Correction: Innovative snail-mucus-extract (SME)-coated nanoparticles exhibit anti-inflammatory and anti-proliferative effects for potential skin cancer prevention and treatment.

[This corrects the article DOI: 10.1039/D4RA00291A.].

The Hybrid Nano-Biointerface between Proteins/Peptides and Two-Dimensional Nanomaterials.

In typical protein-nanoparticle surface interactions, the biomolecule surface binding and consequent conformational changes are intermingled with each other and are pivotal to the multiple functional properties of the resulting hybrid bioengineered nanomaterial. In this review, we focus on the peculiar properties of the layer formed when biomolecules, especially proteins and peptides, face two-dimensional (2D) nanomaterials, to provide an overview of the state-of-the-art knowledge and the current challenges concerning the biomolecule coronas and, in general, the 2D nano-biointerface established when peptides and proteins interact with the nanosheet surface. Specifically, this review includes both experimental and simulation studies, including some recent machine learning results of a wide range of nanomaterial and peptide/protein systems.

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.

Green Nanoformulations of Polyvinylpyrrolidone-Capped Metal Nanoparticles: A Study at the Hybrid Interface with Biomimetic Cell Membranes and In Vitro Cell Models.

Noble metal nanoparticles (NP) with intrinsic antiangiogenic, antibacterial, and anti-inflammatory properties have great potential as potent chemotherapeutics, due to their unique features, including plasmonic properties for application in photothermal therapy, and their capability to slow down the migration/invasion speed of cancer cells and then suppress metastasis. In this work, gold (Au), silver (Ag), and palladium (Pd) NP were synthesized by a green redox chemistry method with the reduction of the metal salt precursor with glucose in the presence of polyvinylpyrrolidone (PVP) as stabilizing and capping agent. The physicochemical properties of the PVP-capped NP were investigated by UV-visible (UV-vis) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies, dynamic light scattering (DLS), and atomic force microscopy (AFM), to scrutinize the optical features and the interface between the metal surface and the capping polymer, the hydrodynamic size, and the morphology, respectively. Biophysical studies with model cell membranes were carried out by using laser scanning confocal microscopy (LSM) with fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) techniques. To this purpose, artificial cell membranes of supported lipid bilayers (SLBs) made with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) dye-labeled with 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD, FRET donor) and/or lissamine rhodamine B sulfonyl (Rh, FRET acceptor) were prepared. Proof-of-work in vitro cellular experiments were carried out with prostate cancer cells (PC-3 line) in terms of cytotoxicity, cell migration (wound scratch assay), NP cellular uptake, and cytoskeleton actin perturbation.

Different In Silico Approaches Using Heterocyclic Derivatives against the Binding between Different Lineages of SARS-CoV-2 and ACE2.

Over the last few years, the study of the SARS-CoV-2 spike protein and its mutations has become essential in understanding how it interacts with human host receptors. Since the crystallized structure of the spike protein bound to the angiotensin-converting enzyme 2 (ACE2) receptor was released (PDB code 6M0J), in silico studies have been performed to understand the interactions between these two proteins. Specifically, in this study, heterocyclic compounds with different chemical characteristics were examined to highlight the possibility of interaction with the spike protein and the disruption of the interaction between ACE2 and the spike protein. Our results showed that these compounds interacted with the spike protein and interposed in the interaction zone with ACE2. Although further studies are needed, this work points to these heterocyclic push-pull compounds as possible agents capable of interacting with the spike protein, with the potential for the inhibition of spike protein-ACE2 binding.

GxxxG Motif Stabilize Ion-Channel like Pores through Cα-H···O Interaction in Aß (1-40).

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.

Combining physical vapor deposition structuration with dealloying for the creation of a highly efficient SERS platform.

