Acs Nano, 2012. 6(4): p. 3080-3091. 4. Guardia, P., et al., One pot synthesis of monodisperse water soluble iron oxide nanocrystals with high values of the specific absorption rate. Journal of Materials Chemistry B, 2014. 2(28): p. 4426-4434. 5. Quarta, A., et al., Targeting FR-expressing cells in ovarian cancer with Fab-functionalized nanoparticles: a full study to provide the proof of principle from in vitro to in vivo. Nanoscale, 2015. 7(6): p. 2336-51.

Resume : Magnetic nanoparticles under alternating magnetic field (AMF) can convert the electromagnetic energy into thermal energy which, in turn can be exploited for hyperthermia (HT) therapy (1). Besides Iron Oxide nanoparticles (IONPs) which are commonly used as heat hubs in magnetic hyperthermia, mixed ferrites formed from substituting few Fe2 ions of these IONPs with Manganese (Mn), Cobalt (Co) or Zinc (Zn) ions are also promising candidates for such magnetic HT treatment due to their improved magnetic properties. Cobalt ferrite nanocubes have shown very high specific adsorption rate (SAR) values (a measure of their heat performances) at medically/clinically acceptable frequency and field conditions (2). However, the use of Co-based nanoparticles for biomedical applications is still under debate. There are numerous evidences of intrinsic toxicity of CoFe2O4 NPs in cellular studies, with a toxicity profile being highly dependent on the cell types and concentration tested (3). Due to the proven in vitro cytotoxicity, not much exploration has been made on cobalt-based nanoparticles in in-vivo conditions. We synthesized 17nm cobalt ferrite nanocubes (CoFe2O4), by using thermal decomposition method, which showed improved SAR values at lower field and frequency conditions compared to standard IONPs of similar size and shape (2). Here, on a xenograft murine mice model, we demonstrate the synergic effects of cobalt ferrite nanocubes as magnetic hyperthermia inducing agents and intrinsic toxic agent with controlled Co ion release. The intra-tumoral injection gave us the control, by specifically inducing toxicity only at to tumor mass and avoiding uncontrolled toxicity to other vital organs. The animals treated with the combination of cobalt-ferrite injection and magnetic hyperthermia showed complete reduction in tumor volume as compared to untreated control and other groups studied (Cobalt ferrite without HT, IONPs with and without HT). Furthermore, the same group showed the longest survival of up to 200 days post treatment. The survived animals showed no signs of pain or distress throughout the whole life span. Histopathological, TEM and elemental analysis were performed on tumor and organs such as kidney, liver and spleen to study in detail the fate of these nanoparticles in vivo. With the results obtained in our study, we demonstrated that the intrinsic toxicity of the Co ions in the cobalt ferrite nanocubes, when exploited in a controlled environment, can replace the usage of other chemotherapeutic drugs and act as a self-standing multi-modal therapy, all working under lower dosage and acceptable hyperthermia limits. Reference: 1. Klaus Maier-Hauff et al., Intracranial Thermotherapy using Magnetic Nanoparticles Combined with External Beam Radiotherapy: Results of a Feasibility Study on Patients with Glioblastoma Multiforme. Journal of Neuro-Oncology, 2007, 81 (1), pp 53?60. 2. Ayyappan Sathya et al., CoxFe3?xO4 Nanocubes for Theranostic Applications: Effect of Cobalt Content and Particle Size. Chem. Mater., 2016, 28 (6), pp 1769?1780. 3. Limor Horev-Azaria et al., "Predictive Toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods of knowledge discovery from data. Particle and Fibre Toxicology 2013, 10:32.

Resume : Early diagnosis and targeted-therapies against cancer are still in a preliminary stage. Currently, promising strategies are being developed that aim at combining diagnosis and therapy capabilities into clinically effective formulations. Magnetic hybrid nanocomposites (mNCs) are being explored to synergistically combine the modified bioactive release provided by the organic encapsulation and the intrinsic physico-chemical properties from the inorganic counterpart. In particular, this approach shows high promise for personalized treatment and real-time monitoring of cancer disease. In this context, we present the preparation of drug loaded magnetic nanocomposites through simple, versatile and scalable melt-emulsification methods. Obtained formulations were fully characterized and showed good multifunctional performance as T2-contrast agents in magnetic resonance imaging (MRI), heat generating sources in magnetic hyperthermia (MH) therapy, and responsive drug delivery vehicles. In vitro, ex vivo and in vivo studies were performed to preclinically validate the potential of these formulations as theranostic agents. Results suggest a synergistic thermo/chemo-therapeutic effect derived from heat generation and controlled drug delivery over cancer growth. The ability to simultaneously encapsulate drugs and magnetic nanoparticles enables an external control over the release profile of the drugs and opens the door to personalized cancer medicine through image-guided drug delivery.

Resume : The majority of studies evaluating the effects of different surgical procedures aimed at defect fill with bone grafts and only employed clinical outcome measures, such as probing pocket depth, probing attachment level, radiological analysis and direct visualization, following surgical re-entry procedures. Such approaches did not facilitate the determination of true bone regeneration, an outcome that requires histologic investigation. A non-invasive and quick method for evaluation of chemical compounds from bone tissues is requested. We suggest a new method, based on the Raman spectroscopy. This non-destructive optical method is able to characterize and differentiate initial normal cortical bone, initial augmentation material and final regenerated bone. Regarding our study, for harvested bone samples were selected 2 patients, before and after maxillary - sinus lift augmentation procedure (cerabone material as bone substitute was used). The healing period was approximatively 8 months for both patients. Bioethitical approval was obtained. Raman Spectroscopy was performed respecting same geometrical conditions for data recording. Corresponding spectra were acquired before and after surgical augmentation procedure. Differences in peaks intensity on raw spectra reflect the differences in the quantities of the chemical components (related to specimens concentration) for investigated specimens. Sensitive information obtained from the Raman spectra, were compared with the histological results (collagen matrix / quantity / bone substitute integration). For both patients? bone samples, higher PPi peak intensities were obtained before treatment (73.04 % - patient #1 and 81.22 % - patient #2; highest value recorded for patient #2 with previous periodontal problems) and lower values after treatment (48.76% - patient #1 and 38.39% ? patient #2). PPi is known acting as a potent inhibitor of HAP crystals precipitation (biological mineralization), aspect that might causes periodontal disease. From histology investigation, morphometric results for ratio areas (bone tissue / implant material) are: 1.6067 - patient #1 and 0.6970 ? patient #2. Histological results confirm Raman evaluation of bone samples. Raman technique is capable to offer a complete bone evaluation (qualitative / quantitative), in the meantime being an independent method.

Resume : One of the major challenges in nanomedical applications is to control the biodistribution of nanoscaled drugs and their delivery of cargo. The deficiency of execution of these nanocarriers is often caused by immediate absorption of proteins onto NPs in physiological environments such as the blood flow. The formation of this so called protein-corona often leads to altered bio-physical properties of the NPs. Therefore, the development of stealth properties for the NP surface to sterically stabilize the particles and to reduce the adsorption of proteins is of utmost interest. Coating of NPs with the mostly established hydrophilic polymer poly(ethylene glycol) (PEG) but also with auspicious novel polymers like poly(2-ethyl-2-oxazoline) (PEtOx) are considered as promising approaches for targeted drug delivery. Therefore, there is a special need for characterization methods that prove stealth properties of newly modified particles. Protein adsorption onto NP can be easily detected by gel electrophoresis. Cellular association of fluorescently-labeled NP can be analyzed by fluorescent microscopy but furthermore it can be optimally detected even by high-throughput fluorescence microscopy such as the Cellomics ArrayScan® Imaging Platform. Application of this automated high-throughput microscopy based approach allows a reliable and reproducible assessment of NP association. Currently, automatically capturing and analyzing of fluorescent images of thousands of cells has made fluorescence microscopy an additional tool applicable for investigating nanobiology. The presented assay principle may also be adapted for a variety of other impacts of NPs onto cells to not only investigate NP association in living cells, but also to identify effects on toxicity and cell viability.

Resume : Rapidly emerging research and clinical studies have shown promising anti-tumor effects of Natural-Killer (NK) cell-based immunotherapy. NK cells are granular lymphocytes that play the key role the innate immune system, and are able to eliminate cancer and viral cells. NK cells express activating receptors that bind their cognate ligands on the target cell surface and regulate the NK cytotoxicity. These ligands are upregulated in cancer and virus-infected cells, yet, they can be also expressed in certain amount by healthy tissues. The cytotoxicity of NK cells is managed by the signaling balance of activating, costimulatory, and inhibitory receptors, and the repertoire of different ligands expressed on the membrane of target cell determines whether it will be attacked or tolerated. Yet, given different levels of expression of NK activating ligands on infected, transformed, and activated immune cells, as well as on several healthy cells, it can be hypothesized that such level of expression alone might determine the cytotoxic activity of NK cells, and that there could be minimal requirements for the expression of activation ligands to stimulate the cytotoxic response of NK cells. To elucidate this role of the composition and spatial distribution of activating ligands in NK cell cytotoxicity, we engineered a nanochip for the controlled activation of human NK cells. The nanochip contained nanopatterned matrices of MHS class I polypeptide related sequence A (MICA) ligands that recognize NKG2D activating receptors in NK cells . The spatial distribution of the ligands was systematically tuned at the nanoscale to encode the receptor clustering in the cell membrane. Each matrix was designed to provide an isolated microenvironment for NK cell activation, in which the MICA spatial distribution was regulated independently of the other matrices. To enable such delicate positioning of ligands, we immobilized them onto nanopatterned metallic nanodots (Fig. 2), thereby creating synthetic vacancies for the recognition by discrete transmembrane receptors. The size of each nanodot is about 10 nm to ensure anchoring of individual receptors. We fabricated the nanodots by the previously reported by us process based on nanoimprint lithography and angle-evaporation shadow masking, and functionalized them nanodots with thiols terminated with Ni-chelated Nitrilotriacetic acid, following the attachment of histidine-conjugated MICA. We verified the selectivity of our functionalization by immunofluorescent staining of immobilized MICA with fluorophore-conjugated antibody, followed by the fluorescent imaging on the arrays on the chip. Using our biochips, we monitored the activation of NK cell in several microenvironments, which were different from each other by spatial distribution of MICA. We found that the average area of the spread NK cell was dependent on the density of MICA, and reached saturation for the matrices with the unit cells of 100 nm or below, corresponding to eh density of 1000 ligated nanodots per square micron. Furthermore, we assessed the degree of NK cell activation by fluorescent imaging of lysosomal-associated membrane protein CD107a, which is a commonly used functional marker for NK cell cytotoxic activity. We found, that whereas MICA density barely influences the average amount of CD107a per cell, it regulates the average probability of whether a cell will be activated or not, with the saturation threshold of 1000 dots per square . Our study provides an important insight on the spatial mechanism of the cytotoxic activity of NK cells, by establishing the ligand distribution within the 100 nm length-scale as a critical barrier for the formation of immune synapse and degranulation. This understanding paves the way to rationally designed immunotherapeutic approaches employing unique NK cytotoxicity against human malignancies.

Resume : Rising criticism of currently available contrast agents for magnetic resonance imaging evoked the need for safer and more versatile agents. In the present study, we demonstrate the suitability of novel dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) for biomedical applications in terms of safety and biocompatibility. We investigated the size-dependent crosslinking process of these particles, as well as the size-dependency of their imaging properties. For the latter purpose, we adopted a simple and easy-to-perform experiment to estimate the relaxivity of the particles. Furthermore, we performed an extensive analysis of particles? storage stability under different temperature conditions, showing their superb stability and the lack of any signs of agglomeration or sedimentation during 12 weeks. Independent of their size, SPIONDex displayed no irritation potential in a chick chorioallantoic membrane assay. Cell uptake studies of ultra-small (30 nm) SPIONDex confirmed their internalization by macrophages, but not by non-phagocytic cells. Additionally, CARPA experiments in pigs treated with SPIONDex indicated the absence of hypersensitivity reactions. These results emphasize the exceptional safety of SPIONDex, setting them apart from the existing SPION-based contrast agents, and making them a very promising candidate for further clinical development.

Resume : Hydrodynamic analysis of bio-fluidic systems such as blood vessels, lymphatic glands, and mucous membranes is a challenging but essential task to treat circulatory and respiratory problems (e.g. thrombosis, lymphedema, asthma). In many cases, the most relevant hydrodynamic parameter linked to the pathological phenomena is the local shear stress and its distribution in microscale. However, no direct method for local shear measurement has existed so far. We developed an innovative technique, using rare-earth doped nanorods (LaPO4:Eu), that permits instant measurement of local shear stress with the spatial resolution equivalent to that of the state-of-the-art confocal microscopy [1]. The idea is based on the spontaneous (and ubiquitous) orientation of colloidal nanorods under flow. The peculiarly polarized luminescence of the rare-earth dopants allows to measure the degree of nanorods orientation which is strongly correlated with the local shear stress. In contrast to the particle imaging velocimetry (PIV) – conventional technique tracking suspended particles – our new method instantly measures the collective orientation of many small nanorods inside the focal volume, which is a key towards its unprecedented dynamic and resolution ranges especially useful for studying non-stationary flows. Based on this approach, we demonstrate a 3D tomographic mapping of shear profiles in microfluidic channels with a complex geometry [1]. [1] J Kim* et al. “Monitoring the orientation of rare-earth doped nanorods for flow shear tomography” Nature Nanotechnology. 12, 914-919 (2017)

Resume : In this study, a controlled drug release platform from natural amyloid-like (AL) protein, bovine serum albumin (AL-BSA) with ampicillin sodium salt (ASS) was developed. Towards accomplishing this target, 5%, 10% and 20% (w/w) ratios of ASS:AL-BSA blends were performed in an electrospinning system corresponding to ultrathin homogeneous nanofibers with an average diameter of 132±69 nm, 159±60 nm and 179±42 nm, respectively. Fourier transform infrared spectroscopy demonstrated that AL-BSA could capable of entrapping large amount of drug inside of nanofibers, which was attributed to the antimicrobial activity of the released drug against Escherichia coli and Staphylococcus aureus. Amount of released drug was measured by using UV-VIS spectrophotometer. The nanofibrous matrix of the electrospun membrane was shown a controlled release behavior of the drug for all ratios of ASS:AL-BSA. The transport mechanism was Fickian for the low ratio of ASS:AL-BSA (5% w/w). However, non-Fickian transport mechanism was observed at high ratios of ASS:BSA (>10% w/w) which was attributed to relatively longer path to the fiber surface due to the larger fiber diameter and non-homogeneous distribution of drug in the matrix as well as high standard deviation of nanofiber distribution. The results were demonstrated that single electrospinning of hydrophilic drugs with natural polymers could potentially be used for controlled release. The core and shell formation of electrospun nanofibers with the same kind of matrices and drugs are still under investigation.

Resume : Organothiolate-protected gold nanoclusters (AuNCs), with precise chemical composition, water solubility and ability to be conjugated to organic materials, were recently developed. Such AuNCs are attractive materials for biomedical applications, because they can be prepared at uniform small sizes allowing diffusion into the intracellular compartment of living cells. Our aim is to produce AuNC-antibody derivatives and to explore their potential as electron microscopy probes for studying the intracellular machinery in a living context. We selected an organothiolate protected AuNC with the chemical formula Au102(SR)44 and a monoclonal antibody directed against the nuclear RNA polII. We then evaluated two routes enabling the conjugation of Au102(SR)44 to unmasked thiols of the antibody hinge area. In one route, the nanoclusters are conjugated by direct ligand exchange to a reduced antibody followed by passivation of the unexchanged SR-ligands. In a second route, the AuNC was first fully reacted with functionalized elements and then conjugated to the antibody. The described conjugation routes will be compared in terms of their synthetic efficiency, as well as in terms of the probe’s ability to selectively bind the intracellular target. This study will provide valuable ground for the development of antibody-gold nanomaterials for biomedical research and particularly for selective targeting and imaging of proteins in the dense and crowded cellular environment.

Resume : Porous silicon (pSi) is a photoluminescent material produced from crystalline silicon wafers by anodization etching in HF solution. They are a good candidate for Nanotheranostics, because they are biodegradable, biocompatible, inert and do not activate the immune response. Their porosity and photoluminescence (PL) make them a very promising platform for drug delivery and bioimaging. To preserve PL quenching in aqueous media, we deposited TiO2 onto their porous surface by ALD in a rotary reactor, to get a uniform thin coating layer. We obtained PL stabilization for almost one year, with an initial small blue shift of the PL, due to the interaction of the coated (pSi-TiO2) microparticles with the aqueous media. By in-vitro tests the pSi-TiO2 microparticles were internalized by the human dendritic cells (DCs), maintaining the PL, as confirmed by two photon absorption experiment in vitro. We observed no decrease of DCs viability, but a priming effect upon co-stimulation with LPS, used as standard immune response activator. These promising results of PL stability and compatibility with human DCs open the way for future developments in the Nanotheranostics and to produce a complete platform for drug delivery and bioimaging.

Resume : Introduction Recent advancements in nanotechnology now also set the stage for improvements in the clinics. CTCs are considered to be of high clinical relevance to diagnose disease and monitor treatment. The molecular profiling of CTCs may allow to uncover novel cancer pathways. However, current CTC detection methods are labor and cost intensive, precluding the analysis of CTCs in large patient cohorts. Methods and Results To overcome these limitations, we are developing miniaturized immuno-nanoparticle-based magnetic flow cytometry chips, allowing the swift and low cost detection of CTCs in blood. We show that magnetic biosensing offers key advantages, a direct electronic read-out, and the option to apply the magnetic cell enrichment directly for cell detection. We will present proof of concept data demonstrating the feasibility of magnetic flow cytometry. However, we found that performance of the technology is dependent of the quality of the used antibody-armed ‘intelligent‘ nanoparticles and plasma proteins adsorbing to nanoprobes. Conclusions Collectively, we report that nanoparticle-based magnetic flow cytometry is highly promising for the further development into an easy to use ‘point of care’ diagnostics, allowing the routine detection of CTCs in HNSCC patients’ blood samples. Applying this technology, prospective (multicenter) clinical studies are needed to clarify the value of CTCs as a surrogate clinical marker for HNSCC.

