SERS nanostructures with engineered active peptides against an immune checkpoint protein.

The immune checkpoint programmed death ligand 1 (PD-L1) protein is expressed by tumor cells and it suppresses the killer activity of CD8+ T-lymphocyte cells binding to the programmed death 1 (PD-1) protein of these immune cells. Binding to either PD-L1 or PD1 is used for avoiding the inactivation of CD8+ T-lymphocyte cells. We report, for the first time, Au plasmonic nanostructures with surface-enhanced Raman scattering (SERS) properties (SERS nanostructures) and functionalized with an engineered peptide (CLP002: Trp-His-Arg-Ser-Tyr-Tyr-Thr-Trp-Asn-Leu-Asn-Thr), which targets PD-L1. Molecular dynamics calculations are used to describe the interaction of the targeting peptide with PD-L1 in the region where the interaction with PD-1 occurs, showing also the poor targeting activity of a peptide with the same amino acids, but a scrambled sequence. The results are confirmed experimentally since a very good targeting activity is observed against the MDA-MB-231 breast adenocarcinoma cancer cell line, which overexpresses PD-L1. A good activity is observed, in particular, for SERS nanostructures where the CLP002-engineered peptide is linked to the nanostructure surface with a short charged amino acid sequence and a long PEG chain. The results show that the functionalized SERS nanostructures show very good targeting of the immune checkpoint PD-L1.

Evaluation of doxorubicin in three-dimensional culture of breast cancer cells and the response in PI3K/AKT/PTEN signaling pathways: a pilot study.

Breast cancer (BC) has a high mortality rate, which is attributed to the absence of effective treatment markers. Doxorubicin (DOX) was evaluated by molecular docking in vitro in cultured BC spheroids and its association with genes involved in the PI3K/AKT/PTEN signaling pathway. Spheroids were obtained from a primary BC. The selected compound was used for molecular docking experiments. Spheroids were treated with DOX for 1 (D1) and 9 (D9) days. qPCR was used to evaluate PIK3CA, HIF-1α, VEGF-A, PTEN expression. Treatment with DOX (1 µM) significantly increased the number of spheroids (D1), whereas exposure to chemotherapy at 2 µM on D9 was more effective. DOX treatment resulted in significantly higher expression of VEGF-A, HIF-1α and PIK3CA by D1 and HIF-1α and PTEN were upregulated by D9. Compared to treatment on D1 with D9 (1 µM) had significantly higher PTEN and lower PIK3CA gene expression. The genes HIF-1α and PTEN were more expressed with 2 µM of DOX while VEGF-A was downregulated. D1 vs. D9 exhibited reduced VEGF-A, HIF-1α, and PIK3CA expression and upregulation of PTEN expression. DOX effects at the molecular mechanisms can be involved the modulation of genes related to angiogenesis cell proliferation and tumor growth in BC tissue spheroids.

Modulation of the 20S Proteasome Activity by Porphyrin Derivatives Is Steered through Their Charge Distribution.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

Protection against proteolysis of a targeting peptide on gold nanostructures.

Cell targeting has been considered an important strategy in diagnostic and therapeutic applications. Among different targeting units, peptides have emerged for their ability to bind to many different cellular targets, their scarce immunogenicity and the possibility of introducing multiple copies on nanosystems, providing high avidity for the target. However, their sensitivity to proteases strongly limits their applications in vivo. Here, we show that when presented on the surface of nanostructures, peptide stability to proteolysis is strongly improved without reducing the targeting activity. We prepared plasmonic nanostructures functionalized with a dodecapeptide (GE11) which targets EGFR, a protein overexpressed on different types of tumors. Two types of nanosystems were prepared in which the targeting unit was either directly linked to gold nanoparticles or through a PEG chain, resulting in a different peptide density on the surface of nanostructures. The peptide was rapidly degraded in 20% human serum or in the presence of isolated serine proteases, whereas no significant proteolytic fragments were detected during incubation of the nanosystems and after 24 h digestion, the nanostructures maintained their targeting activity and selectivity on colon cancer cells. Molecular dynamic calculations of the interaction of the nanostructure with chymotrypsin suggest that the formation of the enzyme-peptide complex, the first step in the mechanism of peptide hydrolysis, is highly unlikely because of the constraint imposed by the link of the peptide to the nanoparticle. These results support the utilization of peptides as active targeting units in nanomedicine.

Orientation-Selective and Frequency-Correlated Light-Induced Pulsed Dipolar Spectroscopy.

We explore the potential of orientation-resolved pulsed dipolar spectroscopy (PDS) in light-induced versions of the experiment. The use of triplets as spin-active moieties for PDS offers an attractive tool for studying biochemical systems containing optically active cofactors. Cofactors are often rigidly bound within the protein structure, providing an accurate positional marker. The rigidity leads to orientation selection effects in PDS, which can be analyzed to give both distance and mutual orientation information. Herein we present a comprehensive analysis of the orientation selection of a full set of light-induced PDS experiments. We exploit the complementary information provided by the different light-induced techniques to yield atomic-level structural information. For the first time, we measure a 2D frequency-correlated laser-induced magnetic dipolar spectrum, and we are able to monitor the complete orientation dependence of the system in a single experiment. Alternatively, the summed spectrum enables an orientation-independent analysis to determine the distance distribution.

Tylopeptin B peptide antibiotic in lipid membranes at low concentrations: Self-assembling, mutual repulsion and localization.

