Improving the Cellular Uptake of Biomimetic Magnetic Nanoparticles

Magnetococcus marinus magnetosome-associated protein MamC, expressed as recombinant, has been proven to mediate the formation of novel biomimetic magnetic nanoparticles (BMNPs) that are successful drug nanocarriers for targeted chemotherapy and hyperthermia agents. These BMNPs present several advantages over inorganic magnetic nanoparticles, such as larger sizes that allow the former to have larger magnetic moment per particle, and an isoelectric point at acidic pH values, which allows both the stable functionalization of BMNPs at physiological pH value and the molecule release at acidic (tumor) environments, simply based on electrostatic interactions. However, difficulties for BMNPs cell internalization still hold back the efficiency of these nano-particles as drug nanocarriers and hyperthermia agents. In the present study we explore the enhanced BMNPs internalization following upon their encapsulation by poly (lac-tic-co-glycolic) acid (PLGA), a Food and Drug Administration (FDA) approved molecule. Inter-nalization is further optimized by the functionalization of the nanoformulation with the cell-penetrating TAT peptide (TATp). Our results evidence that cells treated with the nanofor-mulation [TAT-PLGA(BMNPs)] show up to 80% more iron internalized (after 72 h) compared to that of cells treated with BMNPs (40%), without any significant decrease in cell viability. This nanoformulation showing optimal internalization is further characterized. In particular, the present manuscript demonstrates that neither its magnetic properties nor its performance as a hyperthermia agent are significantly altered due to the encapsulation. In vitro experiments demonstrate that, following upon the application of an alternating magnetic field on U87MG cells treated with BMNPs and TAT-PLGA(BMNPs), the cytotoxic effect of BMNPs was not affected by the TAT-PLGA enveloping. Based on that, difficulties shown in previous studies related to poor cell uptake of BMNPs can be overcome by the novel nanoassembly described here

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors

The light chains (KLCs) of the heterotetrameric microtubule motor kinesin-1, that bind to cargo adaptor proteins and regulate its activity, have a capacity to recognize short peptides via their tetratricopeptide repeat domains (KLC TPR ). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call \u2018Y-acidic\u2019 (tyrosine flanked by acidic residues), in a KLC-isoform specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1 TPR are distinct from those utilized for the recognition of W-acidic motifs found in adaptors that are KLC-isoform non-selective. However, a partial overlap on their receptor binding sites implies that adaptors relying on Y-acidic and W-acidic motifs must act independently. We propose a model to explain why these two classes of motifs that bind to the concave surface of KLC TPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity

Structural studies of a Tremella fuciformis mushroom lectin: a novel protein with antitumoral properties

Lectins are carbohydrate-binding proteins of non-immune origin, widely distributed in all the living organisms. They play a role in different biological processes and, due to their carbohydrate specific binding, some lectins have been found to possess antitumoral properties. A particular case is the recognition of the T-antigenic determinant (Gal\u3b21-3GalNAc\u3b1) on the malignant cell surfaces, which is present in 90% of human carcinomas. Upon binding to the carbohydrate, some lectins can cause apoptosis, cytotoxicity, inhibition of tumour growth, thus preventing the proliferation of tumoral cells. Considering the fact that these carbohydrates are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells. Although the function and the biological properties of several lectins have been determined, there are still many lectins that remain to be structurally and functionally characterized. As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and in the light of this, the crude extract of this fungus was examined to assess the presence of lectins. A lectin of 11 KDa, named TFL, was isolated and purified from the dried fruiting bodies and used for testing several crystal screening conditions. The best crystals were grown in 0.1 M TRIS pH 8.5, 1.5 potassium phosphate dibasic, 1 % DMSO and the final data sets were collected at the ESRF of Grenoble, revealing the three-dimensional structure of the protein. TFL belongs to the space group P1211 and the cell parameters of the crystal are the following: a = 61.62 \uc5, b = 61.83 \uc5, c = 67.84 \uc5, with \u3b2 = 106.87 \ub0. The protein is a monomer, composed of six \u3b2-sheets (from A to F) that arrange to form the so called \u201c\u3b2-barrel\u201d. In addition, two \u3b1-helixes, named H1 and H2 can be recognized in the structure, which is also stabilized by the presence of two disulphide bridges that connect Cys 54 to Cys 70 and Cys 97 to Cys 100, respectively. Thermal protein stability was analyzed by means of differential scanning calorimetry, revealing that TFL does not undergo a two-state unfolding process and its denaturation is reversible, a feature that has been rarely observed for lectins before. In addition, chemical and pH-induced unfolding were investigated using fluorescence spectroscopy, highlighting the high stability of TFL in a wide range of conditions. Exploiting isothermal titration calorimetry, more information about sugar-binding were acquired, identifying N-acetylgalactosamine as the best candidate, which has micromolar affinity for TFL. It has also been observed that Tremella fuciformis lectin shows no cytotoxicity on malignant and healthy cells and interestingly, it seems to reduce skin malignant cells migration and to have a positive effect in the upregulation of certain genes involved in cancer arresting (SBPP1). In addition, immunomodulatory activity of TFL was analyzed by means of ELISA essay, resulting in the up-regulation of the most important inflammatory markers (IL-6, TNF\u3b1), that could be usefully exploited in order to kill intracellular microorganisms and to help the human body to develop a stronger tumoral resistance. In conclusion, this work provides a new and interesting insight into cancer treatment, being an essential prerequisite for future in vitro and in vivo experiments. The encouraging results obtained so far, alongside with the fact that TFL has been produced also heterologously in E.coli, pave the way for further studies, including the feasibility to perform mutagenesis and to explore the possibility to further encapsulate the protein inside tailor-made nanoparticles, with the aim of reducing the amount of protein used, increasing its therapeutical efficiency, and preventing side effects