Resume : Recently, magnetic nanostructures (MNs) obtained a lot of interest in cancer treatment, for hyperthermia therapies and induced cell apoptosis with cell membrane mechanical stimulation [1]. Focusing on hyperthermia applications, when an AC magnetic field is applied to an ensemble of MNs dispersed in a tissue, different physical phenomena can concur to the heat generation, e.g. Néel relaxation, Brownian relaxation, hysteresis and eddy current losses. The relative contribution strongly depends on the size and physical properties of the used MNs [1,2]. Here, we present a micromagnetic modeling analysis and a comparison to experimental data, focusing on Ni80Fe20 nanodisks prepared via a self-assembling technique [3], for application in magnetically mediated hyperthermia. A parametric analysis is performed by varying disk diameter (100-700 nm) and thickness (15-30 nm), to find the optimal conditions for the maximization of the specific heating capabilities. We focus on hysteresis losses, being the predominant heating contribution for such nanosystems, calculating hysteresis loops by means of a GPU-parallelized micromagnetic code [4]. The influence of interdot magnetostatic interactions and relative orientation with the applied field is analyzed. [1] X. L. Liu et al., Adv. Mater. 27, 1939–1944 (2015) [2] A. E. Deatsch and B. A. Evans, J. Magn. Magn. Mater. 354, 163–172 (2014) [3] P. Tiberto et al., J. Appl. Phys. 117, (2015) [4] O. Bottauscio and A. Manzin, J. Appl. Phys. 115, 17D122 (2014)

Resume : Motivated by the functionality and purity demand, laser ablation in liquid ambiance (aqueous or organic solvent) has recently emerged as “green” method to chemical nanomaterial synthesis as it can avoid the use of toxic by-products. By the application of ultrafast laser beam on the interest workpiece (solid target or microparticles powder) material primarily introduced in aqueous medium it can be generated stable colloidal solutions which not contain any protective ligands and contaminated substance. The functionality of the method permits also to work with a great variety of inorganic, metallic and composite materials. Moreover the functionalization of the laser-synthesized with dispersant agent, biomolecules, etc., can be performed in situ during the elaboration process. In this contribution we review our recent results obtained on the elaboration of laser-synthesized nanoparticles based-on silicon (SiNPs) and their potential application as innovative tools in nanomedicine. The physicochemical structural analysis of the fabricated NPs will be reminded. Through systematic in vitro and in vivo tests, the interaction of SiNPs with biological matrix will be presented [1,2,3]. Their potential application as nanotheranostic tools will be discussed. Based-on on our first encouraging results, their incorporation as innovative additives into hybrid electrospun chitosan(PEO) nanofibers for tissue engineering will be also exhibited [4].[1]K. P. Tamarov et al, Sci. Rep., 2014, 4, 7034; [2]T. Baati et al, Sci. Rep., 2016, 6, 25400; [3]A. Al-Kattan et al, J. Mater. Chem. B, 2016, 4, 7852-7858; [4]A. Al-Kattan et al, RSC adv., 2017, 7, 31579-31766

Resume : In recent years, nanotechnology had a great impact in the development of novel drug delivery systems. Polymeric nanostructures gained attention as drug delivery systems due to their biocompatible and biodegradable nature, high drug loading efficiencies and well-controlled release mechanisms. Additionally, conductive and functional polymers facilitate triggered release of drugs. Polymeric nanotubes with their cylindrical shapes demonstrate increased anticancer activity due to longer time of residence in the body. In this study, conductive polymeric nanotubes are fabricated by oxidative chemical vapor deposition (oCVD) which is a vapor phase polymer deposition technique involving step-growth polymerization of a conjugated polymer. Drug loading capacities and controlled release mechanisms of these conductive nanotubes are demonstrated using a model drug under different electrical stimulation methods. Polymeric nanotubes are characterized using SEM, FTIR, ellipsometry, and the release performance is studied using UV-Vis spectroscopy, and 4-point probe conductivity measurements. Finally, to improve the control over drug release, coaxial nanotubes with outer layer of conductive polymer and inner layer of stimuli responsive polymer are prepared, and drug release kinetics is reported.

Resume : A family of histidine-rich peptides LAH4 was designed using linear cationic peptides such as magainins as a template. These designed peptides have been shown to exhibit considerable antimicrobial, nucleic acid transfection as well as cell penetrating activities. The delivery of siRNA and plasmid DNA has been shown highly efficient making the nanostructure complexes interesting for personalized medicine and gene therapeutic approaches. In contrast to their natural templates the membrane interactions of LAH4 peptides are strongly pH dependent. The delivery of cargo by these peptides is complex, involving many steps, which we investigated on a structural and biophysical level. Recently, vectofusin-1, a member of the family of LAH4 peptides has been shown to spontaneously self-assemble into helical coiled-coil structures, spherical aggregates, that further assemble into annular and extended nanofibrils and hydrogels as a function of phosphate and in a pH-dependent manner. This bears considerable interest for the design of biomaterials. Furthermore, the peptide has a strong capacity to enhance the gene transfer by lenti- and adeno associated viruses into the cell interior. Thereby, the fibers formed by this relatively short have therapeutic applications ranging from monogenic and infectious diseases to cancer, by enhancing transduction levels of target cells and reducing the amount of lentivirus for greater safety and reduced costs. Vectofusin-1 promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium, without cytotoxicity. These associate with viral particles allowing them to be easily pelleted. These fibrils have a unique coiled-coil α-helical structure whereas most other viral transduction enhancers form β-amyloid fibrils. Our observations define vectofusin-1 as a member of a new class of α-helical lentiviral transduction enhancers. Its coiled-coil fibril formation is reversible which bears considerable advantages in handling the peptide in conditions well-adapted to gene therapy protocols. References: J. Pep. Sci 21, 346 (2015); J. Phys. Chem. B 119, 9678 (2015); J Biol. Chem., 291, 2161 (2016); Acta Biomat.,64, 259 (2017); Biophys. J. , 113, 2327 (2017)

Resume : Endocytosis is an efficient mechanism to transport macromolecules across cell membranes by taking the polymer in a vesicle. There are two main strategies for delivery vesicles transporting drug. The drug can be encapsulated inside micelles [1][2], like viral vectors [2]. Alternatively, the drug can be transported using polymer-drug conjugates [3]. The drug delivery molecules like peptides are non-viral vectors, which are cheaper and safer, and they can be made in large amount. In the research from Chen et al [4], [6], [7], pseudopeptidic polymers are studied. The pseudopeptides are synthesized by repeating the monomer unit lysine isophthlalamide and form the polymers. Poly (L-lysine isophthalamide) derivatives are one class of anionic polymers, which possess biodegradable amide bonds. This feature can help polymers avoiding alternatives which are non-biodegradable [5]. The essential feature for these pseudopeptidic polymers is pH-activated disruption ability [4][6][7]. When polymers entering the cell, the pH environment is changed from physiological value to endosomal value. The carboxylate groups along the linear backbone of the polymers change from deprotonated at physiological pH values to protonated endosomal values [4]. The membrane will not be disrupted in a physiological environment because of the repulsions between the negative charges. However, when the pH values drop, the negatively charged polymers are protonated, and hydrophobic interactions lead to conformation change [4][6][7]. Subsequently, the polymer disrupts the vesicle membrane before the endosome being a lysosome and could go freely in the cytoplasmic. Actually, the intracellular delivery of biomacromolecules is generally limited, because the cargos could be degraded by the enzymes during the trafficking process from endosomes to lysosomes [4]. A high pH value will interrupt the endocytosis process, and the polymers cannot enter the cell. The low pH could lead that the polymers are delivered to lysosomes and they will not go to the targets. Therefore, the pH value must be modulated carefully. Based on the research of pseudopeptides, hyperbranched poly (L-lysine isophthalamide) [9]and membrane-anchoring polymers [10] are studied. The poly (L-lysine isophthalamide) grafted hydrophobic amino acids or alkyl chains could control its pH-responsive change of conformation and cell membranelytic ability. Hyperbranched topology is introduced to increase the cell membrane penetrating ability. The HPLPs shows the high level of haemolytic activity [9]. HPLPs are the novel cell penetrating peptides and they could be the candidate of drug delivery. The comb-like pseudopeptides [10] are produced by grafting hydrophobic decylamine onto the carboxylic acid groups of the poly (L-lysine isophthalamide). The linear anionic pseudopeptidic polymer shows low cell membrane rupturing ability at 4.5-5.0 pH [4] because of the absence of the aliphatic chains in membrane proteins. By grafting NDA at different degrees of substitution [10], the conformation and haemolytic activity could be changed. The best polymer PLP-NDA 18% [10] displays excellent membrane disruption ability at pH 5.5. The comb-like polymers also have great potential in drug delivery applications. The study of polymers in drug delivery applications is the quantification of the polymer-bilayer interactions. This information is generally difficult to access via experimental techniques. Therefore, computational method is applied. Molecular simulations of these drug delivery vehicles could reveal important information on the interaction between the anionic pseudopeptidic polymers and the cell membrane. Simulations can also provide useful data on elastic properties of lipid membranes. The elastic properties of the cell membrane are essential, which could tell how the transmembrane proteins work. In addition, the existence of thermal undulations controls the elastic behaviour of a fluid lipid bilayer. To describe the undulations, the mechanical parameter, bending modulus, is used. The deformation of lipid bilayer vesicles depends on the bending rigidity. The properties are defined by the fluctuations of membranes and described by using the Helfrich Hamiltonian and compressibility. Membrane bilayer simulation is important in modelling endocytosis. The 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) [11], [12] is used in the research due to the abundance in biological membrane. Molecular dynamics simulation is applied to investigate DPPC which can be constructed by using CG and the Martini force field [12]. In previous studies, Wang and Bresme [8] performed simulations of poly (L-lysine isophthalamide) and DPPC bilayers, to address the impact of phenylalanine content on the polymer-bilayer interactions. Force field parameters were developed that allow linking monomer units to simulate the polymers with a wide range of lengths and compositions [8]. It was found that fully protonated polymers favour the penetration process, which means that the membrane is lytic at endosomal pH [8]. A Markov State Model (MSM) was applied to analyse the polymers conformations next to the bilayer. The protonated grafted polymers have a short time for switching from the water phase conformation to that at the water-lipid interface [8]. The possible reason is that the rapid conformation transition capacity of the polymer is a kinetic factor [8]. The project will be carried out using experiments and computational modelling. This research aims to develop a new pseudopeptidic polymers in experiments and study its interactions with bilayers using Molecular Dynamics simulations. The development of new pseudopeptidic polymers will be of interest to scientists working on polymer synthesis and able to be the foundation of a new polymer. The computational modelling of lipid bilayers and the novel polymers will benefit researchers working on computer simulations of biomolecules and interested in area compressibility modulus of membranes and nanomaterial-biological molecule interactions. This project focuses on drug delivery applications. Industries working on drug delivery will benefit from this work inspired. Reference [1] Y. Huang, D. L. Wang, X. Y. Zhu, D. Y. Yan and R. J. Chen, Polym. Chem., 2015, 6, 2794-2812. [2] J. Y. Liu, W. Huang, Y. Pang, X. Y. Zhu, Y. F. Zhou and D. Y. Yan, Langmuir, 2010, 26, 10585-10592. [3] S. Khormaee, Y. Choi, M. J. Shen, B. Y. Xu, H. T. Wu, G. L. Griffiths, R. J. Chen, N. K. H. Slater and J. K. Park, Adv. Funct. Mater., 2013, 23, 565-574. [4] R. J. Chen, S. Khormaee, M. E. Eccleston and N. K. H. Slater, Biomacromolecules, 2009, 10, 2601-2608. [5] S. Khormaee, R. J. Chen, J. K. Park and N. K. H. Slater, J. Biomater. Sci., Polym. Ed., 2010, 21, 1573-1588. [6] R. J. Chen, S. Khormaee, M. E. Eccleston and N. K. H. Slater, Biomaterials, 2009, 30, 1954-1961. [7] R. J. Chen, M. E. Eccleston, Z. L. Yue and N. K. H. Slater, J. Mater. Chem., 2009, 19, 4217-4224. [8] S. Z. Wang and F. Bresme, J. Phys. Chem. B, 2017, 121, 9113-9125. [9] S. Q. Wang and R. J. Chen, Chem. Mater., 2017, 29, 5806-5815. [10] S. Y. Chen, S. Q. Wang, M. Kopytynski, M. Bachelet and R. J. Chen, ACS Appl. Mater. Interfaces, 2017, 9, 8021-8029. [11] D. Mohammand-Aghaie and F. Bresme, Mol. Simul., 2016, 42, 391-397. [12] Y. W. Zhang, J. W. Carter, A. Lervik, N. J. Brooks, J. M. Seddon and F. Bresme, Soft Matter, 2016, 12, 2108-2117.

Resume : Nanoparticles (NP) from various materials provide promising features for biomedical application as targeting carriers for drugs, genes or as imaging agents. Thanks to surface chemistry, zeta potential, and surface dipoles they can spontaneously adsorb molecules from their environment e.g. proteins, amino acids etc., which can modify properties of NP and even cause loss of specificity in targeting. Thus, attention should be paid to the influence of NP size and surface modification for controlling their biological activity. Detonation nanodiamonds (DND) are being investigated for medical applications thanks to their excellent biocompatibility, chemical and optical properties as well as relatively low-cost synthesis. However, molecular interactions with DNDs are still not fully understood. In this work, we thus characterize composition of protein corona on DNDs with different mean size (2 and 4 nm) and surface chemistry (hydrogen or oxygen groups). The experiments were focused on identification of the proteins adsorbed on the surface of DNDs in dependence on their size and surface chemistry. For this purpose DNDs were incubated in complete cell culture medium with serum, washed by several centrifugation steps, proteins were eluted and then separated by gel electrophoresis. The final protein composition was determined by mass spectrometry/LC-MS/MS. We identified set of proteins common for all types of DNDs, but also proteins unique for DND of different size or surface chemistry.

Resume : In cancer treatment, efficient delivery of active anticancer drugs into cancer cells is highly desirable for maximizing therapeutic effects and alleviating side effects. In this work, nanocarrier consisting of Fe3O4 core, polyglycerol coating, and octalysine functionality (SPION-PG-Lys8) has been designed, synthesized and used to deliver photosensitizer, chlorine e6 (Ce6), into cancer cells for photodynamic therapy (PDT) of cancer cells. SPION-PG-Lys8 is colloidally stable in various aqueous solutions, showing high positive zeta potential of 47.2 ± 6.9 mV in pure water. In vitro characterization reveals that SPION-PG-Lys8 is efficiently taken up by SKOV3 ovarian cancer cells, exhibiting low cytotoxicity, and suppressed autophagy compared to bare SPION. Negatively charged Ce6 is thus loaded on the SPION-PG-Lys8 through electrostatic attraction to yield SPION-PG-Lys8/Ce6 nanocomplex with positive zeta potential of 22.4 ± 4.3 mV. SPION-PG-Lys8/Ce6 is more easily taken up by the cells than free Ce6, and surprisingly, the internalized SPION-PG-Lys8/Ce6 is found to be enriched in the mitochondrial counterpart. SPION-PG-Lys8/Ce6 exhibits almost no cytotoxicity under dark conditions, but strong photocytotoxicity due to the light-triggered production of reactive oxygen species (ROS) destroying the mitochondria. Taken together, our results highlight the great potential of SPION-PG-Lys8 as efficient carrier of Ce6 for photodynamic cancer therapy.

Resume : In recent years gold nanorods (Au NRs) have attracted a great deal of attention due to their excellent optical properties demonstrated in the form of so called localized surface plasmon resonance. This phenomenon combined with biocompatibility of gold makes Au NRs a promising structure for potential use in biomedicine. Variations of nanoparticle properties are usually achieved by changing the shape, size or material of the particles but they can also be altered by creation of a proper shell around a nanoparticle. In this study we fabricated gold-silver and gold-silica (SiO2) core-shell nanorods. The Au-Ag NRs allow tuning the plasmon resonances much more than for bare Au NRs. On the other hand, silica shell is useful for increasing biocompatibility and for fabrication of more complex nanocomposites. Such composite nanoparticles can be used in many applications including theranostics (combining diagnostics and therapy in nanomedicine) or surface enhanced Raman spectroscopy (SERS). The study aims at optimizing the coating process, which is based on seeded growth method, in order to control the shape and thickness of the resultant shells.

Resume : Cancer is one of the dreadful diseases, which claimed many lives in the world. In general, cancer has been (i) diagnosed with X-ray imaging and computerized axial tomography (CAT) scan and (ii) subsequently treated with surgery, chemotherapy, and radiation therapy based on their stages/location. However, these conventional techniques have some serious implications including the exposure of high energy radiations and toxic chemicals, which are again very harmful to the patients and resulting in the severe side effects including organ failure, fatigue and infertility. One more major concern related to cancer is the difficulty in completely diagnosing their extended location and association with the nearby healthy cells for consequent application of suitable therapy. Recently, superparamagnetic iron oxide nanoparticles (SPIONs) - particularly magnetite (Fe3O4)/maghemite (γ-Fe2O3) - have gained a lot of research interest in biomedical applications including simultaneous cancer diagnosis (via magnetic resonance imaging – MRI – as T2 contrast agents – measured in terms of relaxivity) and therapy (via magnetic fluid hyperthermia (MFH) – as heat inducing agents – measured in terms of specific absorption rate – SAR/intrinsic loss power - ILP), since the SPIONs are biocompatible, biodegradable and non-toxic to healthy cells, and also show interesting magnetic properties such as superparamagnetism and high saturation magnetization. Moreover, the SPIONs have the capability to easily enter into leaky vasculatures of tumor tissues, which make the SPIONs based MRI and MFH techniques as highly efficient with very minimal side effects. In addition, the SPIONs are encapsulated along with/without chemotherapeutic drugs (CHDs) into polymeric micelles for multiple therapeutic applications. However, the imaging/heating efficacies (i.e., relaxivity/SAR) of the SPIONs in MRI/MFH based cancer biomedical applications depend majorly on their magnetic properties (for instance, saturation magnetization/anisotropy), which are significantly influenced by their physicochemical properties such as size/shape, surface coatings, and crystallinity. So, these physicochemical/magnetic properties of the SPIONs should be primarily tailored to achieve better relaxivity/SAR values. Moreover, in case of MFH, the concentrations, applied alternating magnetic fields (AMFs – within the biological safety limits to avoid any side effects) and dispersion media should also be optimized for attaining maximum heating effects in cancer treatments. For these purposes, we have initially synthesized, characterized and optimized the physicochemical/magnetic/colloidal properties of the following SPIONs: (i) single-core hydrophilic SPIONs via one-pot co-precipitation/thermolysis methods by using novel π-π conjugated surfactants (individual/combination) such as terephthalic acid (TA)/amino-terephthalic acid (ATA)/trimesic acid (TMA)/pyromellitic acid (PMA)/diaminobenzene (DAB)/amino-benzoic acid (ABA)/diamino-benzoic acid (DABA); (ii) multi-core hydrophilic SPIONs by using ethylene glycol (di-, tri- and tetra-) and ethanolamine (di- and tri-) through thermolysis method; and (iii) single-core hydrophobic SPIONs by using oleic acid (OA) and oleylamine (OM) via thermolysis method. In addition, the hydrophobic SPIONs (with/without CHDs) are encapsulated within the novel polymeric micelles (made of poly(lactic-co-glycolic acid/d-α-Tocopheryl polyethylene glycol 1000 succinate) to form multifunctional SPIONs, which are characterized and optimized for higher SPIONs/drug loading. Then, the as-synthesized single-/multi- core hydrophilic SPIONs and the polymeric micelles encapsulated hydrophobic SPIONs are involved in MRI and MFH experiments, where high relaxivity (up to 735.3 mM–1 s–1) and SAR/ILP (up to 800 W/gFe / 5.2 nHm2/kg) values are attained. Furthermore, these SPIONs have showed high diagnostic/therapeutic efficiencies in in vitro/in vivo cancer (breast/liver) applications.