Nanostructured noble metal thin films are highly studied for their interesting plasmonic properties. The latter can be effectively used for the detection of small and highly diluted molecules by the surface-enhanced Raman scattering (SERS) effect. Regardless of impressive detection limits achieved, synthesis complexity and the high cost of gold restrict its use in devices. Here, we report on a novel two-step approach that combines the deposition of a silver-aluminum thin film with dealloying to design and fabricate efficient SERS platforms. The magnetron sputtering technique was used for the deposition of the alloy thin film to be dealloyed. After dealloying, the resulting silver nanoporous structures revealed two degrees of porosity: macroporosity, associated to the initial alloy morphology, and nanoporosity, related to the dealloying step. The resulting nanoporous columnar structure was finely optimized by tuning deposition (i.e., the alloy chemical composition) and dealloying (i.e., dealloying media) parameters to reach the best SERS properties. These are reported for samples dealloyed in HCl and with 30 atom % of silver at the initial state with a detection limit down to 10-10 mol·L-1 for a solution of rhodamine B.

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.

Green Light-Triggerable Chemo-Photothermal Activity of Cytarabine-Loaded Polymer Carbon Dots: Mechanism and Preliminary In Vitro Evaluation.

Carbon-based nanostructures are attracting a lot of attention because of their very low toxicity, excellent visible light-triggered optical and photothermal properties, and intriguing applications. Currently, the development of multifunctional carbon-based nanostructures for a synergistic chemo-photothermal approach is a challenging topic for the advancement of cancer treatment. Here, we report an unprecedented example of photoresponsive carbon-based polymer dots (CPDs-PNM) obtained by a one-pot thermal process from poly(N-isopropylacrylamide) (PNIPAM) without using organic solvent and additional reagents. The CPDs-PNM nanostructures were characterized by spectroscopic techniques, transmission electron microscopy, and atomic force microscopy. The CPDs-PNM exhibited high photothermal conversion efficiency, lower critical solution temperature (LCST) behavior, and good cytarabine (arabinosyl cytosine, AraC) loading capacity (62.3%). The formation of a CPDs-PNM/AraC adduct and photothermal-controlled drug release, triggered by green light excitation, were demonstrated by spectroscopic techniques, and the drug-polymer interaction and drug release mechanism were well supported by modeling simulation calculations. The cellular uptake of empty and AraC-loaded CPDs-PNM was imaged by confocal laser scanning microscopy. In vitro experiments evidenced that CPDs-PNM did not affect the viability of neuroblastoma cells, while the CPDs-PNM/AraC adduct under light irradiation exhibited significantly higher toxicity than AraC alone by a combined chemo-photothermal effect.

A Multifunctional Conjugated Polymer Developed as an Efficient System for Differentiation of SH-SY5Y Tumour Cells.

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.

A nanosized photothermal responsive core-shell carbonized polymer dots based on poly(N-isopropylacrylamide) for light-triggered drug release.

Core-shell nanocomposites are one of the most important achievements in the fast-growing field of nanotechnology. The combination of multi-responsive nano-shell with luminescent and photothermal core has led to promising applications in various fields such as optics, electronics and medicine. In this work, a nanosized core-shell system composed by carbonized dots core and poly(N-isopropylacrylamide) shell was developed and the photothermal triggered release of doxorubicin was demonstrated. The system was fully characterized by H1-NMR, DLS, Z-potential, AFM, optical absorption and fluorescence measurements. A photothermal conversion efficiency (η) value of about 67.9% and a doxorubicin photo-release rate value of about 1.0% min-1 were measured. Molecular dynamic (MD) simulations data were in agreement with experimental results, at 310 K the coil-to-globule transition and a consequent desorption of doxorubicin from the polymer were observed. Both the radius of gyration and the fluctuation of the distance doxorubicin-PNIPAM pointed that the temperature above the LCST and the acid pH facilitated the polymer transition. Moreover, MD simulations and experimental data suggested an influence on the lower critical solution temperature (LCST) exerted by the number of polymer chains anchored to the carbon core.

