Cisplatin binding to angiogenin protein: new molecular pathways and targets for the drug's anticancer activity.

Cisplatin (CisPt), a platinum-based chemotherapeutic widely used in the treatment of various cancers, has multiple mechanisms of action, including nuclear DNA (nDNA) and mitochondrial DNA (mDNA) damage and cytoskeletal perturbations affecting, in turn, the membrane transporter activity. CisPt binding to proteins and enzymes may modulate its biochemical mechanism of action and is associated with cancer cell resistance to the drug. In this work, we investigate the interaction between cisplatin and angiogenin (Ang), a protein strongly expressed in many types of cancer and a potent angiogenic factor. The adduct formed upon reaction of CisPt with Ang (Ang@CisPt) was characterized by X-ray crystallography to evidence the exact platination site and by UV-visible (UV-vis) absorption and circular dichroism (CD) spectroscopies to shed light on any possible change in the protein conformation. Furthermore, high-resolution electrospray ionization (ESI) mass spectrometry was utilized to evaluate the Ang : CisPt stoichiometry of the Ang@CisPt adduct. The effect of the Ang@CisPt adduct on a prostate cancer cell line (PC-3) was tested by colorimetric assays in terms of cell viability, at both levels of nuclear and mitochondrial damage, and reactive oxygen species (ROS) production. Cellular imaging by laser scanning confocal microscopy (LSM) was utilized to scrutinize the cytoskeleton actin reorganization and the lysosome and mitochondria organelle perturbation. These studies highlight the possibility of new molecular pathways and targets for CisPt activity.

Bioinspired Nanoplatforms Based on Graphene Oxide and Neurotrophin-Mimicking Peptides.

Neurotrophins (NTs), which are crucial for the functioning of the nervous system, are also known to regulate vascularization. Graphene-based materials may drive neural growth and differentiation, and, thus, have great potential in regenerative medicine. In this work, we scrutinized the nano-biointerface between the cell membrane and hybrids made of neurotrophin-mimicking peptides and graphene oxide (GO) assemblies (pep-GO), to exploit their potential in theranostics (i.e., therapy and imaging/diagnostics) for targeting neurodegenerative diseases (ND) as well as angiogenesis. The pep-GO systems were assembled via spontaneous physisorption onto GO nanosheets of the peptide sequences BDNF(1-12), NT3(1-13), and NGF(1-14), mimicking the brain-derived neurotrophic factor (BDNF), the neurotrophin 3 (NT3), and the nerve growth factor (NGF), respectively. The interaction of pep-GO nanoplatforms at the biointerface with artificial cell membranes was scrutinized both in 3D and 2D by utilizing model phospholipids self-assembled as small unilamellar vesicles (SUVs) or planar-supported lipid bilayers (SLBs), respectively. The experimental studies were paralleled via molecular dynamics (MD) computational analyses. Proof-of-work in vitro cellular experiments with undifferentiated neuroblastoma (SH-SY5Y), neuron-like, differentiated neuroblastoma (dSH-SY5Y), and human umbilical vein endothelial cells (HUVECs) were carried out to shed light on the capability of the pep-GO nanoplatforms to stimulate the neurite outgrowth as well as tubulogenesis and cell migration.

Pd-Based Hybrid Nanoparticles As Multimodal Theranostic Nanomedicine.

A nanodelivery system based on palladium nanoparticles (PdNP) and cisplatin (CisPt) was developed by physisorption of the drug onto the PdNP synthesized via a green redox process, using d-glucose and polyvinylpyrrolidone (PVP) as reducing and stabilizing/capping agents, respectively. UV-vis analysis and H2-evolution measurements were carried out to prove the nanoparticles' capability to act as bimodal theranostic nanomedicine, i.e., having both plasmonic and photocatalytic properties. XPS, XRD, and TEM allowed light to be shed on the chemical composition and morphology of the PdNP. The analysis of the UV-visible spectra evidenced plasmonic peak changes for the hybrid nanoparticle-drug assembly (Pd@CisPt), which pointed to a significant interaction of CisPt with the NP surface. The drug loading was quantitatively estimated by ICP-OES measurements, while DLS and AFM confirmed the strong association of the drug with the nanoparticle surface. The test of SOD-like activity in a cell-free environment proved the maintenance of the antioxidant capability of PdNP also in the Pd@CisPt systems. Finally, Pd@CisPt tested in prostate cancer cells (PC-3 line) unveiled the antitumoral action of the developed nanomedicine, related to reactive oxygen species (ROS) generation, with a condition of protein misfolding/unfolding and DNA damage, as evidenced by cytotoxicity and MitoSOX assays, as well as Raman microspectroscopy, respectively. Cell imaging by confocal microscopy evidenced cellular uptake of the nanoparticles, as well as dynamic processes of copper ion accumulation at the level of subcellular compartments. Finally, cell migration studies upon treatment with Pd@CisPt evidenced a tunable response between the inhibitory effect of CisPt and the enhanced rate of cell migration for the metal NP alone, which pointed out the promising potential of the developed theranostic nanomedicine in tissue regeneration.