Resume : Over the last few decades, population aging has become a critical issue and highlighted the well-being of elderly people. As getting older, people are more likely to suffer age-associated disease and those disease can be a great threat to their life expectancy as well as quality of life. Parkinson’s disease (PD), the second most common age related neurodegenerative disease, affects 6.2 million people and was responsible for 117,400 global deaths in 2015. The symptoms of PD includes shaking, rigidity, slowness of movement, difficulty with walking and emotional problems. Currently, the PD management strategy involves delaying its progression while providing relief for symptoms as there’s no available treatment for halting the progression of PD. Once diagnosed, the average life expectancy of patients is between 7 and 14 years. This indicates the earlier PD is diagnosed, the more effective later management can be. However, clinical diagnosis criteria of PD at present is highly dependent on symptoms , by the time at which 40% to 60% of dopaminergic neurons have been destroyed irreversibly and highly restricts the effectiveness of relevant treatment. α-synuclein, a small (~14kDa) presynaptic nerve terminal protein, has been reported closely related to PD and regarded a biomarker in its oligomeric state. Toxic oligomeric pathways include impairment of proteostasis, chronic endoplasmic reticulum (ER) stress, pore formation, and glutamate receptor dysfunction. The monitoring on them can help assessing the risk of having PD even before symptoms are noticed. An effective α-synuclein oligomer detection technique is introduced in this work, which is capable to monitor α-synuclein oligomer level in biological fluids.

Resume : The visualization of mesoscopic structures in cytoplasmic space has been drawing increasing attentions in biology, such as entry of nano-objects (like virus particles) into cell nucleus. We present our recent study aiming to visualize the surface topography and internal structures of frozen-hydrated cells using advanced X-ray microscopy. As the target system, we selected human erythrocytes before and after malaria infection. The surface topography was obtained with lensless coherent X-ray diffraction imaging, unraveling the protein "knobs" expressed on malaria-infected erythrocytes. The hemozoin crystals in a parasite vacuole in an infected cell can be visualized by the combination of 3D X-ray phase tomography and scanning-X-ray microscopy. The use of nano-focused beam enables to localize the location and amount of specific elements, such as K, Ca, S, P, and Fe, with the spatial accuracy of 25 nm. We will present the biomedical relevance of such label-free X-ray imaging techniques and discuss about the potential applications towards visualizing nano-materials in frozen-hydrated human cells.

Resume : Last decades have witnessed a rapid development of living radical polymerizations (LRP) since these techniques enable the synthesis of polymers with unprecedented control over molar mass and architecture along with a very narrow dispersity thus, providing the materials with several intriguing properties. Among those polymerizations, Cu-based Reversible-Deactivation Radical Polymerization (Cu-RDRP), also known as Copper Mediated Radical Polymerization or Atom Transfer Radical Polymerization (ATRP), has been demonstrated as one of the most versatile LRP technique due to the ease to handling and commercial availability of copper catalysts and initiators. Recently, it has been shown that a Cu-RDRP process can be accelerated by the assistance of an ultra-violet source leading to an extremely fast polymerization rate without sacrificing the controlled features of classical Cu-RDRP.1 Interestingly, such approach employs a much more stable catalyst (Cu2+) while offering the possibilities to reduce catalyst amount significantly, which made it be particularly interesting for the preparation of biomaterials. In our laboratory, we demonstrated the applicability of this polymerization technique to prepare several functional co-polymers nanohybrids with stimuli-responsive features, which hold a great potential to be found for biomedical applications. 1. A. Anastasaki et al, J. Am. Chem. Soc., 2014, 136 (3), pp 1141–1149.

Resume : Nanomaterials have been the subject of enormous interest and are at the leading edge of the rapidly developing field of nanotechnology. The two key divisions of medicine, aimed at maintaining the good health of people, are diagnostics and therapeutics. Superparamagnetic iron oxide nanoparticles (SPION) thanks to their unique magnetic properties may successfully link these two medical fields, simultaneously increasing the effectiveness of treatment (e.g. hyperthermia) and improving diagnostic capabilities of magnetic resonance imaging (e.g. MRI). In this report we present the synthesis and physicochemical characterization of superparamagnetic nanoparticles based on iron oxide doped with zinc (SPION-Zn) and modified with cationic derivative of chitosan (CCh) that make them effective agents for anticancer magnetic hyperthermia. SPION-CCh-Zn were synthesized and characterized physicochemically using i.a. transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS) and measurements of zeta potential. Under transmission electronic microscopy, the obtained zinc ferrite nanoparticles were spherical-shaped and monodispersed with a diameter of ~ 10 nm. The SPION-CCh-Zn sample showed a sponge-like morphology with a few nanoparticles embedded in the chitosan matrix. SAXS analysis reveals a lognormal type of size distribution for the iron oxide cores of the particles. Their mean radii are 10.6 nm and 35.9 nm, respectively for the iron oxide core and their aggregates coated by chitosan. The average hydrodynamic radius of the SPION-CCh-Zn was 107.1 nm. In this research we also determined, using XPS. the experimental formulas for each sample and compare them with the theoretical formulas to confirm the amount of zinc incorporated to the structure of the nanoparticles. It was found that the obtained nanoparticles form stable suspensions thanks to the presence of charged polymer coating preserving superparamagnetic properties (the zeta potential value equals to 40 mV). Sedimentation of SPION-CCh-Zn suspensions was not observed even after 6 months of storage at 4ºC, indicating high stability of such suspensions. Magnetic measurements at room temperature showed the prepared sample was superparamagnetic that was also confirmed by Mössbauer spectroscopy. The heating ability of all the obtained hybrid materials was also studied. The temperatures achieved in the hyperthermic experiments exceeded 42°C allowing to conclude that the obtained SPION-CCh-Zn are promising materials for anticancer treatment using magnetic hyperthermia.

Resume : Antibodies can activate a variety of immune cells. This leads to a massive infiltration of activated immune cells into the tumor and lymph nodes. Perfluorocarbones (PFC) which are taken up primarily by monocytes in vivo after intravenous injection can serve as a tracer for immune cell tracking. Imaging was performed on a 9.4 T Bruker animal scanner (Bruker, Ettlingen, Germany). 50 nl of the PFC was placed next to the tumor in a vial as a reference and the 19F-MRI signal intensities were normalized to the reference. A subcutaneous tumor model was generated in mice. Five animals were treated with vehicle, 10 animals were treated with a combination of anti-PD-L1 and anti-CD40 antibodies to stimulate the immune system. Three days after treatment all animals received the contrast reagent PFC (120µl i.v.). Five days after treatment animals were analyzed by MRI. We found a significant difference in the mean normalized signal intensity in tumor between the two groups indicating differences in immune cell infiltration after antibody treatment compared to control in s.c. tumor models in immune competent mice using PFCs as tracer. Analysis of tumor, liver and spleen showed a large difference in the spleen volume between the two groups while there was no significant difference in the spleen/reference intensity or the liver/reference intensity values. Tumor volume was except of one case smaller in the treated group. In conclusion a 19F-MRI surface coil was home built and successfully tested at a small bore animal scanner. In vitro and in vivo test scans using phantoms and mice were performed to test and optimize the MR-protocol. The results showed that it is possible to detect dynamic changes in the migratory pattern and activation of immune cells in tumor models undergoing immune therapy.

Resume : One of the major challenges in nanomedical applications is to control the biodistribution of nanoscaled drugs and their delivery of cargo. The deficiency of execution of these nanocarriers is often caused by immediate absorption of proteins onto NPs in physiological environments such as the blood flow. The formation of this so called protein-corona often leads to altered bio-physical properties of the NPs. Therefore, the development of stealth properties for the NP surface to sterically stabilize the particles and to reduce the adsorption of proteins is of utmost interest. Coating of NPs with the mostly established hydrophilic polymer poly(ethylene glycol) (PEG) but also with auspicious novel polymers like poly(2-ethyl-2-oxazoline) (PEtOx) are considered as promising approaches for targeted drug delivery. Therefore, there is a special need for characterization methods that prove stealth properties of newly modified particles. Protein adsorption onto NP can be easily detected by gel electrophoresis. Cellular association of fluorescently-labeled NP can be analyzed by fluorescent microscopy but furthermore it can be optimally detected even by (high-throughput) microscopy, including novel in vivo imaging techniques. The presented approaches will be useful for a variety of biomedical and biotechnological applications of innovative nanotechnology.

Resume : to be announced

Resume : Biodegradable implants such as vascular grafts are new promising therapeutic biomedical substitutes to restore the functions of diseased organs. In order to monitor the location as well as the postoperative degradation degree using magnetic resonance imaging (MRI), magnetic nanoparticles (MNP) were incorporated in the material of the implants and used as contrast agents. The visualization of the degradation process is linked to the question of how the immobilized and clustered MNP in the implant change the MRI signal. For the assessment of these changes, the behaviour of MNP incorporated in polylactic-co-glycolic acid (PLGA) implants as well as of MNP dispersed in different phantoms were analysed with MRI. The phantoms consisted of either free dispersed or immobilized MNP with different sizes and clusters to mimic their immobilization in PLGA implants during the degradation process. Various degrees of MNP immobilization were realized by incorporating them in hydrogels with different meshes sizes. T2- and T2* relaxation time maps of the implants and phantoms were determined to illustrate the local susceptibility differences in each pixel in the cross section of the implants and phantoms. To conclude, the relaxation times of both phantoms and implants were clearly dependent on MNP size, clustering and the degree of immobilization. These results demonstrate the feasibility of quantitative imaging of the degradation degree of implants in vivo with MRI.

Resume : Designing biocompatible polymer-based nanoparticles has become central in the field of theranostic nanoparticles. A current challenge is the development of simplified approaches with improved properties compared with existing methods in terms of biodegradability, toxicity and processing. Currently, there are very few methods allowing the efficient synthesis of particles made of biomacromolecules especially proteins. In coll. with the Univ. of Melbourne (Prof. F. Caruso), we pioneered an original approach using isobutyramide (IBAM) grafts to assemble non-covalently, protein-based hollow capsules and particles without the need of an additional cross-linking or other adjuvant. The process consists in a single adsorption of proteins onto silica templates prealably grafted with IBAM groups or derivatives (e.g., bromoisobutyramide, BrIBAM) followed by template removal[1]. The driving force is attributed to strong H-bonds between the IBAM interface and the polypeptide chains of the proteins. We applied this method to design bioresponsive hollow capsules and particles made of a range of proteins, including enzymes, insulin and human serum albumin.[2] Furthermore, such carriers were shown to release chemotherapeutic drugs upon biological stimuli e.g. through protease degradation or reductive mimetic cytosolic conditions.[3] This approach was also demonstrated for the design of ca. 100 nm size multifunctional protein-based NPs displaying simultaneously delivery of silencing RNA (siRNA) to cancer cells and magnetic resonance imaging (MRI) by grafting gadolinium complexes.[4] In recent works, we translated this innovative protein nanocoating approach for the design of novel hybrid nanoplatforms made of magnetic cores covered with a mesoporous silica shell. Our aim was the design of new systems for MRI [5], magnetic hyperthermia and drug delivery ensured by alternating magnetic field [6]. The nanoplatforms were loaded with antitumoral agents (doxorubicin), and covered by a tight HSA shell to ensure biocompatibility, stealthiness, biodegradability and efficient encapsulation of DOX. The efficient drug release of such HSA-coated core-shell NPs theranostic NPs in protease media mimicking intracellular lysosomes was shown via enzymatic HSA shell biodegradation.[7] The approach was also translated to carbon nanotubes as core systems for drug release response upon NIR light.[8,9] [1] D. Mertz, P. Tan, Y. Wang, T. K. Goh, A. Blencowe, F. Caruso, Adv. Mater. 2011, 23, 5668-5673. [2] D. Mertz, J. Cui, Y. Yan, G. Devlin, C. Chaubaroux, A. Dochter, R. Alles, P. Lavalle, J. C. Voegel, A. Blencowe, P. Auffinger, F. Caruso, ACS Nano 2012, 6, 7584-7594. [3] D. Mertz, H. Wu, J. S. Wong, J. Cui, P. Tan, R. Alles, F. Caruso, Journal of Materials Chemistry 2012, 22, 21434-21442. [4] D. Mertz, C. Affolter-Zbaraszczuk, J. Barthes, J. Cui, F. Caruso, T. F. Baumert, J.-C. Voegel, J. Ogier, F. Meyer, Nanoscale 2014, 6, 11676-11680. [5] X. Wang, X., D. Mertz, C. Blanco-Andujar , A. Bora, M. Ménard, F. Meyer, G. Giraudeau, S. Bégin-Colin, S. RSC Adv. 2016, 6, 93784−93793. [6] D. Mertz, O. Sandre, S. Bégin−Colin, Biochim. Biophys. Acta BBA - Gen. Subj. 2017, 1861, 1617–1641 [7] M. Ménard, D. Mertz, C.Blanco-Andujar, F. Meyer, S. Bégin-Colin, submitted. [8] V. Fiegel, S. Harlepp, S. Bégin-Colin, D. Bégin, D. Mertz, Design of protein-coated carbon nanotubes loaded with hydrophobic drugs through sacrificial templating of mesoporous silica shells, Chem Eur J.,2018, in revision. [9] C. Wells, O. Bringel, V. Fiegel, S. Harlepp, B. Van der Schueren, S. Begin-Colin, D. Bégin, Mertz D., Engineering of mesoporous silica coated carbon based materials optimized for an ultra-high doxorubicin payload and a drug release activated by pH, T and NIR-light. Adv. Funct. Mater 2018, in revision

Resume : Biological processes leading to tissue formation during embryonic development are characterized by large ‘robustness’, namely stability and reproducibility of events. Also regenerative medicine approaches could be more repeatable and effective if they targeted the recapitulation of molecular pathways typical of tissue development. Within the exemplifying context of cartilage and bone repair, this lecture will introduce and discuss the challenges and opportunities of regenerative concepts based on mimicking developmental processes. Rather than engineering a tissue, the strategy would target the use of cells to engineer temporally staged processes, recapitulating events of development. The product would be a construct containing the necessary and sufficient cues to autonomously remodel into the target repair tissue upon grafting. In this perspective, however, cells in adults may strongly differ from multipotent embryonic cells, and typically reside in an environment, which is tightly regulated by post-natal mechanical conditioning or immune/inflammatory processes. Thus, shouldn’t tissue regeneration strategies be inspired by development but adapted to be effective in a context, which is different from the embryo? This would require to re-design the developmental machinery for regenerative purposes by establishing artificial events or conditions. Will the resulting approach of ‘developmental re-engineering’ offer a chance for enhanced tissue regeneration?

Resume : Chemotherapy seeks to minimize tumor progression and increase patient survival. However, the main problem is to find a balance between the drugs therapeutic effect on cancer cells and their deleterious effect on healthy cells. Due to their high hydrophobicity or rather their high hydrophilicity, these molecules must be injected in high and frequent doses, to avoid a rapid elimination and overcome their lack of specificity. Unfortunately, the high chemotherapeutic doses have side effects that patients find difficult to tolerate. Additionally, the diagnosis and imaging of tumor evolution remain a challenge. In order to overcome the challenges associated with chemotherapy, our research group is engaged in developing smart and responsive drug nanocariers. In this talk, I will be discussing two types of drug delivery systems based on iron oxide nanoparticles. Two different approaches will be presented. The first relies on the surface modification of the nanoparticles with an organic macrocycle (cucurbit[7]uril) which allows the adsorption of a drug such as Doxorubicin. The second strategy makes use of mesoporous iron-oxide nanoparticles as nanocarriers. The efficiency of both systems in vitro and in vivo will be discussed. Moreover, the possibility of these two systems to combine chemo and thermal therapies will also be presented.

Resume : Microbubbles are studied as contrast agents for ultrasound imaging, targeted drug, gene and oxygen delivery.[1-3] The shell of the microbubbles is built from lipids, polymers, nanoparticles, or combinations of those.[4, 5] We report stable fluorocarbon-stabilized microbubbles with a phospholipid shell that incorporate dendronized iron oxide nanoparticles. We investigated Langmuir monolayers of mixtures of phospholipids with dendrons fitted with pegylated chains.[6] We found that the dendrons form microdomains within the monolayers (compression isotherms and AFM after transfer). Fluorocarbon gases, when introduced in microbubbles act as osmotic agents[7] and as cosurfactants to phospholipids.[8, 9] We show that introducing the fluorocarbon in either the gaseous or aqueous phase of microbubble dispersions strongly impacts their size and stability.[10] We also obtained small (2-4 µm) and stable (half-life: 1-2 h) microbubbles with dendronized iron oxide nanoparticles (optical microscopy, static light scattering and acoustical attenuation method). 1. J. R. Lindner, Nat. Rev. Drug Disc., 2004, 3, 527. 2. S. R. Sirsi, M. A. Borden, Adv. Drug Deliv. Rev., 2014, 72, 2. 3. M. P. Krafft, J. Fluorine Chem., 2015, 177, 19. 4. S. Sirsi, M. Borden, Bubble Sci. Eng. Technol., 2009, 1, 3. 5. P. N. Nguyen, G. Nikolova, P. Polavarapu, G. Waton, L. T. Phuoc, G. Pourroy, M. P. Krafft, RSC Adv., 2013, 3, 7743. 6. Dendrimers in Nanomedecine, D. Felder-Flesch (ed.), Pan Stanford, Singapore, 2016. 7. E. S. Schutt, D. H. Klein, R. M. Mattrey, J. G. Riess, Angew. Chem. Int. Ed., 2003, 42, 3218. 8. C. Szijjarto, S. Rossi, G. Waton, M. P. Krafft, Langmuir, 2012, 28, 1182. 9. M. P. Krafft, Biochimie 2012, 94, 11. 10. D. Shi, X.-H. Liu, M. P. Krafft, Soft Matter, 2018, submitted.