TREATMENT OF SKELETAL DISEASES WITH A NATURALLY DERIVED ANTIOXIDANT AND BISPHOSPHONATE EMBEDDED NANOPARTICLES

Skeletal disorders are degenerative diseases causing progressive disability and are becoming more and more prevalent in our society. Bone is a special tissue able to support load and stress and is continuously renewed thanks to bone turnover mediated by the well coordinated activity of osteoblasts and osteoclasts. Many metabolic bone diseases are characterized by an imbalance of the activity of bone cells and bone turnover that could be estimated through the evaluation of laboratory data analysis. In addition to metabolic disorders, bone tissue can be impairs by genetic diseases such as osteogenesis imperfect. In addition, Osteoarthritis (OA), the most prevalent musculoskeletal pathology, is predominantly characterized by the progressive degradation of articular cartilage due to an imbalance between anabolic and catabolic processes. Age-related changes that occur in articular are thought to represent a major risk factor for OA development. Therefore, skeletal diseases have been associated to defective differentiation pathways of progenitor stem cells (PSCs) to produce osteoblasts or chondrocytes. RUNX2 as well as SOX9 are transcription factors 202 responsible of commitment of PSCs in osteoblasts and chondrocytes respectively. In order to evaluate the possibility to affect and improve osteoblasts or chondrocytes commitment we assayed a naturally derived antioxidant and a bisphosphonate (clodronate) embedded nanoparticles in vivo (murine model) and in vitro (cell line) respectively. Antioxidant molecule was embedded into PLGA (poly lactic-co-glycolic acid) with the emulsion evaporation method. Bisphosphonate nanoparticles were prepared using chitosan and hyaluronic acid applying the ionotropic gelation method

Pegylated silica nanoparticles: cytotoxicity and macrophage uptake

Here, we present a thorough study of pegylated silica nanoparticle (SNP) interaction with different biological environments. The SNPs have a mean diameter of about 40 nm and are coated with polyethylene glycol (PEG) of different molecular weights. The physicochemical characterization of SNPs allowed the confirmation of the binding of PEG chains to the silica surface, the reproducibility of the synthesis and the narrow size-dispersion. In view of clarifying the SNP interaction with biological environments, we first assessed the SNP reactivity after the incubation with two cell lines (macrophages RAW 264.7 and primary human fibroblasts), observing a reduced toxicity of pegylated SNPs compared to the bare ones. Then, we investigated the effect of the protein adsorption on the SNP surface using the model serum protein, bovine serum albumin (BSA). We found that the protein adsorption takes place more heavily on poorly pegylated SNPs, promoting the uptake of the latter by macrophages and leading to an increased mortality of these cells. To better understand this mechanism by means of flow cytometry, the dye Ru(bpy)3Cl2 was incorporated in the SNPs. The overall results highlight the SNP potentialities as a drug delivery system, thanks to the low interactions with the macrophages