Anti-Angiogenic and Anti-Proliferative Graphene Oxide Nanosheets for Tumor Cell Therapy.

Graphene oxide (GO) is a bidimensional novel material that exhibits high biocompatibility and angiogenic properties, mostly related to the intracellular formation of reactive oxygen species (ROS). In this work, we set up an experimental methodology for the fabrication of GO@peptide hybrids by the immobilization, via irreversible physical adsorption, of the Ac-(GHHPH)4-NH2 peptide sequence, known to mimic the anti-angiogenic domain of the histidine-proline-rich glycoprotein (HPRG). The anti-proliferative capability of the graphene-peptide hybrids were tested in vitro by viability assays on prostate cancer cells (PC-3 line), human neuroblastoma (SH-SY5Y), and human retinal endothelial cells (primary HREC). The anti-angiogenic response of the two cellular models of angiogenesis, namely endothelial and prostate cancer cells, was scrutinized by prostaglandin E2 (PGE2) release and wound scratch assays, to correlate the activation of inflammatory response upon the cell treatments with the GO@peptide nanocomposites to the cell migration processes. Results showed that the GO@peptide nanoassemblies not only effectively induced toxicity in the prostate cancer cells, but also strongly blocked the cell migration and inhibited the prostaglandin-mediated inflammatory process both in PC-3 and in HRECs. Moreover, the cytotoxic mechanism and the internalization efficiency of the theranostic nanoplatforms, investigated by mitochondrial ROS production analyses and confocal microscopy imaging, unraveled a dose-dependent manifold mechanism of action performed by the hybrid nanoassemblies against the PC-3 cells, with the detection of the GO-characteristic cell wrapping and mitochondrial perturbation. The obtained results pointed out to the very promising potential of the synthetized graphene-based hybrids for cancer therapy.

Hyaluronan-Metal Gold Nanoparticle Hybrids for Targeted Tumor Cell Therapy.

In this study, a novel multifunctional nanoplatform based on core-shell nanoparticles of spherical gold nanoparticles (AuNPs) capped with low and high molecular weight (200 and 700 kDa) hyaluronic acid (HA), was assembled via a green, one-pot redox synthesis method at room temperature. A multitechnique characterization approach by UV-visible spectroscopy, dynamic light scattering and atomic force microscopy pointed to the effective 'surface decoration' of the gold nanoparticles by HA, resulting in different grafting densities of the biopolymer chains at the surface of the metal nanoparticle, which in turn affected the physicochemical properties of the nanoparticles. Specifically, the spectral features of the gold plasmonic peak (and the related calculated optical size), the hydrodynamic diameter and the nanoparticle stability were found to depend on the molecular weight of the HA. The CD44-targeting capability of HA-functionalized gold nanoparticles was tested in terms of antibacterial activity and cytotoxicity. An enhanced inhibitory activity against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus was found, with a HA molecular weight (MW)-dependent trend for the HA-capped AuNPs compared to the bare, glucose-capped AuNPs. Cell viability assays performed on two CD44-positive cell models, namely normal human umbilical vein endothelial (HUVEC) and prostate tumor (PC-3) cells, in comparison with neuroblastoma cells (SH-SY5Y), which do not express the CD44 receptor, demonstrated an increased cytotoxicity in neuroblastoma compared to prostate cancer cells upon the cellular treatments by HA-AuNP compared to the bare AuNP, but a receptor-dependent perturbation effect on cytoskeleton actin and lysosomal organelles, as detected by confocal microscopy. These results highlighted the promising potentialities of the HA-decorated gold nanoparticles for selective cytotoxicity in cancer therapy. Confocal microscopy imaging of the two human tumor cell models demonstrated a membrane-confined uptake of HA-capped AuNP in the cancer cells that express CD44 receptors and the different perturbation effects related to molecular weight of HA wrapping the metallic core of the plasmonic nanoparticles on cellular organelles and membrane mobility.