RESUMEN

We report a water-soluble fluorescence and colorimetric copper probe (LysoBC1); this system can also serve for lysosome labeling and for the dynamic tracking of Cu2+ in living cells. The sensing mechanism takes advantage of the synergic action by the following three components: i) a lysosome targeting unit, ii) the spirolactam ring-opening for the selective copper chelation and iii) the metal-mediated hydrolysis of the rhodamine moiety for fluorescence enhancement. In aqueous environment the molecule acts as a fluorescent reversible pH sensor and as colorimetric probe for Cu2+ at physiological pH; the hydrolysis of the copper targeting unit resulted in a 50-fold increase of the fluorescence intensity. Most importantly, in vitro cell analyses in undifferentiated (SH SY5Y) and differentiated (d-SH SY5Y) neuroblastoma cells, LysoBC1 is able to selectively accumulate into lysosome while the copper binding ability allowed us to monitor intracellular copper accumulation into lysosome.

RESUMEN

Aß (1-40) can transfer from the aqueous phase to the bilayer and thus form stable ion-channel-like pores where the protein has alpha-helical conformation. The stability of the pores is due to the presence of the GXXXG motif. It has been reported that these ion-channel-like pores are stabilized by a Cα-H···O hydrogen bond that is established between a glycine of the GXXXG sequence of an alpha-helix and another amino acid of a vicinal alpha-helix. However, conflicting data are reported in the literature. Some authors have suggested that hydrogen bonding does not have a stabilizing function. Here we synthesized pentapeptides having a GXXXG motif to explore its role in pore stability. We used molecular dynamics simulations, quantum mechanics, and experimental biophysical techniques to determine whether hydrogen bonding was formed and had a stabilizing function in ion-channel-like structures. Starting from our previous molecular dynamics data, molecular quantum mechanics simulations, and ATR data showed that a stable ion-channel-like pore formed and a band centered at 2910 cm-1 was attributed to the interaction between Gly 7 of an alpha-helix and Asp 23 of a vicinal alpha-helix.

Asunto(s)

RESUMEN

Polymer-based systems have been demonstrated in novel therapeutic and diagnostic (theranostic) treatments for cancer and other diseases. Polymers provide a useful scaffold to develop multifunctional nanosystems that combine various beneficial properties such as drug delivery, bioavailability, and photosensitivity. For example, to provide passive tumour targeting of small drug molecules, polymers have been used to modify and functionalise the surface of water-insoluble drugs. This approach also allows the reduction of adverse side effects, such as retinoids. However, multifunctional polymer conjugates containing several moieties with distinct features have not been investigated in depth. This report describes the development of a one-pot approach to produce a novel multifunctional polymer conjugate. As a proof of concept, we synthesised polyvinyl alcohol (PVA) covalently conjugated with rhodamine B (a tracking agent), folic acid (a targeting agent), and all-trans retinoic acid (ATRA, a drug). The obtained polymer (PVA@RhodFR) was characterised by MALDI-TOF mass spectrometry, gel permeation chromatography, thermal analysis, dynamic light-scattering, NMR, UV-Vis, and fluorescence spectroscopy. Finally, to evaluate the efficiency of the multifunctional polymer conjugate, cellular differentiation treatments were performed on the neuroblastoma SH-SY5Y cell line. In comparison with standard ATRA-based conditions used to promote cell differentiation, the results revealed the high capability of the new PVA@RhodFR to induce neuroblastoma cells differentiation, even with a short incubation time and low ATRA concentration.

RESUMEN

Angiogenin (Ang) is a potent angiogenic protein that is overexpressed in many types of cancer at concentration values correlated to the tumor aggressiveness. Here, by means of an integrated multi-technique approach based on crystallographic, spectrometric and spectroscopic analyses, we demonstrate that the anti-cancer drug oxaliplatin efficiently binds angiogenin. Microscopy cellular studies, carried out on the prostate cancer cell (PC-3) line , show that oxaliplatin inhibits the angiogenin prompting effect on cell proliferation and migration, which are typical features of angiogenesis process. Overall, our findings point to angiogenin as a possible target of oxaliplatin, thus suggesting a potential novel mechanism for the antineoplastic activity of this platinum drug and opening the avenue to novel approaches in the combined anti-cancer anti-angiogenic therapy.