Resume : Microbubbles are studied as contrast agents for ultrasound imaging, targeted drug, gene and oxygen delivery.[1-3] The shell of the microbubbles is built from lipids, polymers, nanoparticles, or combinations of those.[4, 5] We report stable fluorocarbon-stabilized microbubbles with a phospholipid shell that incorporate dendronized iron oxide nanoparticles. We investigated Langmuir monolayers of mixtures of phospholipids with dendrons fitted with pegylated chains.[6] We found that the dendrons form microdomains within the monolayers (compression isotherms and AFM after transfer). Fluorocarbon gases, when introduced in microbubbles act as osmotic agents[7] and as cosurfactants to phospholipids.[8, 9] We show that introducing the fluorocarbon in either the gaseous or aqueous phase of microbubble dispersions strongly impacts their size and stability.[10] We also obtained small (2-4 µm) and stable (half-life: 1-2 h) microbubbles with dendronized iron oxide nanoparticles (optical microscopy, static light scattering and acoustical attenuation method). 1. J. R. Lindner, Nat. Rev. Drug Disc., 2004, 3, 527. 2. S. R. Sirsi, M. A. Borden, Adv. Drug Deliv. Rev., 2014, 72, 2. 3. M. P. Krafft, J. Fluorine Chem., 2015, 177, 19. 4. S. Sirsi, M. Borden, Bubble Sci. Eng. Technol., 2009, 1, 3. 5. P. N. Nguyen, G. Nikolova, P. Polavarapu, G. Waton, L. T. Phuoc, G. Pourroy, M. P. Krafft, RSC Adv., 2013, 3, 7743. 6. Dendrimers in Nanomedecine, D. Felder-Flesch (ed.), Pan Stanford, Singapore, 2016. 7. E. S. Schutt, D. H. Klein, R. M. Mattrey, J. G. Riess, Angew. Chem. Int. Ed., 2003, 42, 3218. 8. C. Szijjarto, S. Rossi, G. Waton, M. P. Krafft, Langmuir, 2012, 28, 1182. 9. M. P. Krafft, Biochimie 2012, 94, 11. 10. D. Shi, X.-H. Liu, M. P. Krafft, Soft Matter, 2018, submitted.

Resume : The continuous growth of nanotechnology has brought challenging innovations in the synthesis of multifunctional nano-objects for medicine, able to revolutionize the field of diagnosis and therapy. Today’s aim is to develop multifunctional theranostic (i.e. including therapeutic and diagnostic functions) nanoparticles (NPs) which can both identify disease states and deliver therapy and allow thus following the effect of therapy by imaging. Lots of efforts are focused on iron oxide NPs that already demonstrated efficiency as biodegradable and nontoxic T2 contrast agent for MRI along a strong potential for treatment through magnetic hyperthermia (MH). Yet theranostic applications are currently limited by the low magnetic properties of usual magnetic NPs, requiring a local injection of large quantities of NPs. One strategy for increasing the magnetic properties of the NPs is to play on their shape to add shape anisotropy, which can also enable cooperative magnetism. Our knowledge in the area of nanoparticles synthesis through thermal decomposition allowed us to successfully synthesize several anisotropic shapes such as cubes, octopods and plates. After fine structural and magnetic properties characterizations, the various NPs were made biocompatible with a Dendron molecule. The effect of the shape on MH and MRI properties has been investigated in solution but also in vitro and in vivo in order to identify the best design of NPs, promising for theranostic biomedical applications.

Resume : The limited response rate to currently available therapies for patients suffering from colorectal cancer (CRC) motivates the development of novel, more effective treatments. A promising therapeutic approach is currently offered by the use of nanoparticle (NP)-mediated drug delivery, allowing the achievement of high concentrations of anti-cancer therapeutics at the target site, while sparing healthy tissues. However, the possibility to assess the NPs’ impact on tumor cells is hampered by the lack of preclinical systems reliably mirroring the complexity of the in vivo tumor microenvironment (TME). Here, a simple, one-pot and reagent-free method for the preparation of human serum albumin (HSA) NPs using high frequency ultrasound was used1. The prepared HSA-NPs showed high stability and biocompatibility. Our results showed that the newly developed HSA-NPs have a diameter of 150 nm, a uniform size distribution and that they are non-toxic. They can be efficiently loaded with 5-Fluorouracil, a conventional chemotherapeutic drug extensively used in CRC treatment. Our results showed that at early time-point (5 days) HSA-NPs were rapidly accumulated in the stromal component instead of within tumor cells. However, after 24 h of incubation, the HSA-NPs were completely internalized by both the cell fractions. In addition, the HSA-NPs can be efficiently perfused through an in vitro engineered tumor tissue able to mimic the interaction between malignant and non-transformed cells of the TME. The HSA-NPs might be an innovative drug-delivery system, and their delivery efficacy could be tested in a controlled fashion by exploiting the perfusion-based bioreactor TME in the in vitro model. 1. Francesca Cavalieri, Enrico Colombo, Eleonora Nicolai, Nicola Rosato, Muthupandian Ashokkumar, Mater. Horiz., 2016 ,3, 563-567

Resume : Introduction Recent advancements in nanotechnology now also set the stage for improvements in the clinics. CTCs are considered to be of high clinical relevance to diagnose disease and monitor treatment. The molecular profiling of CTCs may allow to uncover novel cancer pathways. However, current CTC detection methods are labor and cost intensive, precluding the analysis of CTCs in large patient cohorts. Methods and Results To overcome these limitations, we are developing miniaturized immuno-nanoparticle-based magnetic flow cytometry chips, allowing the swift and low cost detection of CTCs in blood. We show that magnetic biosensing offers key advantages, a direct electronic read-out, and the option to apply the magnetic cell enrichment directly for cell detection. We will present proof of concept data demonstrating the feasibility of magnetic flow cytometry. However, we found that performance of the technology is dependent of the quality of the used antibody-armed ‘intelligent‘ nanoparticles and plasma proteins adsorbing to nanoprobes. Conclusions Collectively, we report that nanoparticle-based magnetic flow cytometry is highly promising for the further development into an easy to use ‘point of care’ diagnostics, allowing the routine detection of CTCs in HNSCC patients’ blood samples. Applying this technology, prospective (multicenter) clinical studies are needed to clarify the value of CTCs as a surrogate clinical marker for HNSCC.

Resume : Bioorthogonal chemistry represents a challenging approach in pretargeted radioimmunotherapy (PRIT) based on fast, strong and biocompatible covalent interaction between trans-cyclooctene (TCO) conjugated antibodies (mAbs) and radiolabelled probes linked to tetrazine (TZ). Herein, we reported a process to modify mAbs with TCO bearing polyethylene glycol (PEG) spacers of variable length on three different mAbs: rituximab (anti-CD20), Ts29.2 (anti-TSPAN8) and 35A7 (anti-CEA). We first synthesized TCOPEG derivatives, while controlling the isomerization rate of TCO to its non-reactive form cis-cyclooctene (CCO) by HPLC, then mAb-PEGn-TCO conjugates. MALDI-TOF MS analyses showed that PEGn-TCO units were grafted reproducibly, the number of TCO moieties grafted ranging from 1-2 to 13-14 respectively to the PEGn-TCO equivalents added in the reaction. mAbs-PEGn-TCO functionality was also checked using fluorescent TZ probes through gel electrophoresis and also by immunofluorescence and/or flow cytometry experiments on corresponding cell models (GRANTA-519 for CD20, HT29 for TSPAN8 and A431-CEA-Luc for CEA). We were able to demonstrate that the control of the isomerization rate of PEGn-TCO solution before mAb grafting is crucial to maximize the interaction with TZ. We also observed that PEGylation of mAbs-TCO induced a loss of interaction with fluorescent TZ in two models. These results are prerequisites for our incoming PRIT experiments and radiosensitization using nanoparticles.

Resume : Polymeric micelles (PMs) are nanostructures formed upon the spontaneous self-assembly of amphiphilic copolymers blocks above the critical micellar concentration and they emerged as one of the most versatile nanotechnology platforms for drug encapsulation, delivery and targeting owing to the high diversity of hydrophilic and hydrophobic blocks and the chemical flexibility to tailor the amphiphilic architecture. The low physical stability of PMs upon dilution in the biological environment is the most striking drawback. Moreover, PMs were mainly utilized for the intravenous administration of antitumorals drugs and not for mucosal routes because of two main limiting drawbacks: weak interaction with mucus and inability to sustain the release of the encapsulated payload over time. Finally, despite their high chemical functionality, PMs are not often designed to actively target specific cells populations (e.g., cancer cells). My research group dedicates dauntless efforts to design novel amphiphilic nanomaterials with advanced features and thus, to extend the application of PMs pharmaceutical research and development. To expand the application of PMs in drug delivery, we have developed a new type of non-covalently crosslinked nanogels based on the aggregation of amphiphilic graft copolymers that display a multifunctional hydrophilic backbone and hydrophobic side blocks. One of the striking advantages of these novel nanobiomaterials is its modular nature.

Resume : Multivalency is a ubiquitous phenomenon in nature involving complex binding mechanisms for achieving non-covalent strong yet reversible interactions. Interfacial multivalent interactions at pathogen-cell interfaces (left) can be competitively inhibited by multivalent scaffolds (right) that prevent pathogen adhesion to the cells during the initial stages of infection, while monovalent inhibition fails to inhibit the biological pathway (middle). The lack in understanding of complex biological systems makes the design of an efficient multivalent inhibitor a toilsome task and is the reason why as of yet no multivalent anti-infective has emerged on the market until now. This talk will focus on the design and application of dynamic 2D and 3D multivalent nanosystems as potent inhibitors for pathogens. Bhatia, Lauster, Wolff, Hamann, Böttcher, Herrmann, Haag, et al. Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo. Biomaterials 2017, 138, 22-34.

Resume : Nowadays, multimodal diagnosis by combining several imaging techniques, such as mammography, magnetic resonance imaging (MRI) and computed X-ray tomography (CT) is playing a leading role in the diagnosis of breast cancer. However clinical application of this approach is still problematic due the lack of appropriate commercial contrast agents showing capabilities to distinguish between malignant tumours and benign abnormal masses. Hybrid nanoparticles, which contain in the same structure magnetic and radiopaque elements are a promising alternative for the generation of multimodal contrast agents due to their unique characteristics, such as: high biocompatibility, contrast enhancement efficacy, cost effectiveness and colloidal stability in the physiological environment. Unfortunately, conventional chemical procedures in liquid media used to synthesize multimodal nanoparticles are not well standardized and cost-effective methods to produce large quantities of them. In order to overcome these limitations, Liquid-Assisted Pulsed Laser Ablation (LA-PLA) was used to synthesize water stable colloidal dispersions of ‎Fe2WO6, FeBiO3, FeLaO3, Fe3WLaO9 and Fe2BiLaO6. Standard MTT and cellular uptake assays on HeLa cells and hemolysis tests were conducted to determine the cytotoxicity of the synthesized NPs. In vitro MRI studies were conducted to demonstrate that these nanoparticles produced MRI contrast enhancement at the concentrations usually used in clinical analysis. Moreover, in vitro CT analysis showed that these nanoparticles generated different X-ray contrast enhancement as function of the radiopaque element present in their formulas and the voltage applied to the X-ray tube during the analysis.

Resume : Lanthanides have emerged over the last years as attractive probes for bioimaging.[1] Their exceptionally long lifetime as well as their large Stokes shift can be used to overcome autofluorescence issues in biological samples. However, they are strongly limited by their low extinction coefficients providing a limited brightness. Here we propose an approach to design probes with superior brightness by encapsulating large amounts of a lanthanide complex inside polymer nanoparticles (NPs).[2] We previously described charge-controlled nanoprecipitation for making ultrasmall polymer NPs with efficient encapsulation of high amounts of hydrophobic fluorescent dyes.[3,4] Using this approach, we assembled three series of poly(methyl methacrylate) based NPs with different sizes (10, 20 and 30 nm) encapsulating up to 40 wt% of an Eu3 complex (>200 complexes per particle). The resulting NPs were characterized with respect to their size, absorbance and luminescence properties. The polymer matrix provides an efficient protection of the encapsulated Eu3 complex, increasing its lifetime and reaching quantum yields of up to 25%. The resulting particles are bright enough to be imaged at the single particle level and are readily internalized by cells with excellent stability for cellular imaging. References: [1] Sy, M.; et al. Chem. Commun. 52, (2016), 5080. [2] Reisch, A.; Klymchenko, A.S. Small 12, 15 (2016), 1968. [3] Runser, A. et al. ACS Nano 9, 5 (2015), 5104. [4] Reisch, A. Nat Commun. 2014, 9, 4089.

Resume : The combination of imaging and therapeutic modalities in a unique formulation through nanotechnological approaches bears thus an enormous potential for the diagnosis of cancers and their treatment. Indeed the explosive growth of nanotechnology has brought challenging innovations in the synthesis of multifunctional nano-objects for medicine, able to revolutionize the field of diagnosis and therapy. Dendronized Iron oxide (IO) nanoparticles (NPs) have been widely studied for the last decades as T2 contrast agent for magnetic resonance imaging (MRI). The dendron molecules are ensuring biocompatibility, solubility and colloidal stability but also small size bio-distribution, and few non-specific interactions, while the magnetic inorganic core, synthesized by thermal decomposition, is providing high r2 relaxivity making dendronized NPs suitable as contrast agent for MRI. Furthermore the thermal decomposition synthesis method allows tuning the size, shape and composition of NPs enabling them to be efficient for therapy by magnetic hyperthermia and to combine several imaging modalities by elaborating core-shell NPs. Indeed the amount of heat generated by magnetic hyperthermia by iron oxide NPs strongly depends on their size distribution but also on their magnetic properties and high saturation magnetization and anisotropy energy are required. One strategy to increase these properties is to synthesize iron oxide NPs doped with Cobalt, Zinc or Rare Earth (RE) Element. However such doping is challenging as the different thermal decomposition temperature of precursors led often to heterogeneous distribution of doping element. By designing suitable precursors, we were able to synthesize doped iron oxide NPs with enhanced magnetic properties leading to high heating values. Then by synthesizing core-shell NPs with an iron oxide–base core and a shell integrating RE featuring fluorescent properties or by combining such NPs with quantum dots, we succeeded in designing nanoplatforms providing multimodal imaging and therapy by magnetic hyperthermia. Their different properties were further confirmed by in vitro studies and preliminary in vivo exepriments.

Resume : In this paper we present a new and patented technology of physical nanocapsules (functionalized or not) relying on nanotechnology processes and aiming to provide novel materials for clinical protocols that will combine diagnostic testing, imaging and therapy. Indeed, the fabricated nanocapsules are smart and multi-functional, and display three potentialities: functionalization for localized delivery (targeting), contrast properties for imaging (diagnostic and therapy monitoring), and in vivo targeted activation (hyperthermia and active agent delivery). Our technology presents an alternative to the well-established and known chemical encapsulation that suffers from several drawbacks such as: - the specificity of the encapsulation for the molecule to be encapsulated, - the bad control of capsule size (height and width) - the low concentration of the encapsulated active product in the resulting capsules, Indeed, in our technology, the encapsulated active product can reach 50% of the capsule volume, the same process can be used for any compound and the encapsulating materials can be chosen independently of the encapsulated ones. The fabrication of the nanocapsules uses top down techniques based on a nanotechnology process and is compatible with a future mass production. Moreover, the independent choice of the encapsulation material, the compound and the caps, offers many possibilities. At this point the pre-structured open cavity can be filled with any compound at any state of the matter (dry powder, wet powder, liquid). Following this step the capsule can be closed with a variety of types of cap made from digestible materials (enzyme sensitivity), physically sensitive to exterior modifications (ultrasound, Infra red, other radiation such as ultra violet,?) or other.

Resume : Immunotherapies consist of enhancing the response of the immune system to treat a specific disease, and are widely predicted to dominate next-generation cancer therapy. Many different cells of the immune system can be targeted to enhance its efficiency such as T lymphocytes, B lymphocytes, Dendritic Cells or Natural Killer cells. A range of approaches are being developed, including nano-vaccines and artificial antigen-presenting cells that activate T cells. Nanotechnology is specifically deployed in many of these approaches, and methods of biofunctional nanomaterials synthesis are thus critical to success. This poster presents new approaches to develop biofunctionalized nanoparticles that specifically target immune cells to strengthen the immune response. The efficacy of these approaches will be compared with existing methods.

Resume : Multifunctional nanoconstructs are particle-based nano-scale systems designed for the ‘smart’ delivery of therapeutic and imaging agents. The Laboratory of Nanotechnology for Precision Medicine at IIT-GE synthesizes polymeric nanoconstructs with different sizes, ranging from a few tens of nanometers to a few microns; shapes, including spherical, cubical and discoidal; surface properties, with positive, negative, neutral coatings; and mechanical stiffness, varying from that of cells to rigid, inorganic materials, such as iron oxide. These are the 4S parameters – size, shape, surface, stiffness – which can be precisely tuned in the synthesis process enabling disease- and patient-specific designs of multifunctional nanoconstructs. In this talk, the role of manipulating these 4S parameters over different temporal and length scales, with particular emphasis on mechanical stiffness, will be elucidated in the context of future nanomedicines.

Resume : Enteric bacteria cause severe diseases, including gastric cancer-associated Helicobacter pylori. Their infection paths overlap with the oro-gastrointestinal uptake route for nanoparticles, increasingly occurring during environmental or consumer/medical exposure. Most studies focused on antimicrobial, metal-based nanoparticles engineered to interact with bacteria. By comprehensive independent analytical methods, such as live cell fluorescence, electron as well as atomic force microscopy and elemental analysis, we show that a wide array of nanoparticles (NPs) but not microparticles form complexes with H. pylori and enteric pathogens. NP-assembly occurred rapidly (< 30 sec), was not affected by variations in temperature (4-55°C), though dependent on the NPs' physico-chemical characteristics. Improved binding was observed for small nanoparticles with negative surface charge, whereas binding was reduced by surface 'stealth' modification. NP-bacteria assembly did not follow the rules of colloidal electrostatics, and was strongly enhanced by the low pH of the gut. NP-binding affected the (patho)biological identity of both, NPs and bacteria, including the antimicrobial activity of silver nanoparticles, uptake into epithelial target cells or phagocytes as well as NP-induced cell toxicity. In gastric epithelial cells and human 3D-organoid models of the stomach, NP-coating did not inhibit H. pylori's cellular attachment. However, even assembly of non-bactericidal silica nanoparticles attenuated H. pylori infection by reducing CagA phosphorylation, cytoskeletal rearrangement, and IL-8 secretion. We demonstrate that nanoparticle binding to enteric bacteria systematically impact their pathobiology particularly in the stomach. Non-toxic nanoparticles may be further exploited even as potential protective food additives.

