Antonio Topete

Fluorescent drug-loaded, polymeric-based, branched gold nanoshells for localized multimodal therapy and imaging of tumoral cells.

Here we report the synthesis of PLGA/DOXO-core Au-branched shell nanostructures (BGNSHs) functionalized with a human serum albumin/indocyanine green/folic acid complex (HSA-ICG-FA) to configure a multifunctional nanotheranostic platform. First, branched gold nanoshells (BGNSHs) were obtained through a seeded-growth surfactant-less method. These BGNSHs were loaded during the synthetic process with the chemotherapeutic drug doxorubicin, a DNA intercalating agent and topoisomerase II inhibitior. In parallel, the fluorescent near-infrared (NIR) dye indocyanine green (ICG) was conjugated to the protein human serum albumin (HSA) by electrostatic and hydrophobic interactions. Subsequently, folic acid was covalently attached to the HSA-ICG complex. In this way, we created a protein complex with targeting specificity and fluorescent imaging capability. The resulting HSA-ICG-FA complex was adsorbed to the gold nanostructures surface (BGNSH-HSA-ICG-FA) in a straightforward incubation process thanks to the high affinity of HSA to gold surface. In this manner, BGNSH-HSA-ICG-FA platforms were featured with multifunctional abilities: the possibility of fluorescence imaging for diagnosis and therapy monitoring by exploiting the inherent fluorescence of the dye, and a multimodal therapy approach consisting of the simultaneous combination of chemotherapy, provided by the loaded drug, and the potential cytotoxic effect of photodynamic and photothermal therapies provided by the dye and the gold nanolayer of the hybrid structure, respectively, upon NIR light irradiation of suitable wavelength. The combination of this trimodal approach was observed to exert a synergistic effect on the cytotoxicity of tumoral cells in vitro. Furthermore, FA was proved to enhance the internalization of nanoplatform. The ability of the nanoplatforms as fluorescence imaging contrast agents was tested by preliminary analyzing their biodistribution in vivo in a tumor-bearing mice model.

Hydration effects on the fibrillation process of a globular protein: the case of human serum albumin

In this work, we have studied the fibrillation process of human serum albumin (HSA) under different solution conditions. In particular, aggregation kinetics, fibril morphology, and composition structural changes were investigated under varying experimental conditions such as pH (2.0 and 7.4), temperature (at 25 and 65 °C), and solvent polarity (ethanol/water mixtures, 10–90% v/v). The characterization was carried out by means of static and dynamic light scattering (SLS and DLS), ThT fluorescence, circular dichroism (CD) and Fourier Transform Infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The aggregation process and the α-helix to β-sheet transitions were found to be favored by temperature and physiological pH. Also, pH was observed to influence both the fibrillation pathway and aggregation kinetics, changing from a classical fibrillation process with a lag phase under acidic conditions to a downhill polymerization process at physiological pH in the presence of the alcohol. Regarding protein structural composition, at room temperature and physiological pH ethanol was found to promote an α-helix to β-sheet conformational transition at intermediate alcohol concentrations, whereas at low and high ethanol contents α-helix prevailed as the predominant structure. Under acidic conditions, ethanol promotes an important fibrillation at high cosolvent concentrations due to screening of electric charges and a decrease in solvent polarity. On the other hand, important differences in the morphology of the resulting fibrils and aggregates are observed depending on the solution conditions. In particular, the formation of classical amyloid-like fibrils at physiological pH and high temperature is observed, in contrast to the usual curly morphology displayed by HSA fibrils under most of the solution conditions. Although the high temperature and pH are the main parameters influencing the protein structure destabilization and subsequent aggregation upon incubation, ethanol helps to regulate the hydrogen bonding, the attractive hydrophobic interactions, and the protein accessible surface area, thus, modifying the packing constraints and the resulting aggregate morphologies.