Asunto(s)

RESUMEN

Angiogenesis plays a key role in the wound healing process, involving the migration, growth, and differentiation of endothelial cells. Angiogenesis is controlled by a strict balance of different factors, and among these, the angiogenin protein plays a relevant role. Angiogenin is a secreted protein member of the ribonuclease superfamily that is taken up by cells and translocated to the nucleus when the process of blood vessel formation has to be promoted. However, the chemical signaling that activates the protein, normally present in the plasma, and the transport pathways through which the protein enters the cell are still largely unclear. Copper is also an angiogenic factor that regulates angiogenin expression and participates in the activation of common signaling pathways. The interaction between angiogenin and copper could be a relevant mechanism in regulating the formation of new blood vessel pathways and paving the way to the development of new drugs for chronic non-healing wounds.

Asunto(s)

RESUMEN

Nowdays, neurodegenerative diseases represent a great challenge from both the therapeutic and diagnostic points of view. Indeed, several physiological barriers of the body, including the blood brain barrier (BBB), nasal, dermal, and intestinal barriers, interpose between the development of new drugs and their effective administration to reach the target organ or target cells at therapeutic concentrations. Currently, the nose-to-brain delivery with nanoformulations specifically designed for intranasal administration is a strategy widely investigated with the goal to reach the brain while bypassing the BBB. To produce nanosystems suitable to study both in vitro and/or in vivo cells trafficking for potential nose-to-brain delivery route, we prepared and characterized two types of fluorescent poly(ethylene glycol)-methyl-ether-block-poly(lactide-co-glycolide) (PLGA-PEG) nanoparticles (PNPs), i.e., Rhodamine B (RhB) dye loaded- and grafted- PNPs, respectively. The latter were produced by blending into the PLGA-PEG matrix a RhB-labeled polyaspartamide/polylactide graft copolymer to ensure a stable fluorescence during the time of analysis. Photon correlation spectroscopy (PCS), UV-visible (UV-vis) spectroscopies, differential scanning calorimetry (DSC), atomic force microscopy (AFM) were used to characterize the RhB-loaded and RhB-grafted PNPs. To assess their potential use for brain targeting, cytotoxicity tests were carried out on olfactory ensheathing cells (OECs) and neuron-like differentiated PC12 cells. Both PNP types showed mean sizes suitable for nose-to-brain delivery (<200 nm, PDI < 0.3) and were not cytotoxic toward OECs in the concentration range tested, while a reduction in the viability on PC12 cells was found when higher concentrations of nanomedicines were used. Both the RhB-labelled NPs are suitable drug carrier models for exploring cellular trafficking in nose-to-brain delivery for short-time or long-term studies.

RESUMEN

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes' geometry to modulate peptides' activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.

Asunto(s)

RESUMEN

In this work, we aimed to develop a hybrid theranostic nano-formulation based on gold nanoparticles (AuNP)-having a known anti-angiogenic character-and the angiogenin (ANG), in order to tune the angiogenesis-related phases involved in the multifaceted process of the wound healing. To this purpose, spherical were surface "decorated" with three variants of the protein, namely, the recombinant (rANG), the wild-type, physiologically present in the human plasma (wtANG) and a new mutant with a cysteine substitution of the serine at the residue 28 (S28CANG). The hybrid biointerface between AuNP and ANG was scrutinized by a multi-technique approach based on dynamic light scattering, spectroscopic (UV-visible, circular dichroism) and microscopic (atomic force and laser scanning confocal) techniques. The analyses of optical features of plasmonic gold nanoparticles allowed for discrimination of different adsorption modes-i.e.; predominant physisorption and/or chemisorption-triggered by the ANG primary sequence. Biophysical experiments with supported lipid bilayers (SLB), an artificial model of cell membrane, were performed by means of quartz crystal microbalance with dissipation monitoring acoustic sensing technique. Cellular experiments on human umbilical vein endothelial cells (HUVEC), in the absence or presence of copper-another co-player of angiogenesis-were carried out to assay the nanotoxicity of the hybrid protein-gold nanoassemblies as well as their effect on cell migration and tubulogenesis. Results pointed to the promising potential of these nanoplatforms, especially the new hybrid Au-S28CANG obtained with the covalent grafting of the mutant on the gold surface, for the modulation of angiogenesis processes in wound care.