Resume : The rational design of safe and effective drug delivery systems requires deep understanding of the processes that govern the interaction between cells and nanocarriers (NCs). Here, we investigate the mechanisms involved in the nano-bio interplay to find correlation between physical-chemical properties of NCs and their intracellular fate. In general, internalization of NCs mainly occurs through endocytosis, resulting in their endo-lysosomal localization. However, to improve the safety and therapeutic efficacy of the NCs, cytosolic delivery is often desirable, which is challenging. In particular, we show that the ability of membranotropic peptides to transport cargos across cell membrane, escaping endocytosis, is strongly dependent on NC size as well as on their dispersion/aggregation status in culture media. On the other side, a potential therapeutic improvement of NCs confined in lysosomal vesicles can be achieved by prolonging their intracellular retention time, so to increase the effective intracellular dose of the transported drug. We show that such a general strategy, named “exocytosis engineering”, enables strong improvement of the therapeutic action of drug-loaded NCs in several tumor cell lines and in 3D tumor spheroids.

Resume : Applications of nanoparticles (NPs) are rising in biotechnology and biomedicine. However, the field has just started to explore the complex interplay of NPs with microbes, as well as its (patho)biological consequences. Based on recent insights, we here present our knowledge on the interaction of NPs with pathogenic microbes and its analytical investigation. We comment on how the NPs' characteristics influence complex formation with pathogens, present the underlying physico-chemical forces, and how this knowledge can be used to rationally control NP-microbe interaction. We conclude by debating the role of the biomolecule corona on NP-microbe crosstalk, and by discussing the impact of NP-microbe complex formation on the (patho)biological outcome and fate of infections. The presented insights will support the design of smart NPs with improved anti-microbial activity.

Resume : There is an acute shortage of organs due to disease, trauma, congenital defects, and most importantly, age related maladies. The synthetic materials used in tissue engineering applications today are typically composed of millimeter or micron sized particles and/or fiber dimensions. Although human cells are on the micron scale, their individual components, e.g. proteins, are composed of nanometer features. By modifying only the nanofeatures on material surfaces without changing surface chemistry, it is possible to increase tissue growth of any human tissue by controlling the endogenous adsorption of adhesive proteins onto the material surface. In addition, our group has shown that these same nanofeatures and nano-modifications can reduce bacterial growth without using antibiotics, which may further accelerate the growth of antibiotic resistant microbes. Inflammation can also be decreased through the use of nanomaterials. Finally, nanomedicine has been shown to stimulate the growth and differentiation of stem cells, which may someday be used to treat incurable disorders, such as neural damage. This strategy also accelerates FDA approval and commercialization efforts since new chemistries are not proposed, rather chemistries already approved by the FDA with altered nanoscale features. This invited talk will highlight some of the advancements and emphasize current nanomaterials approved by the FDA for human implantation.

Resume : Glioblastoma (GBM) is the most frequent and malignant brain tumor with a high mortality rate. The tumor-associated macrophages (TAM) closely interact with the GBM cells (GC) to promote the survival, progression and therapy resistance of the GBM. Various therapeutic strategies have been devised either targeting the GC or the TAM but few have addressed the cross-talks between the two cell populations. The present study was carried out to explore the possibility of exploiting the cross-talks between the GC and TAM for regulating the tumor microenvironment through using Nano-DOX, a drug composite based on nanodiamonds bearing doxorubicin. In the in vitro work, Nano-DOX-loaded TAM were first shown to be viable and able to infiltrate three-dimensional GC spheroids and release cargo drug therein. GC were then demonstrated to encourage Nano-DOX-loaded TAM to unload Nano-DOX back into GC which consequently emitted damage-associated molecular patterns (DAMPs) that are powerful immunostimulatory agents as well as indicators of cell damage. As a result, Nano-DOX-damaged GC exhibited an enhanced ability to attract both TAM and Nano-DOX-loaded TAM. Most remarkably, Nano-DOX-damaged GC reprogrammed the TAM from a pro-GBM phenotype to an anti-GBM phenotype that suppressed GC growth. Finally, mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mouse TAM which were found releasing drug in the GBM xenografts 24 h after injection. GC damage was evidenced by the induction of DAMPs emission within the xenografts and a shift of TAM phenotype was detected as well. Taken together, our results demonstrate a novel way with therapeutic potential to harness the cross-talk between GBM cells and TAM for modulation of the tumor immune microenvironment.

Resume : A new surgery area is rising: the Augmented Surgery. It aims to augment surgeon vision, surgeon gesture and surgeon decision, introducing the Augmented Surgery concept. Augmented surgical vision is based on 3D/4D patient-specific modelling. The first step consists in the Visible Patient online 3D modelling of organs and pathologies from a patient’s medical image (CT or MRI). Preoperatively, the resulting virtual clone can be used to plan and simulate the surgical procedure thanks to user-friendly mobile software. Intraoperative assistance will then consist in Augmented Reality that provides a kind of virtual transparency of the patient. Main limits of this technique are linked to organ movement and deformation between the preoperative image and the intraoperative position and shape. To overcome this limit, the introduction of 3D-medical imaging systems in the Operating Room is then mandatory. The intraoperative medical image is registered with the preoperative image in order to correct organ deformations. By adding the laparoscopic image analysis, it is then possible to compute in real-time the precise location and shape of organs and pathology. This information can be combined with a robotic system to develop the next generation of automated robots linked to Artificial Intelligence. A.I. will so not only assist surgeons in therapy definition, but also control and assist them intraoperatively just like a pilot during a flight. Luc Soler is president of Visible Patient, Scientific Director of IRCAD and part of the surgical team of Prof. Didier Mutter as invited professor at the Medical Faculty of Strasbourg. He is one of Prof. Jacques Marescaux’s close collaborators since 1995. He is an acknowledged expert in image-guided computer-assisted surgery.

Resume : Complete tumor removal during surgery has a great impact on patient survival rate. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient1. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. We present here the design, syntheses and characterization of three polymeric Turn-ON probes activated by cathepsin B enzymatic degradation to generate a fluorescent signal. Two polymeric backbones are composed of biodegradable poly-L-glutamic acid (PGA) polymer, loaded with self-quenched (homo-FRET) near-infrared (NIR) Cy5 molecules or hetero-FRET-quenched Cy5 and dark quencher moieties. The third polymeric Turn-ON probe was recently reported by us2 and composed of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer bearing self-quenched Cy5 fluorescent dyes. We studied the kinetics of the polymeric nano-probe on orthotopic breast cancer model in mice and showed an improved tumor-to-background ratio. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling a more accurate resection. This “smart” polymeric Turn-ON probe can potentially assist surgeons to decide in real time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.

Resume : Active immunotherapy is at the forefront of anti-cancer therapies because it combines both a high degree of specificity to general high effectiveness and fewer side effects on healthy cells. Nevertheless, successful therapeutic anticancer treatments are not yet been obtained with current immunotherapeutic strategies. Gold nanoparticles (AuNPs) are very useful biocompatible antigen scaffolds and they can be engineered to present a high degree of multivalency.[1] The extracellular domain of the mucin-1 (MUC1) glycoprotein is an attractive target for the development of therapeutic cancer vaccines. Tumor-associated MUC1 (TA-MUC1) is found overexpressed on epithelial cancer cells and markedly underglycoslyated compared to MUC1 on healthy cells[2], which results in the display of new peptide and carbohydrate epitopes. In this contribution, we describe the efficaciously development of novel vaccine formulations using PEGylated AuNPs[3] as scaffolds for the multivalent presentation of TA-MUC1 and adjuvating B-cell epitopes. We show that our AuNP-based vaccine formulations elicit not only a robust humoral immune response but also a cellular immune response in wild-type mice. Our results show the great potential of TA-MUC1 vaccine candidates based on PEGylated AuNPs, especially considering their high biocompatibility and good immunogenicity. [1] Irvine DJ et al, Chem. Rev. 2015, 115, 11109-11146 [2] Nath S et al, Trends Mol Med 2014, 20, 332-342 [3] Maus L et al, ACS Nano 2010, 4, 6617-6628

Resume : Polydopamine (PDA) modified superparamagnetic core shell nanoparticles (NPs) are highly robust and biocompatible MRI contrast agents for the detection of tumors. Reactive quinone on the surface enhances the binding efficiency of various enzymes and biomolecules for targeted delivery. Glutathione disulphide (GTH) was thus immobilised on the surface to treat prostate cancer by carrying docetaxel (DTX) drug. To achieve maximum accumulation of the developed therapeutics at the targeted site, GTH immobilised NPs can manifests a successful biomarker for selecting tumors potentially responsive to chemotherapeutic regimens.1 The NPs were sequentially characterised using FTIR, XPS, TGA, zeta potential, UV, and Raman spectroscopies. Thus, the developed NPs offers dual anticancer mode as NO release 2 using GTH and by DTX release which is also analysed using in-vitro studies. References (1) Traverso, N.; Ricciarelli, R.; Nitti, M.; Marengo, B.; Furfaro, A. L.; Pronzato, M. A.; Marinari, U. M.; Domenicotti, C. Oxid Med Cell Longev., 2013, 2013. (2) Singh, N.; Patel, K.; Sahoo, S. K.; Kumar, R. Colloids Surf., B 2017.

Resume : Photodynamic therapy (PDT) is an emerging non-invasive technique for the treatment of cancer. It involves the administration of a photosensitizer (PS) that, after its excitation, can produce reactive oxygen species (ROS), causing damage to targeted cancer cells. Even if some important developments have been achieved, some problems still exist and there has been extensive research into the design of alternative PSs. Polymethine dyes deserve to be counted among innovative potential PSs for their strong absorption in the NIR region perfectly matching the biological tissues’ transparency window (600-900 nm). Unfortunately, they are characterized by a very low solubility in physiological media and by the easy formation of non-fluorescent aggregates. In this context, the incorporation of these dyes in nanoparticles (NPs) is extremely important. In this work we designed and synthesised a new series of NIR absorbing polymethine dyes with different substitution groups to investigate how the structure may influence the capacity of these molecules to produce singlet oxygen, which was accessed in vitro. On the most promising PSs, ROS generation, cytotoxicity, cell death and DNA damage analyses were performed after PDT treatment. In particular, two of these squaraine dyes showed very interesting PDT performances as well as co-localization in mitochondria. Here we present the results obtained along with a structure-activity relationship discussion of these new photosensitizers for PDT.

Resume : Among a variety of drug delivery systems, liposomes are very promising candidates considering their biocompatibility, biodegradability, and their drug loading capacity for both hydrophobic and hydrophilic molecules. These nanocarrier-based systems have been successfully translated to clinical applications (Doxil®, Ambisome®, DepoDurTM). However, their major drawback is their instability in the gastro-intestinal environment and their enzymatic degradation by lipases(1). Lots of efforts have been made to overcome this major hurdle and design stable orally deliverable liposomes. One approach to increase their stability is the layer-by-layer coating with oppositely charged polymers to obtain structures called ?layersomes?(2). So far, a couple of poly-electrolytes combinations have been tested for various therapeutic applications such as poly(acrylic acid) and poly(allyl amine), poly(glutamic acid) and poly(allyl amine), chitosan and dextran sulfate ?(3), (4), (5). Based on previous work, the poly-L-lysine (PLL) and poly-L-glutamic acid (PGA) combination was chosen by our group for layersome formulation. Indeed, PLL and PGA coating was shown to increase the robustness of liposomes(6). Small unilamellar liposomes (Lp) composed of phosphatidylcholine, phosphatidylglycerol and cholesterol were used as starting material. Alternative deposition of positively and negatively charged polymers was achieved by slowly dropping the liposomes into the PLL solution and then by slowly dropping the obtained Lp-PLL suspension into the PGA solution. Excess of polymer was removed after each coating step by filtration. We first demonstrated that the alternative deposition of positively and negatively charged PLL and PGA, respectively leads to an increase of the average diameter and a variation of the zeta potential values compared to primarily liposomes, which is consistent with a layer-by-layer coating. The formulation procedure was optimized to obtain homogenous, stable and quite small (< 200 nm) layersomal structures. We tuned various parameters such as the formulation temperature, the concentration of both poly-electrolytes and the filtration technique. We succeeded in developing stable and monodisperse layersome formulations of about 100 nm average diameter (half-value width: 15 nm). In parallel to formulation, we performed the synthesis of PLL and PGA covalently coupled to rhodamine B and aminofluorescein, respectively, to demonstrate the alternative coating of liposomes by Fluorescence Resonance Energy Transfer (FRET). So far, both PLL-rhodamine B and PGA-fluorescein with different grafting percentages were synthetized and tested for layer-by-layer coating of liposomes. A positive FRET signal was observed twice with two formulations. These are preliminary encouraging results that we are currently tuning. (1) Rowland, R. N.; Woodley, J. F. The stability of liposomes in vitro to pH, bile salts and pancreatic lipase. Biochim. Biophys. Acta 1980, 620, 400-409. (2) Sukhorukov, G., Fery, A., Mohwald, H., 2005. Intelligent micro- and nanocapsules. Prog. Polym. Sci., 885?897. (3) Marc Michel, Youri Arntz, Guillaume Fleith, Julien Toquant, Youssef Haikel, Jean-Claude Voegel, Pierre Schaaf, and Vincent Ball. Layer-by-layer self assembled polyelectrolyte mutlilayers with embedded liposomes : immobilized submicronic reactors for mineralization. Langmuir 2006, 22, 2358-2364 (4) Harshad Harde, Ashish Kumar, Agrawal and Sanyog Jain. Development of dual toxoid-loaded layersomes for complete immunostimulatory response following peroral administration. Nanomedicine 2015, 10(7), 1077?1091. (5) Sergio Madrigal-Carballo, Seokwon Lim, Gerardo Rodriguez, Amparo O. Vila, Christian G. Krueger, Sundaram Gunasekaran, Jess D. Reed. Biopolymer coating of soybean lecithin liposomes via layer-by-layer self-assembly as novel delivery system for ellagic acid. Journal of Functionnal Foods 2010, 2, 99-10. (6) Ciobanu, M.; Heurtault, B.; Schultz, P.; Ruhlmann, C.; Muller, C. D.; Frisch, B. Layersome: development and optimization of stable liposomes as drug delivery system. Int. J. Pharm. 2007, 344, 154-157.

Resume : Nanoparticle targeting remains one of the important aspects of modern nanomedicine, which continue to attract the attention of researchers. To achieve recognition of specific cells nanoparticles are functionalized by ligands, aptamers or antibodies capable of specific interactions with cell surface receptors. The main difficulties are that experimental testing of selectivity can be challenging and there might not be a universal solution of the problem, as the result depends on both properties of the carrier and cell surface. Understanding how the design can influence the selectivity of targeting becomes increasingly important as development of nanoparticulate formulations progresses from the laboratory stage to clinical testing. Using computer modeling we investigate the influence of the nanoparticle properties (nanoparticle size, polymer tether length and density, ligand density and valence) on the selectivity of nanoparticle-cell surface interactions and make predictions regarding favorable nanoparticle design for achieving nanoparticle attachment to cells with high receptor density while sparing healthy cells with a low density of receptors. Based on the obtained data, we make experimentally testable predictions regarding the ways to enhance selectivity of nanoparticle-cell surface interactions by optimizing the nanoparticle architecture, which will be discussed during the talk.

Resume : The design of selective drug delivery nanocarriers is a promising approach to release active agents in cellular districts, counteracting central nervous system disorders [1]. However, the use of hydrophilic drugs presents the drawback of unwanted rapid diffusion, limiting the therapeutic effects in the target site. To overcome this aspect, we proposed conjugated nanogels where the drug was linked to the network through a thiol-sensitive bond. These nanodevices were composed by polyethylene glycol (PEG) and polyethyleneimine (PEI) both chemically functionalized: PEG was linked to rhodamine (RhB, used as drug mimetic) through a disulfide bond and PEI modified with Cy5 dye using click chemistry to ensure the nanogel traceability. The selectivity as drug delivers is related to the S-S bond that can be disrupted by glutathione or cysteine cell component, releasing RhB as loaded. We tested the RhB release in glutathione solution, representative of its human body concentration, obtaining a faster release than in PBS. Moreover we tested our nanogels in microglia culture: the colocalization of Cy5 and RhB signals showed that the mimetic drug was internalized. After 4 days, the delocalization of RhB signal than Cy5 confirmed its release from nanogel, within cytosol [2], suggesting that these nanogels were able to carry hydrophilic molecules towards specific target and release them selectively. [1] Papa et al., ACS Nano, 2013, 7, 9881-9895 [2] Mauri et al., RSC Adv., 2017, 7:30345-30356

Resume : A new method to deliver exogeneous biomolecules into targeted cells using a laser and metallic nanoparticles will be presented. Irradiating gold nanoparticles by an ultrafast laser beam produces highly localised processes on the nanoscale in the biological surrounding medium, yielding to the optoporation of the cells. These gold nanoparticles could be functionalised to target specific biological entities, thus performing multiple targeted optoporation on the nanoscale. As an example, the laser technique was employed to perform in vitro gene transfection in living cell with an efficiency as high as 80%. Complete physical model was developed to determine the basic mechanism underlying this new nanosurgery process. Our laser technique shows promises as an innovative tool for fundamental research in biology and medicine as well as an efficient alternative nanosurgery technology that could be adapted to therapeutic tools in the clinic. It is now being developed in close collaboration with researchers in hospital for applications in cancer treatment, ophthalmology, cardiology and neurology. Recent in vivo experiments in rat eye model show that this new technique could be efficiently used to optoporate retinal ganglion cell using conjugated gold nanoparticles. A provisional patent was recently submitted.

Resume : Despite an aggressive conventional therapy for newly diagnosed glioblastoma (GB) including surgery, radio- and chemotherapy, the median survival remains poor. Hypoxia, a hallmark of GB, is among the main causes of resistance to treatments, and a new therapy able to alleviate hypoxia specifically in the tumour is hence highly desired. However, while hypercapnic/hyperoxic gas mixture breathing or injection of artificial oxygen carriers (such as microparticles or perfluorocarbon emulsions) were designed to raise oxygen pressure, it is not satisfactory for brain tumor applications because of their lack of specificity or their large size. Thanks to the permeable nature of blood vessels of GB, nanoparticles (NP) are investigated to specifically target the tumour. Here, we report a new approach based on the use of metal containing microporous crystalline nanoparticles (zeolite) as a specific hyperoxic gases carrier. The zeolite nanoparticles act as a vasoactive agent for a targeted reoxygenation of the tumour. Furthermore, the low amount of metals (either gadolinium or iron) introduced in the zeolite nanoparticles is used as a contrast agent for further visualization of the tumour and tracking of the nanocrystals by magnetic resonance imaging (MRI). The absence of acute and chronic toxicity of zeolites nanocrystals to living animals (mice, rats and non-human primates) is confirmed. The set of experimental data unambiguously proves the efficiency of metal containing nanosized zeolites as a theranostic tool for (i) diagnosis and (ii) vectorization of hyperoxic gases.