One-Dimensional Magnetic Nanowires Obtained by Protein Fibril Biotemplating

Magnetic nanowires were obtained through the in situ synthesis of magnetic material by Fe-controlled nanoprecipitation in the presence of two different protein (human serum albumin (HSA) and lysozyme (Lys)) fibrils as biotemplating agents. The structural characteristics of the biotemplates were transferred to the hybrid magnetic wires. They exhibited excellent magnetic properties as a consequence of the 1D assembly and fusion of magnetite nanoparticles as ascertained by SQUID magnetometry. Prompted by these findings, we also checked their potential applicability as MRI contrast agents. The magnetic wires exhibited large r(2)* relaxivities and sufficient contrast resolution even in the presence of an extremely small amount of Fe in the magnetic hybrids, which would potentially enable their use as T(2) contrast imaging agents.

Micellisation of triblock copolymers of ethylene oxide and 1,2-butylene oxide: Effect of B-block length

We have used pyrene fluorescence spectroscopy and isothermal titration calorimetry (ITC) to investigate the effect of hydrophobic-block length on values of the critical micelle concentration (cmc) for aqueous solutions of triblock poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers (B(n)E(m)B(n), where m and n denote the respective block lengths) with hydrophobic block lengths in the range n=12-21. Combined with results from previous work on B(n)E(m)B(n) copolymers with shorter B blocks, plots of log(10)(cmc) (cmc in molar units and reduced to a common E-block length) against total number of B units (n(t)=n for diblock or n(t)=2n for triblock copolymers) display transitions in the slopes of the two plots, which indicate changes in the micellisation equilibrium. These occur at values of n(t)which can be assigned to the onset and completion of collapse of the hydrophobic B blocks, an effect not previously observed for reverse triblock copolymers. The results are compared with related data for diblock E(m)B(n) copolymers.

Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia

Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.

NIR-light active hybrid nanoparticles for combined imaging and bimodal therapy of cancerous cells

We report the synthesis of a multifunctional biocompatible theranostic nanoplatform consisting of a biodegradable PLGA matrix surface-functionalized with indocyanine green (ICG), a near-IR fluorescent dye, and co-loaded with superparamagnetic iron oxide nanoparticles (SPIONs) and the anticancer drug doxorubicin (DOXO). Combination of chemo- and photothermal therapeutic efficacy as well as magnetic resonance and optical fluorescence imaging performance were successfully tested in vitro on a tumoral cervical HeLa cell line. Magnetic in vitro guided targeting of these nanoplatforms was also proven. These nanoconstructs also enabled to monitor their in vivo biodistribution by fluorescence imaging in a mice model, which revealed their effective accumulation in the tumor and, unexpectedly, in the brain area. A lower presence of nanoplatforms was noted in the reticulo-endothelial system. The present observations suggest the nanoplatforms ability to possibly overcome the blood brain barrier. These results open up new possibilities to use our multifunctional nanoplatforms to treat brain-located diseases.

Easy and efficient cell tagging with block copolymers based contrast agents for sensitive MRI detection in vivo.

Superparamagnetic iron oxide nanoparticles (MNPs) together with magnetic resonance imaging (MRI) are the preferred tools for monitoring the fate and biodistribution of administered cells in stem cell therapy studies. Commercial MNPs need transfection agents and long incubation times for sufficient cell labeling and further in vivo cell detection. In this work, we have synthesized MNPs coated with pluronic F127 and tetronic 908, and validated their applicability as contrast agents for MRI cell detection on two different cell types: rat mesenchymal stem cells (MSCs) and multipotent neural progenitor cell line from mice (C17.2). No transfection agent was needed for a complete MNP internalization, and the uptake was only dependent on MNP concentration in medium and limited on the incubation time. By combining in vivo MRI and ex vivo histology microscopy, we have demonstrated the MRI signal detected corresponded exclusively to labeled cells and not to free particles. Pluronic F127- and tetronic 908-coated MNPs represent promising contrast agents for stem cell tracking due to their ease of use in preparation, their efficiency for cell labeling, and their high sensitivity for in vivo cell detection.