Conjugation to gold nanoparticles of methionine gamma-lyase, a cancer-starving enzyme. Physicochemical characterization of the nanocomplex for prospective nanomedicine applications.

The pyridoxal 5'-dependent enzyme methionine γ-lyase (MGL) catalyzes the degradation of methionine. This activity has been profitable to develop an antitumor agent exploiting the strict dependence of most malignant cells on the availability of methionine. Indeed, methionine depletion blocks tumor proliferation and leads to an increased susceptibility to anticancer drugs. Here, we explore the conjugation of MGL to gold nanoparticles capped with citrate (AuNPs) as a novel strategy to deliver MGL to cancer cells. Measurements of Transmission Electron Microscopy, Dynamic Light Scattering, Asymmetrical Flow Field-Flow Fractionation, X-ray Photoelectron Spectroscopy, and Circular Dichroism allowed to achieve an extensive biophysical and biochemical characterization of the MGL-AuNP complex including particle size, size distribution, MGL loading yield, enzymatic activity, and impact of gold surface on protein structure. Noticeably, we found that activity retention was improved over time for the enzyme adsorbed to AuNPs with respect to the enzyme free in solution. The acquired body of knowledge on the nanocomplex properties and this encouraging stabilizing effect upon conjugation are the necessary basis for further studies aimed at the evaluation of the therapeutic potential of MGL-AuNP complex in a biological milieu.

Enhancing pancreatic ductal adenocarcinoma (PDAC) therapy with targeted carbon nano-onion (CNO)-mediated delivery of gemcitabine (GEM)-derived prodrugs.

Nanotechnology's potential in revolutionising cancer treatments is evident in targeted drug delivery systems (DDSs) engineered to optimise therapeutic efficacy and minimise toxicity. This study examines a novel nanocarrier constructed with carbon nano-onions (CNOs), engineered and evaluated for its ability to selectively target cancer cells overexpressing the hyaluronic acid receptor; CD44. Our results highlighted that the CNO-based nanocarrier coupled with hyaluronic acid as the targeting agent demonstrated effective uptake by CD44+ PANC-1 and MIA PaCa-2 cells, while avoiding CD44- Capan-1 cells. The CNO-based nanocarrier also exhibited excellent biocompatibility in all tested pancreatic ductal adenocarcinoma (PDAC) cells, as well as healthy cells. Notably, the CNO-based nanocarrier was successfully loaded with chemotherapeutic 4-(N)-acyl- sidechain-containing prodrugs derived from gemcitabine (GEM). These prodrugs alone exhibited remarkable efficacy in killing PDAC cells which are known to be GEM resistant, and their efficacy was amplified when combined with the CNO-based nanocarrier, particularly in targeting GEM-resistant CD44+ PDAC cells. These findings demonstrate the potential of CNOs as promising scaffolds in advancing targeted DDSs, signifying the translational potential of carbon nanoparticles for cancer therapy.

Morphological and lipid metabolism alterations in macrophages exposed to model environmental nanoplastics traced by high-resolution synchrotron techniques.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs' presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP-immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 µg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0-100 µg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

Protein-corona formation on aluminum doped zinc oxide and gallium nitride nanoparticles.

The interaction of semiconductor nanoparticles with bio-molecules attracts increasing interest of researchers, considering the reactivity of nanoparticles and the possibility to control their properties remotely giving mechanical, thermal, or electrical stimulus to the surrounding bio-environment. This work reports on a systematic comparative study of the protein-corona formation on aluminum doped zinc oxide and gallium nitride nanoparticles. Bovine serum albumin was chosen as a protein model. Dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy techniques have been used to demonstrate the formation of protein-corona as well as the stability of the colloidal suspension given by BSA, which also works as a surfactant. The protein adsorption on the NPs surface studied by Bradford Assay showed the dependence on the quantity of proteins adsorbed to the available sites on the NPs surface, thus the saturation was observed at ratio higher than 5:1 (NPs:Proteins) in case of ZnO, these correlating with DLS results. Moreover, the kinetics of the proteins showed a relatively fast adsorption on the NPs surface with a saturation curve after about 25 min. GaN NPs, however, showed a very small amount of proteins adsorbed on the surface, a change in the hydrodynamic size being not observable with DLS technique or differential centrifugal sedimentation. The Circular Dichroism analysis suggests a drastic structural change in the secondary structure of the BSA after attaching on the NPs surface. The ZnO nanoparticles adsorb a protein-corona, which does not protect them against dissolution, and in consequence, the material proved to be highly toxic for Human keratinocyte cell line (HaCaT) at concentration above 25 µg/mL. In contrast, the GaN nanoparticles which do not adsorb a protein-corona, show no toxicity signs for HaCaT cells at concentration as high as 50 µg/mL, exhibiting much lower concentration of ions leakage in the culture medium as compared to ZnO nanoparticles.

Highly specific targeting of human acute myeloid leukaemia cells using pharmacologically active nanoconjugates.

In this study we used 5 nm gold nanoparticles as delivery platforms to target cancer cells expressing the immune receptor Tim-3 using single chain antibodies. Gold surfaces were also covered with the cytotoxic drug rapamycin which was immobilised using a glutathione linker. These nanoconjugates allowed highly specific and efficient delivery of cytotoxic rapamycin into human malignant blood cells.