Resume : Porous silica materials and capsules have attracted a lot of interest since they combine their rigid and stable silica framework with an emptiness that allow the entrapment and release of desired molecules. They can therefore be used as a delivery system, as sequestering agents and as catalytic substrate. Our interest in these materials is mainly in the biomedical area and we have to face the problem related to the fate of the inorganic nanocontainers in in vitro and in vivo applications. Indeed the issue related to the use of materials for therapy and imaging in living organism, is their accumulation in vital organs that often prevent their use in clinical use. Recently, a new generation of breakable hybrid nanoparticles, able to response and degrade upon external stimuli (e.g. reductive agents, pH, etc.), have been developed in our group1,2. The insertion of responsive linkers in the framework of these particles, results not only in the destruction and safe excretion of the nanoparticles from the cells, but also in a faster and better delivery of the payloads. Moreover, to expand the breakability properties of this material for other purpose, the possibility to entrap proteins into a breakable silica shell has also been realized in our laboratory3. It has been shown that the activity of different proteins remains intact after their delivery into cancer cells. In addition mesoporous silica has been used as component of hybrid hydrogels. I will illustrate biocompatible hydrogel able to release the chemo-attractant Stromal cell-Derived Factor-1α (SDF-1α), for the recruitment of stem cells. The hydrogel proved to provide optimal structural support for infiltration and proliferation and chemotaxis of stem cells in vitro, and subcutaneous implantation of SDF-1α-releasing hydrogel in mice resulted in a modulation of the inflammatory and fibrotic reaction in vivo, suggesting an improvement of the tissue response towards the implant.4 Encouraged by these results the hybrid hydrogels have also been injected in the stomach of pigs with the final aim to study the gelation kinetic in vivo. References. 1. Maggini.L et al, Nanoscale, 2016 , 7240-7247 2. Maggini.L et al, Chem. Eu. J., 2016, 22, 3697-3703 3. Prasetyanto, E.A. et al, Angew. Chem. Int. Ed. 2016, 3323–3327 4. Fiorini F. et al. Small, 2016, 12, 4881

Resume : Owing to their specific optical properties, gold nanoparticles (NPs) are promising devices for plasmonic photothermal therapy (PPTT) and cancer treatment. To be used in PPTT, it is necessary to match the GNPs plasmon band and the biological tissues transparency window (700 to 1500 nm). Hence nanorods, cages, stars or other complex architectures have been developed in the past decades [1,2] as their plasmon band are shifted towards higher wavelengths compared to nanospheres (525 to 550 nm). However, the syntheses of these architectures are complex, barely scalable and often require the use of non-biocompatible reagents. As those therapeutic agents are not completely cleared by the kidneys after the treatment and can persist in the body for months, this type of nanostructures can be toxic [3]. Furthermore, these objects often exceed the size of 50 nm, limiting the biodegradability inside the body, whereas small nanoparticles have been shown to be degraded over time [4]. To combine biocompatibility and photothermal properties, several studies propose the use of aggregates of easily producible 10-nm nanospheres, that can reach heating capacities similar to bigger and more complex nanostructures [5,6]. Our purpose in this project is to control this aggregation, through the association of gold NPs with different polyelectrolytes, to optimize the optical and photothermal capacities of this kind of objects. For that matter, we synthetize a wide range of aggregates varying the NPs diameter (D=4, 13, or 40 nm), the polyelectrolyte, and the concentration ratio between those two. A multimodal characterization of this nanostructures, based on DLS, cryo-TEM, SAXS, UV visible spectroscopy and photothermal measurements, enable us to enlight structure-properties relationship. We have been able to show that interesting heating properties can be reach with really small nanoparticles, by a simple synthesis, and with a safer-by-design approach. Moreover, in vitro experiments have been performed to validate their use for biology and medicine. References 1- N. Li et al, Ang. Chem. Int. Ed. 53, 1756 (2014) 2- X. Huang et al, Lasers Med, Sci. 23, 217 (2008) 3- S. Wang et al, Chem. Phys. Let. 463, 145 (2008) 4- J. Kolosnjaj-Tabi et al, ACS Nano 9, 7925 (2015) 5- M. Sun et al, Nanoscale 8, 4452 (2016) 6- S. Kang et al, ACS Nano 9, 9678 (2015)

Resume : The interest of mesoporous silica nanoparticles (MSNs) as drug delivery system rises from their advantageous structural properties, biocompatibility, high loading capacities and the possibility to selectively functionalize the material both on the external surface and within the pores. However, MSNs, tends to accumulate into the different organs in the body hindering their commercialization as a medical tool. To overcome this issue, we developed disulfide-doped large pore breakable mesoporous silica nanoparticles (BPBPs) characterized by large pores of ca 10 nm and able to degrade in small pieces upon exposure to reduced glutathione (GSH), a natural reducing agent overexpressed in cancer cell. Compared to standard MSNs (MCM-41 type, 2.5 nm pores), BPBPs are able to incorporate a double strand siRNA within the pores, therefore protecting the gene from nucleases and allowing its safe transfection within the cells. We developed a system able to deliver siRNA, silencing the expression of the polo-like kinase 1 gene (PLK1), which regulates cell proliferation and is overexpressed in many cancer types such as the hepatocellular carcinoma Huh-7 cells. The BPBPs have then been coated with a linear polyethylemimine (jetPEI®), in order to protect then the siRNA and increase the cellular uptake. We demonstrated that our system was able to release efficiently the biomolecule, and to reduce tumor growth in xenograph….

Resume : Porous silicon (PSi) nanoparticles (NPs) are known to be biocompat-ible, biodegradable, and promising for diagnostics and therapy of can-cer. The present work is devoted to experimental study of a combined effect of PSi nanoparticles (NPs), which were loaded with anti-tumor drug, and activated by therapeutic ultrasound (US) to suppress cancer tumor growth in vivo. PSi NPs were prepared from mesoporous PSi films grown by electrochemical etching of crystalline Si wafers fol-lowed with high-energy ball milling. The prepared NPs were loaded by doxorubicin (DOX) and tested to suppress cancer tumor growth in mice. The US treatment with DOX-loaded PSi NPs was found to result in a strong suppression of the tumor growth and prolongation of mice’ lifetime. The same US-treatment with unloaded NPs or injection of DOX–loaded NPs without US treatment induced weaker effects on the tumor growth inhibition. The obtained results indicate that PSi NPs are promising for applications in sonodynamic and combined therapy of cancer.

Resume : Among inorganic colloids, silica nanoparticles own a variety of unique features, including low cytotoxicity and scalable synthetic availability, but also wide possibility to precisely control particle size, porosity, crystallinity, and shape to tune their nanostructure for diverse applications. In particular, a number of silica-based nanomaterials with engineered shape, structure and surface modification have been prepared and evaluated for applications in the biomedical area.1 One approach consists in the development of functional nanoparticles that can directly interact with cells or tissues.2 Yet the understanding of silica reactivity in biological conditions remains highly challenging, especially because the question whether mammalian cells exhibit specific biological mechanisms to degrade silica remains open. Alternatively silica nanomaterials can be used to construct composite hydrogels where they can play a dual function of mechanical strengthening and drug delivery.3 However more work is needed to optimize the silice-polymer interface and interplay. We addressed the first question by preparing three types of PEGylated fluorescent silica nanoparticles with various internal structures (core-shell bio-composite, multilayered and hollow mesoporous) and compared their degradation in abiotic conditions and in contact with human dermal fibroblasts.4 All particles were uptaken by cells with limited cytotoxic effect. Their intracellular degradation occurred faster than in solution but following almost similar dissolution mechanisms. These results strongly suggest that silica nanoparticles must be considered as bioresorbable but not biodegradable, a point of importance for their future application in drug delivery. In particular, the ability of silica-based nanostructured particles for cancer cell imaging and killing will be presented. Considering the bionanocomposite approach, we have prepared silica nanorods and combined them with type I collagen to obtain novel hydrogels with tunable mechanical properties. This allowed to study the proliferation and migration of cells on materials with similar mechanical properties but different compositions. These results provide fruitful guidelines for further development of such nanocomposites as biomaterials. References 1. Z. X. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart and J. I. Zink, Chem. Soc. Rev., 2012, 41, 2590-2605. 2. A. M. Mebert, C. Aimé, G. S. Alvarez, Y. Shi, S. A. Flor, S.E. Lucangioli, M. F. Desimone and T. Coradin, J. Mater. Chem. B, 2016, 4, 3135-3144 3. X. Wang, C. Hélary, T. Coradin, ACS Appl. Mater. Interfaces, 2015, 7, 2503-2511 4. Y. Shi, C. Hélary, B. Haye and T. Coradin, Langmuir, 2018, 34, 406-415.

Resume : Silica nanoparticles are attractive material for cancer imaging and therapy. These type of particles showed low cytotoxicity and their silica matrix can be easily modified to encapsulate drugs or to add new functional groups and bioactive elements. Functionalization with vectors, ligands, fluorophores and/or drugs is often achieved by multistep chemical reactions (e.g. amine-NHS ester, click chemistry, thiol-maleimide) requiring the prior surface modification of NPs. In this context, we first describe a straightforward method relying on the synthesis of trialkoxysilyl hybrid building blocks (i.e peptides, fluorophores, polymers) to yield in one step multifunctional NPs using in sol-gel conditions. We applied our methodology to the synthesis of multifunctional, cancer targeting, fluorescent NPs, presenting integrin ligands and other tumor specific binders as well as polyethylene glycol chains for furtivity. Starting fluorescent NPs were obtained by co-condensation method followed by post-synthetic grafting with various triethoxysilyl hybrid peptides and polymers. To achieve accurate control of the grafting of the different elements, we incorporated a fluorinated probe into the hybrid building blocks. It allows their easy quantification by 19F NMR with ERETIC Method (Electronic Reference To access In vivo Concentrations). The synthesis of hybrid silylated peptides, fluorophores and polymers will be presented as well as the cancer targeting results obtained by the co-presentation of several different ligands on the surface.

Resume : Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs, uncontrolled use of these drugs has promoted the development of resistance to current antifungals. The clinical implication of resistant strains has led to the search for safer and more effective drugs as well as alternative approaches, such as controlled drug release using liposomes and photodynamic inactivation (PDI), to eliminate pathogens by combining light and photosensitizers. In this study, we used layer-by-layer (LBL) assembly to immobilize triclosan and acridine orange encapsulated in liposomes and investigated the possibility of controlled release using light. The effects of laser irradiation were investigated by fluorescence microscopy, atomic force microscopy, and release kinetics. Liposomes were successfully prepared and immobilized using the self-assembly LBL technique. Triclosan was released more quickly when the LBL film was irradiated. The release rate was approximately 40% higher in irradiated films (fluence of 15 J/cm2) than in nonirradiated films. The results of the susceptibility experiments and surface morphological analysis indicated that C. albicans cell death is caused by PDI. Liposomes containing triclosan and acridine orange may be useful for inactivating C. albicans using light. Our results lay the foundation for the development of new clinical strategies to control resistant strains.

Resume : Nanomedicine continues to gain significant interest, to which dendrimer has emerged as a promising candidate for many applications, thanks to its various advanced functionalities. We report herein the synthesis and characterization of dendrimer-coated magnetic nanoparticles (NPs) which aim to meet dual purposes: early diagnosis and targeted therapy. Our nanoparticles are made of a spherical iron oxide core synthesized by thermal decomposition and coated with functional hydrophilic dendrons via a biphosphonic tweezer. The presence of discrete polyethylene glycol chains at their periphery allows enhancing the aqueous solubility, thus the interaction with water molecules and then MRI contrast enhancement properties, while the branched structure allows for long-term stability of the dendronized NPs in physiological media. Those nanoprobes displaying a small hydrodynamic size (smaller than 30mn), they show excellent biodistribution and bioelimination post i.v. injection with complete clearance after 24 hours via renal and hepatobiliary pathways, and without any unspecific uptake (no RES uptake). By conjugating those dendronized NPs with a small specific ligand targeting melanine granules, we highlighted their specific accumulation in tumor cells of Melanoma B16F0 mice via confocal microscopy, MRI and PET imaging studies.

Resume : The aim of the study was the fabrication of a new immunochromatographic strip for rapid quantitative detection of myoglobin in human serum, an early cardiac protein biomarker important in Acute Myocardial Infarction (AMI) diagnosis. Determination of myoglobin is important in estimating the severity of AMI followed by setting the treatment as soon as possible. The immunochromatographic strip is assembled from nitrocellulose membrane (test pad), fiberglass membrane coated with antibody– fluorescent nanoparticles bioconjugates (conjugate pad) and cellulose membranes (sample and absorbent pad). We used fluorescent nanoparticles, cadmium telluride (CdTe) hydrophilic quantum dots coated with carboxyl groups for bioconjugation with specific antibodies. A commercially available inkjet printer was used to deposit specific antibodies (a solution of monoclonal mouse anti-human cardiac myoglobin antibodies to a concentration of 1 mg/mL for Test Line and a solution of goat anti-mouse IgG polyclonal antibodies to a concentration of 1.5 mg/mL for Control Line) which enables wide-area printing, in a uniform and thermally controlled atmosphere. Test and control lines were measured in fluorescence by a portable dedicated platform, designed and fabricated for immunochromatographic strips. The results show that the linear fitting algorithm gave a value of 0.975 for the regression coefficient R2 in the calibration range 50 – 400 ng/mL. These results (design, fabrication and tests) allowing the development of a point-of-care system based on fluorescent nanoparticles will be presented.

Resume : The aim of research is the microfabrication of an immunosensor. All stages of functionalization were characterized by SEM (Electronic Scanning Microscopy), FTIR and Electrochemical Impedance Spectroscopy in order to understand the contribution of each layer, initially using flat gold surfaces, at each stage changing the electrochemical parameters ΔEp and Rct. Preparing and cleaning the flat surfaces and the electrodes represents one of the most important stage in the development of immunosensors as it affects the stability and homogeneity of the SAMs monolayer. In this case, the chemical cleaning was performed by simple immersion followed by an electrochemical cleaning. Then a mixed SAMs (mSAMs) consisting of a mixture of 11 MUA and 6 mercaptohexanol (6 MPOH) was made, compared with a self assembled 11 MUA (sSAMs) layer. The quality of the obtained layers was determined by cyclic voltammetry, FTIR spectroscopy (for studying the chemical bond configurations from the thiol samples deposited on the gold substrate) and SEM. The adhesion of the thiols to the surface of the gold substrate is confirmed in the 2600-2400 cm-1 region and in the 750-700 cm-1 area, with the possibility of creating Au-S bonds. The spectrum samples are dominated by the presence of the absorption bands in the 3000-2800 cm-1 region, which can be attributed to the symmetrical and asymmetric vibration mode of the C-H bonds from the methylene groups, suggesting the possibility of arranging the hydrocarbon chains in the gauche conformation (sin-intercalated). The Nyquist diagrams are made by the Randles circuit - which describes the electrochemical properties of the electrode at its interface. The greatest decrease was recorded in the formation of the self-assembled layer of 11MUA thiol molecules case due to the high containment capacity of this film compared to the SAMs where the isolation is lower.

Resume : A novel strategy to directly separate chiral drugs by using modified MOF materials which were functionalized through cation exchange process was developed. Considering the stability and the pore size, the follow anionic MOFs were selected: MIL-101-SO3H(Cr), UiO-66-SO3H and [Et4N]3[In3(BTC)4] (H3BTC = benzenetricarboxylic acid). Chiral cations, N-benzylcinchonidinium, (S)-butan-2-aminium and (−)-N,N-dimethylephedriniumwere incorporated into these anionic achiral MOF scaffolds respectively by taking place of the endogenous cations to give chiral cation@MOF structure. These MOF materials were characterized through NMR, XRD and ICP and were used as stationary phase in gas chromatography to efficiently separate common racemates such as ephedrine and ibuprofen. These results provide a platform for the development of modification of MOFs for chiral resolutions by direct exchange of chiral cation into anionic MOFs.

Resume : Lipid nanocarriers emerged as promising candidates for drug delivery and cancer targeting because of their low toxicity, biodegradability and capacity to encapsulate a drug or a contrasting agent. However, because of poor understanding of their in vivo fate and integrity, their translation from laboratory to biomedical applications is limited. In this work, we exploited the Förster Resonance Energy Transfer (FRET) technique for real time monitoring of their stability in vivo. Using our recently developed approach of hydrophobic counterion tetraphenyl borate (TPB), we encapsulated two lipophilic NIR cyanine dyes (Cy 5.5/TPB and Cy 7.5/TPB) inside a lipid nanocarrier of 100 nm size. [1] Using two-color whole animal NIR imaging with these nanocarriers, we could quantify their integrity directly in blood circulation, liver and tumor xenografts of living mice. This methodology reveals that the particles remain stable in the blood circulation for at least 6h. They accumulate in tumor rapidly in mostly entire form (77% after 2h) through enhanced permeability and retention (EPR), and then disintegrate with half-life of 4.4 h. In conclusion, we showed that lipid nanocarriers can deliver their cargo to tumors in nearly intact form. 1) Bouchaala, R.; Mercier, L.; Andreiuk, B.; Mély, Y.; Vandamme, T.; Anton, N.; Goetz, J.G. Klymchenko, A.S. J. Controlled Release, 2016, 236, 57. Acknowledgements: This work is supported by ERC consolidator grant Brightens 648528

Resume : Drug delivery systems who has property of encapsulating both hydrophilic and hydrophobic in double emulsion system (e.g. W/O/W) contents are of great interest for scientists. In our study, we have proposed the formation of such a system in the form of double emulsion at nano scale. Reaching such a small level can lead to loss of encapsulation efficiency of system. Herein we proposed a new technology that reinforce the inner water droplet with silica shell, resulting in the improvement of the encapsulation efficiency. Firstly, primary W/O emulsion is prepared by ultra-sonication and its characterization and optimization for size distribution and dispersity index is done to select the best candidate for making the double emulsions. Then, double emulsion at macro-scale are obtained by mechanical mixing and at nano-scale by spontaneous emulsification. Morphological studies by optical microscopy is done to visualize the double structure at micro scale. Encapsulation efficiency and stability against severe temperature conditions has been analyzed and proved that system having silica are much more effective regarding encapsulation as compared to control. Morphological studies by transmission electron microscopy are done to visualize appearance of the nano double emulsion droplets. Then, encapsulation studies proved that systems having certain amount of silica are more protective as compared to control even at nano scale. Finally, release studies proved that system can release almost all of encapsulated materials at 37⁰C.