A novel facile one-pot synthesis of photothermally responsive carbon polymer dots as promising drug nanocarriers.

Luminescent and photothermic carbon polymer dots (CPDs-PNM), composed of a carbonized core and cross-linked chains of poly(N-isopropylacrylamide), were synthetized by a novel, simple, solvent- and reagent-free method. The formation of CPDs-PNM was controlled by both temperature and heating time. The CPDs-PNM exhibited LCST behaviour, high photothermal conversion efficiency, curcumin loading capacity and no toxicity to eukaryotic cells. Proof of concept experiments confirmed an excellent thermally induced drug release activity to be used for photothermally controlled drug release.

Oxaliplatin inhibits angiogenin proliferative and cell migration effects in prostate cancer cells.

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.

Enhanced plasmonic processes in amino-rich plasma polymer films for applications at the biointerface.

A new plasmonic biosensor was developed in a planar chip-based format by coupling the plasmonic properties of gold nanoparticles (Au NPs) with the mechanical and bioadhesive features of unconventional organic thin films deposited from plasma, namely primary amine-based plasma polymer films (PPFs). A self-assembled layer of spherical Au NPs, 12 nm in diameter, was electrostatically immobilized onto optically transparent silanised glass. In the next step, the Au NP layer was coated with an 18 nm polymeric thick PPF layer via the simultaneous polymerization/deposition of a cyclopropylamine (CPA) precursor performed by radio frequency discharge, both in pulsed and in continuous wave modes. The CPA PFF surface plays the dual role of an adsorbent towards negatively charged chemical species as well as an enhancer of plasmonic signals. The biosensor was tested in a proof-of-concept series of experiments of human serum albumin physisorption, and chosen as a model system for blood serum. The peculiar surface features of CPA PPF, before and after the exposure to buffered solution of fluorescein isothiocyanate-labelled human serum albumin (FITC-HSA), were investigated by a multi-technique approach, including UV-visible and X-ray photoelectron spectroscopies, atomic force microscopy, scanning electron microscopy, contact angle and surface free energy measurements. The results showed the very promising potentialities from both bioanalytical and physicochemical points of view in scrutinizing the macromolecule behavior at the biointerface.

Angiogenin and Copper Crossing in Wound Healing.

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.

mPEG-PLGA Nanoparticles Labelled with Loaded or Conjugated Rhodamine-B for Potential Nose-to-Brain Delivery.

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.

Peptides Derived from Angiogenin Regulate Cellular Copper Uptake.

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.

ß-amyloid monomers drive up neuronal aerobic glycolysis in response to energy stressors.

Research on cerebral glucose metabolism has shown that the aging brain experiences a fall of aerobic glycolysis, and that the age-related loss of aerobic glycolysis may accelerate Alzheimer's disease pathology. In the healthy brain, aerobic glycolysis, namely the use of glucose outside oxidative phosphorylation, may cover energy demand and increase neuronal resilience to stressors at once. Currently, the drivers of aerobic glycolysis in neurons are unknown. We previously demonstrated that synthetic monomers of ß-amyloid protein (Aß) enhance glucose uptake in neurons, and that endogenous Aß is required for depolarization-induced glucose uptake in cultured neurons. In this work, we show that cultured cortical neurons increased aerobic glycolysis in response to the inhibition of oxidative phosphorylation by oligomycin or to a kainate pulse. Such an increase was prevented by blocking the endogenous Aß tone and re-established by the exogenous addition of synthetic Aß monomers. The activity of mitochondria-bound hexokinase-1 appeared to be necessary for monomers-stimulated aerobic glycolysis during oxidative phosphorylation blockade or kainate excitation. Our data suggest that, through Aß release, neurons coordinate glucose uptake with aerobic glycolysis in response to metabolic stressors. The implications of this new finding are that the age-related drop in aerobic glycolysis and the susceptibility to Alzheimer's disease could be linked to factors interfering with release and functions of Aß monomers.