The Tim-3-galectin-9 Secretory Pathway is Involved in the Immune Escape of Human Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a severe and often fatal systemic malignancy. Malignant cells are capable of escaping host immune surveillance by inactivating cytotoxic lymphoid cells. In this work we discovered a fundamental molecular pathway, which includes ligand-dependent activation of ectopically expressed latrophilin 1 and possibly other G-protein coupled receptors leading to increased translation and exocytosis of the immune receptor Tim-3 and its ligand galectin-9. This occurs in a protein kinase C and mTOR (mammalian target of rapamycin)-dependent manner. Tim-3 participates in galectin-9 secretion and is also released in a free soluble form. Galectin-9 impairs the anti-cancer activity of cytotoxic lymphoid cells including natural killer (NK) cells. Soluble Tim-3 prevents secretion of interleukin-2 (IL-2) required for the activation of cytotoxic lymphoid cells. These results were validated in ex vivo experiments using primary samples from AML patients. This pathway provides reliable targets for both highly specific diagnosis and immune therapy of AML.

Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability.

Highly Flexible Platform for Tuning Surface Properties of Silica Nanoparticles and Monitoring Their Biological Interaction.

The following work presents a simple, reliable and scalable seeding-growth methodology to prepare silica nanoparticles (SiO2 NPs) (20, 30, 50 and 80 nm) directly in aqueous phase, both as plain- as well as fluorescent-labeled silica. The amount of fluorescent label per particle remained constant regardless of size, which facilitates measurements in terms of number-based concentrations. SiO2 NPs in dispersion were functionalized with an epoxysilane, thus providing a flexible platform for the covalent linkage of wide variety of molecules under mild experimental conditions. This approach was validated with ethylenediamine, two different amino acids and three akylamines to generate a variety of surface modifications. Accurate characterization of particle size, size distributions, morphology and surface chemistry is provided, both for as-synthesized particles and after incubation in cell culture medium. The impact of physicochemical properties of SiO2 NPs was investigated with human alveolar basal epithelial cells (A549) such as the effect in cytotoxicity, cell internalization and membrane interaction.

Multi-Functionalized Carbon Nano-onions as Imaging Probes for Cancer Cells.

Carbon-based nanomaterials have attracted much interest during the last decade for biomedical applications. Multimodal imaging probes based on carbon nano-onions (CNOs) have emerged as a platform for bioimaging because of their cell-penetration properties and minimal systemic toxicity. Here, we describe the covalent functionalization of CNOs with fluorescein and folic acid moieties for both imaging and targeting cancer cells. The modified CNOs display high brightness and photostability in aqueous solutions and their selective and rapid uptake in two different cancer cell lines without significant cytotoxicity was demonstrated. The localization of the functionalized CNOs in late-endosomes cell compartments was revealed by a correlative approach with confocal and transmission electron microscopy. Understanding the biological response of functionalized CNOs with the capability to target cancer cells and localize the nanoparticles in the cellular environment, will pave the way for the development of a new generation of imaging probes for future biomedical studies.

Measuring protein structure and stability of protein-nanoparticle systems with synchrotron radiation circular dichroism.

We measure the structural and stability changes of proteins at nanomolar concentration upon interaction with nanoparticles. Using synchrotron radiation circular dichroism (SRCD), we measure a decrease of 6 °C in the thermal unfolding of human serum albumin upon interaction with silver nanoparticles while this does not happen with gold. The use of SRCD allows measuring critical parameters on protein-nanoparticle interactions, and it will provide experimental data on the relative stability of key biological proteins for nanotoxicology.

pH-dependent immobilization of proteins on surfaces functionalized by plasma-enhanced chemical vapor deposition of poly(acrylic acid)- and poly(ethylene oxide)-like films.

The interaction of the proteins bovine serum albumin (BSA), lysozyme (Lys), lactoferrin (Lf), and fibronectin (Fn) with surfaces of protein-resistant poly(ethylene oxide) (PEO) and protein-adsorbing poly(acrylic acid) (PAA) fabricated by plasma-enhanced chemical vapor deposition has been studied with quartz crystal microbalance with dissipation monitoring (QCM-D). We focus on several parameters which are crucial for protein adsorption, i.e., the isoelectric point (pI) of the proteins, the pH of the solution, and the charge density of the sorbent surfaces, with the zeta-potential as a measure for the latter. The measurements reveal adsorption stages characterized by different segments in the plots of the dissipation vs frequency change. PEO remains protein-repellent for BSA, Lys, and Lf at pH 4-8.5, while weak adsorption of Fn was observed. On PAA, different stages of protein adsorption processes could be distinguished under most experimental conditions. BSA, Lys, Lf, and Fn generally exhibit a rapid initial adsorption phase on PAA, often followed by slower processes. The evaluation of the adsorption kinetics also reveals different adsorption stages, whereas the number of these stages does not always correspond to the structurally different phases as revealed by the D- f plots. The results presented here, together with information obtained in previous studies by other groups on the properties of these proteins and their interaction with surfaces, allow us to develop an adsorption scenario for each of these proteins, which takes into account electrostatic protein-surface and protein-protein interaction, but also the pH-dependent properties of the proteins, such as shape and exposure of specific domains.

Fouling and non-fouling surfaces produced by plasma polymerization of ethylene oxide monomer.

This paper presents the results of plasma polymerization using diethylene glycol dimethyl ether as a precursor in a capacitively coupled radio frequency system. The chemical structure of the coatings was characterized using several analysis techniques (X-ray photoelectron spectroscopy, Fourier transform-infrared spectroscopy, ellipsometry), while the biological response of these coatings has been tested by protein adsorption and cell culture experiments. The modulation of the input plasma power controls the concentration of polyethylene oxide groups in the coatings and allows the production of films with opposite protein and cell repellent properties. The study of the stability of these coatings in different media (water, acetone, phosphate-buffered saline) reveals that these films could be involved in classical lift-off processes for the production of patterned surfaces.