Resume : Doxorubicin (DOX) is a widely used anti-cancer drug. The important factor limiting the clinical use of DOX is its toxicity. The DOX entrapped in liposomal formulation have shown reduced toxicity. The objective of this study is to develop a novel efficient pH-sensitive liposomal formulation able to encapsulate doxorubicin and to ensure targeted and controlled delivery of DOX to the cancerous area, resulting in reduced toxicity of DOX. The major challenge for encapsulating DOX in pH sensitive liposomes is its aqueous solubility, i.e., it is soluble at acidic pH and its solubility decreases with increasing pH, while on the other hand the pH must be alkaline to formulate the pH sensitive liposomes. The first part of this study focuses on the formulation of carboxyfluorescein (CF) loaded pH-sensitive liposomes and studying the impact of the formulation parameters (chemical nature of lipids and the lipid/stabilizing agent ratio) on the pH sensitivity of the liposomes. The pH-sensitivity was assessed by measuring the release of CF from liposomes at different pH values and for different incubation periods by using a fluorescent methodology based on self-quenching of CF. Optimized liposome formulations were then selected for the encapsulation of DOX by active loading procedure, through a pH gradient between the aqueous phase inside and outside the liposomes. Numerous experimental conditions were explored, which allowed identifying critical parameters for the efficient DOX encapsulation in pH-sensitive liposomes. The results have shown that these liposomes are stable at physiological pH and collapse in slightly acidic medium to release their DOX load, thus providing an efficient targeted delivery system for anticancer drugs.

Resume : Recently, we directly fabricated the graphene-based composite from graphite in the presence of chlorin e6 (Ce6) by sonication in aqueous phase [1]. The composite (G/Ce6) was found to deliver the Ce6 into cancer cells and work as a photosensitizer for cancer photodynamic therapy (PDT). In this paper, we used h-BN in place of graphite and realized much higher Ce6 concentration in the composite of h-BN/Ce6 than that of G/Ce6. 　Aqueous dispersion of h-BN/Ce6 was prepared by use of commercially available h-BN and Ce6 under the same conditions as those in the preparation of G/Ce6. The formation of h-BN/Ce6 was confirmed by the red shift of Q band in the absorption spectra and quenching of the fluorescence. In comparison of these dispersions, the contents of Ce6 and the carrier were 10 and 20 times larger in h-BN/Ce6 than G/Ce6, respectively. Since the size of h-BN is much smaller than that of graphite, it is concluded that h-BN was exfoliated much more easily than graphite in the presence of Ce6 in water. This is supported by the phenomenon that the loading capacity of Ce6 on h-BN is half of that on graphene. 　The cytotoxicity of h-BN/Ce6 to HeLa cells was confirmed under irradiation of 660 nm LED light. While Ce6 without carrier exhibited no cytotoxicity, viability of the cells significantly decreased to less than 20% at the Ce6 concentration of 0.2 µg/mL. This PDT effect by use of h-BN/Ce6 is quite similar to that of G/Ce6, indicating no difference between h-BN and graph ene as a drug carrier. [1] G. Liu, H. Qin, T. Amano, T. Murakami, N. Komatsu, ACS Appl. Mater. Interfaces. 7, 23402 (2015)

Resume : Lanthanide-doped nanomaterials have emerged as a promising candidate for bio-imaging applications because they allow low-energy excitation for minimized autofluorescent background in bio-tissues. Although upconverting nanomaterials were widely developed, the low quantum yield of upconversion process and their visible emissions are major challenges for deep tissue bio-imaging. This paves an attractive way to explore near infrared-to-near infrared downconverting nanomaterials for achieving better imaging penetration depth. In this work, tetrahedral KY3F10 nanoparticles (NPs) singly doped with Nd3+ ions were synthesized by the co-precipitation method. Surprisingly, the size at sub-10 nm and morphology of these NPs exhibited negligible change as the Nd3+ dopant concentration increased from 1% to 15%. Nd3+ ions simultaneously acted as a sensitizer and emitter, displaying strong downconverion luminescence at 1054 nm upon 808 nm excitation. With the wavelengths falling within the biological windows, the NPs are highly favorable for deep tissue optical imaging. In addition, they showed excellent water dispersibility but little cytotoxicity towards HeLa cells after surface modification with poly(acrylic acid). Taking advantage of the superior optical property and small size, the NPs may also be potential for in-vivo bio-imaging.

Resume : In the past two decades, several novel superparamagnetic nanoparticles (MNPs) were shown to have great potential in biomedical applications. One of the most promising such applications seems to be in cancer treatment using magnetic hyperthermia (MH). Lately, NiCu based nanoparticles (especially with the composition Ni67.5Cu32.5), coated in a thin layer of silica, attracted an increasing interest for dual therapeutic interventions in cancer treatment. Namely, the prepared materials can not only induce MH, but can at the same time act as an efficient delivery system for drugs to the cell interior. In one of latest studies, we have demonstrated a concept to design a novel innovative drug delivery system based on Ni67.5Cu32.5 nanoparticles in a silica matrix that can deliver a model fluorescent drug rhodamine (RHO6G) to various human cells (human skin fibroblasts, and model cancer cells, e.g. HeLa, Caco-2). The drug release performance was assessed using in vitro drug release studies. The combination of different physicochemical and morphological methods with biocompatibility studies served as a general evaluation of the novel formulations safety and efficiency.

Resume : To reduce the risk of desiccation and evaporative cooling, during both open and laparoscopic surgery , humidifying and heating systems are currently used especially to maintain moist and warm the peritoneum ( e.g. F&P HumiGard? System ). This maybe an innovative delivery device for drugs or other substances to prevent the spreading of tumoral seeds in the tissues and organs nearby the resected one ( adjuvant or concurrent therapy). Nanotech approaches can be exploited to tailor suitable nanocarriers. In order to reach this goal, apart from the intrinsic drug properties, the nanocarrier should be non toxic, biocompatible and biodegradable, microbiologically sterile, of dimensions lower than equipment filters, with buoyancy properties which make it able to join the vapour flow along the delivering tube and stable enough to avoid disruption. All the above requirements can be satisfied by nanometric ( < 100 nm) droplets shelled by chitosan and filled with Perfluorocarbons vaporizing at body temperature and therefore producing very low weight nanobubbles, which are also monitorable with clinical sonographers (1). Such nanostructures may contain drugs or other substances either in the inner core or at the interface, and are therefore very versatile and can virtually carry any of them. Preliminary results are presented, showing their physico-chemical characterization, stability, and mechanical resistance in the vapour current at large shear stress conditions. (1) 2H,3H-Decafluoropentane-Based Nanodroplets: New Perspectives for Oxygen Delivery to Hypoxic Cutaneous Tissues M Prato et al. PloS one Volume: 10 Issue: 3 Pages: e0119769 2015

Resume : Lumiphores that emit within the red or NIR are highly desirable for biological imaging applications, as longer wavelengths of light penetrate deeper through tissue compared to shorter wavelengths. Square planar Pt(II) polypyridyl complexes,[1] that are capable of forming red to NIR emissive self-assembled aggregates, have recently emerged as candidates for imaging applications. However, a common drawback of these complexes is poor water solubility that precludes their used for imagining. Judicious ligand selection is often required to provide sufficient solubility for use in imaging. Here we report a series of water insoluble square planar Pt(II) complexes that display red to NIR emission upon aggregation. Sublimation of these complexes onto mesoporous silica nanoparticles resulted in highly emissive, water dispersible nanoparticles. The emission from sublimed Pt(II) complexes was hypsochromically shifted compared to the fully aggregated species, though significant emission was still observed beyond 650 nm. This technique represents a novel means of generating emissive materials for biological imaging applications, which takes advantage of the known emission properties of self-assembled Pt(II) complexes. 1. Ly. Kiet Tuong; C.-C. Ren-Wu; L. Hao-Wu; S. Yu-Jeng; L. Shih-Hung; C. Pi-Tai; T. Cheng-Si; H. Yu-Ching; C. Yun, Nature Photonics, 2017, 11, 63–68.

Resume : Due to their ability to carry anticancer drugs and generate localized heat when exposed to an alternating magnetic field, superparamagnetic iron oxide (SPIO) nanoparticles (NPs) can be used as multimodal cancer therapy agent by combining chemotherapy and hyperthermia1. Core/shell Fe3O4@copolymer nanoparticles were synthesized with covalent grafting of a thermos-responsive biocompatible copolymer based on 2-(2-methoxy) ethyl methacrylate (MEO2MA) and oligo (ethyleneglycol) methacrylate (OEGMA) on a superparamagnetic NPs surfaces. The lower critical solution temperature (LCST) of grafted copolymer was tuned in physiological media in order to release the cancer drug at controlled temperatures. The 41-42°C LCST was obtained with a copolymer composed with 60% MEO2MA and 40% OEGMA. Another class of responsive NPs was obtained by the functionalization the Fe3O4@copolymer by folic acid (FA) which led to the Fe3O4@copolymer-FA. Both types of NPs (Fe3O4@copolymer and Fe3O4@copolymer-FA) were loaded with the anticancer agent doxorubicin (DOX). In vitro, DOX release kinetics was investigated at 42°c: 25% at 5h, 50% at 24h and 100% at 56h of DOX release were measured for both types of NP. At 37°C, NPs were found stable until 24h (<10% DOX release)2–4. Further, the viability of human ovarian cancer cells (Skov3) exposed to NPs, free DOX or DOX-NPs for 24h at 41°C or 37°C for control cells, was assessed by measuring cell metabolic activity (MTT test). Results showed that NPs (without DOX) preserved cell viability (> 78.44 ± 9.48 %) irrespective of the temperature and the NP concentration. This study demonstrates the potential of these nanoparticles for cancer treatment combining chemotherapy and hyperthermia.

Resume : In this work, hybrid assemblies of plasmonic gold nanoparticles (AuNPs) and angiogenin peptides were investigated in their interaction with artificial membranes of supported lipid bilayers (SLBs) and cellular membranes of cancer (neuroblastoma) and normal (fibroblasts) cell lines. The peptides used contained the angiogenin (60-68) sequence, i.e., the putative cellular binding site of the protein. Three analogous fragments were employed, namely Ang(60-68), the cysteine derivative Ang(60-68)Cys, and the fluorescein-labelled Fam-Ang(60-68). These fragments allowed to compare the purely physisorption mechanism of the peptide onto gold to the covalent immobilization and to the interaction affected by steric and charge effects. The peptide-gold nanoparticles was characterized by means of UV-visible, AFM and CD, to address the plasmonic changes, the nanoparticle coverage and conformational features at the hybrid biointerface. Lateral diffusion measurements on SLBs after their interaction with the peptide-functionalised AuNPs pointed to a stronger membrane interaction in comparison with the uncoated nanoparticles. Cell viability and proliferation assays indicated a slight nanotoxicity, only for the longer incubation times, in neuroblastoma cells and a proliferative activity in fibroblasts. The cytoskeleton features, observed by confocal microscopy imaging of actin staining, pointed out different levels of interaction between the different AuNP-Ang peptides and the cell membranes.

Resume : Biosensor having electrical detection method can decrease the time and cost needed for sample preparation due to labeling-free. Also, electrochemical biosensor has advantages that it can be fabricated with miniaturization and portable device based on simplicity, rapid response time and high sensitivity. For fabrication of biosensor, various materials have been used, and Carbon Nanotube (CNT) and Graphene have mainly been utilized because of exceptional electrical property. However, these have critical disadvantage as low reproducibility. On the other hand, Indium Tin Oxide (ITO) shows reproducibility as well as remarkable electrical property that were proved by plenty of studies and paper. In this research, we fabricated ITO-based biosensor having detection of tumor marker (Alpha-fetoprotein; AFP) in blood serum, and report a number of results using biosensor. Electrochemical biosensor that has micro scale consists of 10 ITO channels fabricated by RF sputtering system. The surface and cross-section of biosensor were analyzed through Field Effect Scanning Electron Microscope (FE-SEM) and Atomic Force Microscope (AFM). Then, we investigated stability of biosensor device based on electrical properties between source and drain, identifying voltage-current curve using Probe station. Additionally, we introduced outstanding detection ability and selective detection depending on concentration of blood serum with tumor marker.

Resume : The Alzheimer′s disease (AD) is challenging to develop early diagnosis of AD due to the lack of a decisive biomarker in blood. Recent reports on the amyloid-β (Aβ) as a biomarker demonstrated its possibility for identifying early onset of AD in patients, but its low concentration in blood requires highly reliable detection techniques. In this work, we developed an Indium Tin Oxide (ITO) microchannel array-based biosensors for AD diagnosis based on impedimetric detection by Aβ, which is a biomarker for AD. The biosensors ware fabricated using conventional micro-fabrication technique. Electivity of the reaction due to the affinity of Aβ to the antibody and the sensitivity according to the concentration of Aβ were also demonstrated. This microchip consists of 10 ITO microchannels fabricated by photolithographic process and sputtering system. Consequently, the biosensors has strong potential as a feasible system for use in the diagnosis of AD with a fast and easy immunoassay process, since the suggested platform can be automated with ease for point-of-care testing as well as high-throughput diagnostic equipment

Resume : The main challenges are the design of NPs which will allow, in one nano-object, combining imaging and efficient therapy, the design of an organic coating bearing different functions allowing optical imaging, furtivity, biodistribution, and targeting, while keeping NPs at the nanoscale and their in vitro and then in vivo validation. In that context, different types of nano-objects such as carbon nanotubes filled with ferrite NPs, cubic shaped and core-shell NPs coated with dendron molecules and silica coated NPs will be shown to be promising to combine therapeutic and imaging properties.

Resume : In the field of the synthesis and functionalization of magnetic nanoparticles (NPs) for biomedical applications, most researches aim at developing multifunctional theranostic NPs which can both identify disease states and deliver therapy and allow thus following the effect of therapy by imaging. The main challenges are the design of NPs which will allow, in one nano-object, combining imaging and efficient therapy, the design of an organic coating for colloidal stability and their in vitro and then in vivo validation. In that context, we have developped magnetic carbon nanotubes combining both diagnotic and therapeutc properties. Carbon nanotubes (CNTs) were filled with a very high loading with ferrite NPs to develop their magnetic manipulation and theranostic applications. The originality of the filling process was the use of CNTs as nanoreactors and which provide also stable containment of nanomagnets. Cellular studies showed very promising results in terms of magnetic handling and hyperthermia at the subcellular level. The magnetic CNTs thus prepared are adjustable within the cell by an external magnetic field. By rotating them such a nano-drilling on the cells, they increase their uptake by tumor cells and potentiate the cytotoxic effect of light irradiation. They are capable of absorbing and efficiently converting NIR light into heat to generate thermoablative temperatures and cell lysis. They can be used as T2 agents for MR image-guided photothermal therapy. These multifunctional magnetic CNTs visible by MRI, activated by light and manipulated magnetically are good candidates for local activation and control of biological processes in the body.

Resume : The superparamagnetic behavior of Gadolinium that appears when its size is reduced to nanoscale arouses great interest and is used as magnetic resonance imaging contrast agent as well as in treatments against cancer by hyperthermia. Laser ablation of solids in liquids (LASL) is a very powerful technique because allows for controlling size and shape of nanoparticles by tuning processing parameters. At same time, let us also to obtain pure nanoparticles with no need of chemical precursors or chemical reactions which can contaminate the obtained material, of special importance in the case of biomedical applications. In this work, two laser sources with 1064 and 532nm of wavelength were employed to ablate a gadolinium plate submerged in two different solvents (acetone and methyl alcohol). Size, morphology and crystalline phases of the obtained nanoparticles were studied by means of transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDS) and UV/VIS absorption spectroscopy. The influence of the wavelength and the solvent in the formation mechanism was studied and discussed. The obtained colloidal solutions consisted of Gd nanoparticles showing homogenous and rounded shape with diameters ranging from few nanometers to 50nm, presenting a greater degree of agglomeration those processed in acetone.

Resume : The design and development of magnetic core-porous silica shell nanocomposites for nanomedicine applications has known a tremendous interest this last decade. Such nanomaterials can be defined as one or several magnetic core NPs embedded in a porous silica inorganic matrix. The combination of these two inorganic components affords new and complementary properties for biomedical applications. Indeed, while the magnetic core brings the remote magnetic features ensuring imaging by magnetic resonance imaging (MRI) and/or therapy by magnetic hyperthermia (MH) and magnetic manipulation, the porous silica shell brings: a high colloidal stability in aqueous solution, a high degree of surface functionalization and capacity of therapy by drug delivery. In a first work, we focused on a new strategy for covalent grafting of quantum dots using large pores (ca 15 nm) mesoporous silica with a stellate morphology to add a new imaging property. The quantum dots were encapsulated with a high efficiency (90%). To prevent the toxic release of quantum dots, two coating were investigated. i) a coating of a further MS shell having small pores (SP) (ca. 2.5 nm) or ii) a tight polysaccharide shell deposited on the surface of these STMS NPs particles via the isobutyramide(IBAM)-mediated method. These two ways were shown very efficient to coat the STMS@QDs and to ensure QDs protection, luminescence properties and colloidal stability. In a second work, 20 nm iron oxide nanoparticles made by thermal decomposition were combined with the previous fluorescent large pore stellate silica to form original magneto/luminescent composites for biological fluorescent and MRI imaging.

Resume : Conventional hyperthermia applied through microwaves or radiofrequency is an established adjuvant in oncology, combined with radiotherapy or chemotherapy. Using superparamagnetic iron oxide nanoparticles (SPIONs), magnetically-mediated hyperthermia may help to treat difficult-to-reach and deep-seated tumors[1]. A challenge towards clinical translation lies in the high SPION concentrations required at the tumor site to achieve an effective heat delivery to the tumor. We discuss herein two distinct routes of administration, local injection to the tumor site and subcutaneous injection of targeted nanoparticles. Local deposition of SPIONs embedded in formulations capable of forming a depot at tumor site combines tumor embolization with subsequent, repeatable mild hyperthermia. We investigated this approach for soft tissue and bone tumors. Following intratumoral injection of SPIONs suspended in a polymer solution, hyperthermia above 45 °C was obtained in mice xenografted with human colorectal tumors leading to a 45% one-year survival[2]. Similarly, using SPIONs embedded in an acrylic polymer used for spine stabilization (vertebroplasty), we demonstrated the safety of the procedure in sheeps. To treat vertebral metastases as the cause of spine destabilization, sustained delivery of doxorubicine over months was also shown, in view of a combined chemo-thermotherapy. In order to diagnose and potentially treat early prostate cancer metastases, SPIONs might be injected subcutaneously, trafficking towards sentinel lymph nodes to detect metastases. In this view, we decorated SPIONs with PSMA (prostate surface membrane antigen)-targeting ligands such as aptamer or small urea-like molecule. Specific binding to PSMA-positive cells (LNCaP) was demonstrated for both types of ligands. The nanocarriers could be detected by magnetic resonance imaging (MRI) in vivo, using optimized MRI sequences. The approach is therefore promising for the detection of specific cancer metastases. Still, in order to achieve therapeutic hyperthermia at the tumor site, efforts towards more effective heating and/or higher SPIONs accumulation are needed. References [1] Datta N.R., et al. (2016). Magnetic nanoparticle-induced hyperthermia with appropriate payloads: Paul Ehrlich’s “magic (nano)bullet” for cancer theranostics? Cancer Treatment Reviews, 50:217-227. [2] Le Renard P.E., et al. (2009). Local magnetic induced moderate hyperthermia treatment through an implant formed in situ in a mouse tumor model. Int J Hyperthermia, 2009.