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Engineered graphene-based derivatives are attractive and promising candidates for nanomedicine applications because of their versatility as 2D nanomaterials. However, the safe application of these materials needs to solve the still unanswered issue of graphene nanotoxicity. In this work, we investigated the self-assembly of dityrosine peptides driven by graphene oxide (GO) and/or copper ions in the comparison with the more hydrophobic diphenylalanine dipeptide. To scrutinize the peptide aggregation process, in the absence or presence of GO and/or Cu2+, we used atomic force microscopy, circular dichroism, UV-visible, fluorescence and electron paramagnetic resonance spectroscopies. The perturbative effect by the hybrid nanomaterials made of peptide-decorated GO nanosheets on model cell membranes of supported lipid bilayers was investigated. In particular, quartz crystal microbalance with dissipation monitoring and fluorescence recovery after photobleaching techniques were used to track the changes in the viscoelastic properties and fluidity of the cell membrane, respectively. Also, cellular experiments with two model tumour cell lines at a short time of incubation, evidenced the high potential of this approach to set up versatile nanoplatforms for nanomedicine and theranostic applications.

RESUMEN

The opossum is a peculiar model of immunity to prion diseases. Here we scrutinised the bis-decarepeat peptide sequence of the opossum prion (Op_bis-deca) protein by a multitechnique approach, with a combined experimental (potentiometry, UV-visible, circular dichroism, NMR and EPR spectroscopy, quartz crystal microbalance with dissipation monitoring and confocal microscopy) and simulation (DFT calculations) approach. Results showed that the macrochelate structures formed upon the binding to Cu(ii) by the analogous bis-octarepeat peptide sequence of human prion (Hu_bis-octa) are not found in the case of Op_bis-deca. At physiological pH and equimolar amount of copper ions, the [CuLH-2] is the major species formed by Op_bis-deca. In this species one imidazole and two amide nitrogen atoms are involved in metal coordination and its stability constant value is lower than that of the analogous species formed by Hu_bis-octa, due to the presence of an extra proline residue. Moreover, the study on the interaction of the peptides or the peptide/Cu(ii) complexes with the model cell membranes made of supported lipid bilayers disclosed different levels of interaction, monitored by the viscoelastic changes of the membranes, which exhibited a similar viscoelastic response at the interface of the two complexes, while in the absence of Cu(ii), the Hu_bis-octa/SLB interface was more viscoelastic than the Op_bis-deca one.

Asunto(s)

RESUMEN

Angiogenin (ANG), an endogenous protein that plays a key role in cell growth and survival, has been scrutinised here as promising nanomedicine tool for the modulation of pro-/anti-angiogenic processes in brain cancer therapy. Specifically, peptide fragments from the putative cell membrane binding domain (residues 60-68) of the protein were used in this study to obtain peptide-functionalised spherical gold nanoparticles (AuNPs) of about 10 nm and 30 nm in optical and hydrodynamic size, respectively. Different hybrid biointerfaces were fabricated by peptide physical adsorption (Ang60-68) or chemisorption (the cysteine analogous Ang60-68Cys) at the metal nanoparticle surface, and cellular assays were performed in the comparison with ANG-functionalised AuNPs. Cellular treatments were performed both in basal and in copper-supplemented cell culture medium, to scrutinise the synergic effect of the metal, which is another known angiogenic factor. Two brain cell lines were investigated in parallel, namely tumour glioblastoma (A172) and neuron-like differentiated neuroblastoma (d-SH-SY5Y). Results on cell viability/proliferation, cytoskeleton actin, angiogenin translocation and vascular endothelial growth factor (VEGF) release pointed to the promising potentialities of the developed systems as anti-angiogenic tunable nanoplaftforms in cancer cells treatment.