Resume : Dermatological problems are a major part of pathological conditions that need to be addressed, and the way to deal with them evolves constantly. An attempt is being made for more specialized treatment of burns and skin regeneration to cosmetic use. The need to create a specialized method that can controllably transport medicine only where is needed, has led to the construction of biodegradable and non-toxic, localized, controlled-release drug delivery scaffolds. In this study, a Drug Delivery Nanoplatform of polymeric Polylactic acid (PLA) and Chitosan scaffolds, loaded with curcumin drug and aloe vera extract has been fabricated via Electrospinning process. The surface structure of the scaffolds was observed using Scanning Electron Microscope and Atomic Force Microscope. The cytotoxicity of drug–loaded scaffolds on fibroblasts was investigated in vitro, using MTT and brdU proliferation assay and Methylene Blue staining, showing excellent compatibility. The release behavior of both drugs from the nanoid platform exhibited a biphasic release pattern and the degradation rate of the drug loaded scaffold was higher than blank scaffold. To get the research work one step further, GFP protein immobilization on the nanofibers was successfully accomplished by EDC-NHS chemical method, thus achieving to prove that the biofunctionalization of the fibers can occur. This felicitous approach may be the priming treatment of dermatological malfunctions and alleviate patients’ needs.

Resume : Due to the ultrasensitivity, low operation potential <1V and the possibility to operate in aqueous media, Electrolyte-Gated Organic Field Effect Transistors (EGOFETs) are emerging as ideal candidates for the next generation of biosensors suitable for the detection and quantification of biomarkers. Functionalising the gate electrode with specific recognition moieties, like antibodies or aptamers, enable the EGOFETs to sense their binding with the target antigens in the solution. In this study the possibility of integrating multiple top-gate electrodes to the same semiconductive channel is proposed and validated. First, a redox sensor has been built exploiting two top gates, one exposing bare gold to the electrolyte and the other functionalised with an electrochemically active Self-Assembled Monolayer (SAM). Changes in the redox state of the SAM are shown to strongly affect the characteristics of the EGOFET with respect to the characteristic obtained addressing the bare gate. Second, a lab-on-chip device is obtained by the integration between EGOFET and a microfluidic channel featuring perfusion controlled by a peristaltic pump. The proposed novel approach of patterning four top gold gate electrodes and functionalising three of them with specific recognition moieties allows the sensing of different concentrations of the inflammatory biomarker Tumor Necrosis Factor (TNF-α) in buffer solution (lowest concentration 1pM), in a ratio metric approach with an internal reference. The configuration with multiple top gate electrodes allows statistical robustness as well as continuous monitoring of the stability of the organic electronic device. The choice of different sensing moieties with a specific sensing function on each electrode allows the creation of a multi-sensor device capable of performing simultaneous detection of different analytes in complex electrolytes.

Resume : Mercaptobenzoic acid (MBA) monolayer-protected nanoclusters of average formula Au102MBA44 are attractive nanometric materials because of their precise formula, small sizes and ease of functionalization via exchange of the MBA ligands with thiols but not with disulfide. The small size renders the materials compatible for diffusion in water and dense and filament-rich tissue and cytosol. Moreover, the exclusive sensitivity of the monolayer coverage to thiolated molecules can be exploited for selective release of the cluster’s coverage inside the cytosol since this compartment contains large amount of reduced gluthione. To evaluate the diffusion ability and stability of the Au-thiol bond into the cell, we prepare and characterized various peptide-coated gold nanoclusters by exchanging some MBAs to thiolated peptides selective to intracellular subcellular organelles. The peptide-covered gold nanoclusters are then electroprated inside living HeLa cells and their fates are determined by detecting the gold clusters. Our data show that these nanoparticles diffuse into the dense and filament-rich cytosol without impacting the cell viability. We also provide data on the stability of the peptide-coated gold nanoclusters within living cells. Altogether, our results will help to conceive novel nanometric materials with biomedical application.

Resume : Active immunotherapy is at the forefront of anti-cancer therapies because it combines both a high degree of specificity to general high effectiveness and fewer side effects on healthy cells. Nevertheless, successful therapeutic anticancer treatments are not yet been obtained with current immunotherapeutic strategies. Gold nanoparticles (AuNPs) are very useful biocompatible antigen scaffolds and they can be engineered to present a high degree of multivalency.[1] The extracellular domain of the mucin-1 (MUC1) glycoprotein is an attractive target for the development of therapeutic cancer vaccines. Tumor-associated MUC1 (TA-MUC1) is found overexpressed on epithelial cancer cells and markedly underglycoslyated compared to MUC1 on healthy cells[2], which results in the display of new peptide and carbohydrate epitopes. In this contribution, we describe the efficaciously development of novel vaccine formulations using PEGylated AuNPs[3] as scaffolds for the multivalent presentation of TA-MUC1 and adjuvating B-cell epitopes. We show that our AuNP-based vaccine formulations elicit not only a robust humoral immune response but also a cellular immune response in wild-type mice. Our results show the great potential of TA-MUC1 vaccine candidates based on PEGylated AuNPs, especially considering their high biocompatibility and good immunogenicity. [1] Irvine DJ et al, Chem. Rev. 2015, 115, 11109-11146 [2] Nath S et al, Trends Mol Med 2014, 20, 332-342 [3] Maus L et al, ACS Nano 2010, 4, 6617-6628

Resume : Nanodiamonds (NDs) are promising material for medical field due to its huge specific surface area, ease of surface modification and biocompatibility. Previously, our group demonstrated that polyglycerol-functionalized fluorescent NDs having a 50 nm diameter (FND50) was taken up by cancer cells and showed strong fluorescence in the lysosomes [1]. In this research, we further extend the usage of NDs as cancer therapy agents utilizing the nature of passive targeting to cancer cell. First, we tried to apply FNDs to in vivo tumor imaging. We have adopted FNDs with a diameter of 100 nm to maximize targeting ability and fluorescence of each particle. FNDs are functionalized by polyglycerol (PG) to make the FNDs have more dispersibility in the medium. The resulting material, FND100-PG, shows good stability in both water and physiological environment as the medium. We succeeded in induction of FND100-PG to a mouse by intravenous injection. After 4 hours, the mouse did not die and strong fluorescence was observed from the liver and the tumor by in vivo and ex vivo photoimaging, respectively. A point worthy of special mention, strong fluorescence from the tumor was observed in the young tumor because uptake ability seems to attenuate as the tumor grows. NDs could be delivered to early- stage cancer considering this result. Next, we are going to apply NDs as DDS carrier. By conjugating anti-cancer drug such as cisplatin, NDs could be applied to cancer therapy. [1] L. Zhao. et. al. Adv. Funct. Mater., 24, 5348 (2014).

Resume : Single molecule imaging of biomolecules inside living cells using fluorescence microscopy is of key importance for the understanding of biological processes at the molecular level. The speed and resolution with which the molecules can be tracked depends strongly on the performance of the fluorescent probe. Ideal probes should combine high brightness with small size and absence of non-specific interactions. Dye-loaded polymer nanoparticles appeared recently as systems with exceptional brightness.[1] In particular, we used cationic dyes together with bulky hydrophobic counterions to achieve efficient fluorescence with various emission colors.[2?5] In this work we designed polymers with different types and degrees of functionalization in order to control the size and surface properties of nanoparticles made through nanoprecipitation. In this way nanoparticles with diameters of less than 10 nm were obtained that allowed encapsulation of large amounts of dyes, making them up to 10 times brighter than quantum dots. These particles were used for intracellular imaging with improved accessibility to crowded cellular regions. Grants: ERC consolidator grant BRIGHTSENS and ANR JC/JC grant supertrack. [1] A. Reisch and A. S. Klymchenko, Small 2016, 12, 1968. [2] A. Reisch et al. Nat Commun 2014, 5. [3] A. Reisch et al. ACS Nano 2015, 9, 5104. [4] A. Reisch, K. Trofymchuk et al. ACS Appl. Mater. Interfaces 2017, 9, 43030. [5] B. Andreiuk et al. Small 2017, 13, 1701582.

Resume : The interactions between nanomaterials and the immune system has become a hot topic in nanobiology. During our investigations of the immunological effects of various nanomaterials and the underlying mechanisms, we find that the interactions of these nanomaterials with immune factors/immune cells could be modulated through regulating their surface chemistry. Our recent work have shown that upconversion nanoparticles (UCNPs) with certain surface modifications can serve as an effective nano-adjuvant for dendritic cell (DC) based cancer vaccine and induce strong cellular immune responses, suggesting promising applications in cancer immunotherapy as well as DC tracking. We further load UCNPs with chlorin e6 (Ce6), a photosensitizer, and imiquimod (R837), a Toll-like-receptor-7 agonist. The obtained multitasking UCNP-Ce6-R837 nanoparticles under near-infrared (NIR) irradiation with enhanced tissue penetration depth would enable effective photodynamic destruction of tumors to generate a pool of tumor-associated antigens, which in the presence of those R837-containing UCNPs as the adjuvant are able to promote strong antitumor immune responses. More significantly, photodynamic therapy (PDT) with UCNP-Ce6-R837 in combination with the cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) checkpoint blockade not only shows excellent efficacy in eliminating tumors exposed to the NIR laser but also results in strong antitumor immunities to inhibit the growth of distant tumors left behind after PDT treatment. Furthermore, such a cancer immunotherapy strategy has a long-term immune memory function to protect treated mice from tumor cell rechallenge. Our work presents an immune-stimulating UCNP-based PDT strategy in combination with CTLA-4 checkpoint blockade to effectively destroy primary tumors under light exposure, inhibit distant tumors that can hardly be reached by light, and prevent tumor reoccurrence via the immune memory effect. Our results also highlight the critical roles of surface chemistry for the rational design of nanomaterials for immunotherapy.

Resume : Endoscopic Luminescent Imaging for Oncologic Surgery: the ELIOS project Fluorescence Imaging Guided Surgery (FIGS) is an optical navigation modality that enables the visualization of unapparent structures at the naked eye, and the evaluation of metabolic activities, such as organ perfusion. Fluorescence is obtained through injection of a fluorescent dye, which can emit a fluorescent signal after being excited by Near-Infrared light sources. ELIOS (Endoscopic Luminescent Imaging for Oncologic Surgery) is a project funded by the French Foundation ARC (Association for Cancer Research) and led by the IHU-Strasbourg, Institute of Image-Guided Surgery. The focus of the ELIOS project is on optimizing radical removal and reducing complications by means of FIGS applied to cancers of the gastrointestinal (GI) tract. Innovations are expected at various levels: 1) new smart fluorophores, 2) innovative hardware, and 3) optimization of current devices performance through software-based image analysis. Additionally, an extensive activity on education and dissemination around FIGS is being carried out, with the aim to increase the widespread adoption and to standardize the procedures with the creation of patient registry and the organization of consensus conferences. The structure of the ELIOS project and some of the ongoing and future preclinical and clinical trials will be outlined in the lecture.

Resume : The use of fluorescent imaging is a valuable tool for tracking and identifying different cell populations. Indeed, cell labelling techniques can help to answer key questions in cancer research, cell differentiation or regenerative medicine. Here we present a new approach for long term multicolor cell labelling. This method is based on fluorescent polymer nanoparticles (NPs) encapsulating large quantities of dyes, so-called dye-loaded polymer NPs. These new nanomaterials can combine biodegradability and low toxicity with superior brightness (1). Extending our approach of counterion-controlled encapsulation of dyes to different cyanine dyes allowed creating NPs with three distinct absorption and emission bands but identical size and surface properties that are endocytosed equally well by living cells. Mixing NPs of three colors in different proportions generates cell populations of any desired color code (2). The color codes are homogeneous within one cell population and transmitted through many generations of daughter cells. This technology is validated on numerous cell lines and up to 13 color codes and enables simultaneous tracking of co-cultured color-coded cell populations for >2 weeks. The technique was applied to measuring adhesion of multiple glioblastoma cell lines simultaneously and tracking of cells in 3D tumor models and living zebrafish. References : [1] A. Reisch, A. S. Klymchenko, Small 2016, 12, 1968. [2] B. Andreiuk et al. Small 2017, 13, 1701582.

Resume : Dye-loaded fluorescent polymer and lipid nanoparticles (NPs) appear as an attractive alternative to inorganic NPs, such as quantum dots [1]. Here, we address several challenges in the field: (a) making small and bright NPs of any desired color; (b) their application for long-term cell tracking in vitro and in vivo; and (c) understanding their integrity in animals when they reach the target tumor. Small size of polymer NPs (<40 nm) can be achieved through a nanoprecipitation of biocompatible polymers modified with 1-2 charged groups per chain [2,3]. To achieve high brightness, we proposed to use bulky hydrophobic counterions that prevent dye self-quenching inside polymer matrix [2].The obtained NPs are brighter than quantum dots and their color can be tuned from blue to near-infrared. These NPs internalize by endocytosis, which enables long-term labeling of cancer cells and their tracking in vitro and in vivo using >6 color codes [4]. Finally, we developed lipid nano-droplets encapsulating a FRET couple of near-infrared dyes [5], which enabled visualizing integrity of nanocarriers in blood circulation of mice and show that they can accumulate in tumors in nearly intact form. References: 1) Reisch, A.; Klymchenko, A.S. Small 12 (2016) 1968. 2) Reisch, et al Nat. Commun. 5 (2014) 4089. 3) Reisch et al, ACS Nano, 9 (2015) 5104. 4) Andreiuk et al, Small 2017, 13, 1701582 5) Bouchaala et al, J. Controlled Release, 236 (2016) 57. Acknowledgements: ERC consolidator grant BrightSens 648528.

Resume : The onset of resistance to chemotherapy drugs and radiotherapy, is a severe challenge that can be tackled using multi-modal strategies. A single nanostructure designed to perform multiple therapies at once at the site of the tumor could allow a significant reduction of doses and of systemic side effects [1]. Our research focuses on the treatment of deep solid tumors by the combined application of Magnetic Hyperthermia (MHT) and of the X-ray triggered generation of reactive oxygen species (ROS), a therapy known as Self-Lighted Photodynamic Therapy (SLPDT). In the already established Photodynamic Therapy (PDT), a photosensitizing agent is activated by UV or visible light to generate ROS, such as singlet oxygen ( 1 O 2 ); this induces oxidative stress in cancer cells with consequent DNA damage, apoptosis or necrosis. The applicability of PDT is limited to superficial tumors by the short penetration depth of light. SLPDT extends PDT to deep tumors by nanostructures that act as localized light sources when activated by highly penetrating radiation, typically 6MeV X-rays used in radiotherapy. The nanostructure transfers the absorbed energy to its photosensitizing constituent, which generates ROS leading to localized oxidative stress [2]. We already developed a nanostructure for SLPDT made of a nano-sized matrix of the photo- sensitizing material ZnO, that embeds scintillating CeF 3 nanoparticles [3]. The SLPDT efficiency has been proved on human adenocarcinoma cells (A549). Irradiation with low doses (< 2Gy, 6MeV) of X-rays from a radiotherapy source triggers ROS and singlet oxygen generation; this reduces the viability of cancer cells and blocks the cellular cycle before mitosis [4]. We are now extending its range of action by adding MHT functionality by means of magnetite superparamagnetic nanoparticles. MHT performances of the pristine magnetite nanoparticles are well characterized [5]; the MHT efficiency of the multi-material nanostructure are currently being tested. Finally, we plan to test in vitro the combined efficiency of sequential sessions of SLPDT and MHT on cellular lines of deep solid tumors (e.g. A549 cells). [1] Kemp et al. Advanced Drug Delivery Reviews 98, 1 pp 3-18 (2016) [2] Fan et al. Chemical Society Review, 45 pp.6488 (2016) [3] Rimoldi et al., Journal of Materials Science, Materials in Medicine 27,10, 159 (2016) [4] Orsi et al., submitted (2017) [5] Campanini et. Al., Nanoscale 7, pp. 7717 (2015)

Resume : Mesenchymal stem cells (MSC), either manipulated ex vivo to secrete antineoplastic compounds or relying or their endogenous immunomodulatory capacity, have been extensively investigated as cell carriers or cell therapies for treating a wide range of malignant and inflammatory diseases. Despite promising preclinical results, MSCs have largely failed to translate into broadly-applicable clinical application. This failure was majorly attributable to MSCs’ susceptibility to host-induced changes, limiting their ability to deliver a long-lasting effect. Our group have developed MSC membrane nano-vesicles, produced though a scalable and potentially cGMP compliant technological process, and which require no synthetic functionalization to achieve active targeting. These inherently-targeted MSC nano-vesicles were termed Nano-Ghosts (NGs). The NGs were shown to retain the cells’ membrane asymmetry and their surface-associated ability to target a wide range of tumor models. The NGs could be effectively loaded and used to selectively deliver diverse therapeutics including biological drugs, small molecules, and nucleic acids. Their abundance of natural targeting mechanisms allows the NGs to penetrate the entire tumor bulk and rapidly deploy their payload directly into the target cells’ cytoplasm and nucleus led to unprecedented tumor growth inhibition and increased animals’ survival in prostate and an established metastatic lung cancer models. The NGs led to no off-target effects.

Resume : Nanomedicines are 1-100(0) nm-sized carrier materials designed to improve the biodistribution and the target site accumulation of systemically administered (chemo-) therapeutic drugs. By delivering drug molecules more efficiently to pathological sites, and by preventing them from accumulating in healthy tissues, nanomedicines are able to improve the balance between efficacy and toxicity. Nanomedicines rely on the Enhanced Permeability and Retention (EPR) effect for efficient target site accumulation, which is notoriously known to be highly variable, both in animal models and in patients. To overcome this high heterogeneity in EPR, and to improve the (pre-) clinical performance of anticancer nanomedicines, we are working on systems and strategies to modulate and monitor tumor-targeted drug delivery. In the present lecture, several of these strategies will be highlighted, including pharmacological and physical modulation of tumor blood vessels and the microenvironment, and theranostic concepts for individualized and improved nanomedicine treatment.