The medium-length peptide Tylopeptin B possesses activity against Gram-positive bacteria. It binds to bacterial membranes altering their mechanical properties and increasing their permeability. This action is commonly related with peptide self-assembling, resulting in the formation of membrane channels. Here, pulsed double electron-electron resonance (DEER) data for spin-labeled Tylopeptin B in palmitoyl-oleoyl-glycero-phosphocholine (POPC) model membrane reveal that peptide self-assembling starts at concentration as low as 0.1 mol%; above 0.2 mol% it attains a saturation-like dependence with a mean number of peptides in the cluster = 3.3. Using the electron spin echo envelope modulation (ESEEM) technique, Tylopeptin B molecules are found to possess a planar orientation in the membrane. In the peptide concentration range between 0.1 and 0.2 mol%, DEER data show that the peptide clusters have tendency of mutual repulsion, with a circle of inaccessibility of radius around 20 nm. It may be proposed that within this radius the peptides destabilize the membrane, providing so the peptide antimicrobial activity. Exploiting spin-labeled stearic acids as a model for free fatty acids (FFA), we found that at concentrations of 0.1-0.2 mol% the peptide promotes formation of lipid-mediated FFA clusters; further increase in peptide concentration results in dissipation of these clusters.

Melatonin ameliorates degenerative alterations caused by age in the rat prostate and mitigates high-fat diet damages.

Imbalance of sexual steroids milieu and oxidative stress are often observed during aging and correlated to prostate disorders. Likewise, high-fat intake has been related to prostate damage and tumor development. Melatonin (MLT) is an antioxidant whose secretion decreases in elderly and is also suggested to protect the gland. This study evaluated the impact of a long-term high-fat diet during aging on prostate morphology and antioxidant system of rats and tested the effects of MLT supplementation under these conditions. Male rats were assigned into four groups: control, treated with MLT, high-fat diet and high-fat diet treated with MLT. The high-fat diet was provided from the 24th week of age, MLT from the 48th (100 µg/kg/day) and rats were euthanized at the 62nd week. The high-fat diet increased body weight, retroperitoneal fatness, glycaemia, and circulating estrogen levels. It aggravated the aging effects, leading to epithelial atrophy (∼32% reduction of epithelial height) and collagen fibers increase (83%). MLT alone did not alter biometric and physiological parameters, except for the prostate weight decrease, whereas it alleviated biometric as well as ameliorated acinar atrophy induced by high-lipid intake. Systemic oxidative stress increased, and prostatic glutathione peroxidase activity decreased fivefold with the high-fat diet despite the indole. Regardless of the diet, MLT triggered epithelial desquamation, reduced androgen receptor-positive cells, increased smooth muscle layer thickness (12%), decreased at least 50% corpora amylacea formation, and stimulated prostatic gluthatione-S-transferase activity. In conclusion, MLT partially recovered prostate damage induced by aging and the long-term high-fat diet and ameliorated degenerative prostate alterations.

Light-Induced Triplet-Triplet Electron Resonance Spectroscopy.

We present a new technique, light-induced triplet-triplet electron resonance spectroscopy (LITTER), which measures the dipolar interaction between two photoexcited triplet states, enabling both the distance and angular distributions between the two triplet moieties to be determined on a nanometer scale. This is demonstrated for a model bis-porphyrin peptide that renders dipolar traces with strong orientation selection effects. Using simulations and density functional theory calculations, we extract distance distributions and relative orientations of the porphyrin moieties, allowing the dominant conformation of the peptide in a frozen solution to be identified. LITTER removes the requirement of current light-induced electron spin resonance pulse dipolar spectroscopy techniques to have a permanent paramagnetic moiety, becoming more suitable for in-cell applications and facilitating access to distance determination in unmodified macromolecular systems containing photoexcitable moieties. LITTER also has the potential to enable direct comparison with Förster resonance energy transfer and combination with microscopy inside cells.

Nanoparticles Engineering by Pulsed Laser Ablation in Liquids: Concepts and Applications.

Laser synthesis emerges as a suitable technique to produce ligand-free nanoparticles, alloys and functionalized nanomaterials for catalysis, imaging, biomedicine, energy and environmental applications. In the last decade, laser ablation and nanoparticle generation in liquids has proven to be a unique and efficient technique to generate, excite, fragment and conjugate a large variety of nanostructures in a scalable and clean way. In this work, we give an overview on the fundamentals of pulsed laser synthesis of nanocolloids and new information about its scalability towards selected applications. Biomedicine, catalysis and sensing are the application areas mainly discussed in this review, highlighting advantages of laser-synthesized nanoparticles for these types of applications and, once partially resolved, the limitations to the technique for large-scale applications.

Biomimetic Nanoarchitectures for Light Harvesting: Self-Assembly of Pyropheophorbide-Peptide Conjugates.

The biological light-harvesting process offers an unlimited source of inspiration. The high level of control, adaptation capability, and efficiency challenge humankind to create artificial biomimicking nanoarchitectures with the same performances to respond to our energy needs. Here, in the extensive search for design principles at the base of efficient artificial light harvesters, an approach based on self-assembly of pigment-peptide conjugates is proposed. The solvent-driven and controlled aggregation of the peptide moieties promotes the formation of a dense network of interacting pigments, giving rise to an excitonic network characterized by intense and spectrally wide absorption bands. The ultrafast dynamics of the nanosystems studied through two-dimensional electronic spectroscopy reveals that the excitation energy is funneled in an ultrafast time range (hundreds of femtoseconds) to a manifold of long-living dark states, thus suggesting the considerable potentiality of the systems as efficient harvesters.

Correction: An EPR study of ampullosporin A, a medium-length peptaibiotic, in bicelles and vesicles.

Correction for 'An EPR study of ampullosporin A, a medium-length peptaibiotic, in bicelles and vesicles' by Marco Bortolus et al., Phys. Chem. Chem. Phys., 2016, 18, 749-760.

PreS1 peptide-functionalized gold nanostructures with SERRS tags for efficient liver cancer cell targeting.

Early detection is the most effective mean of improving prognosis for many fatal diseases such as cancer. In this context, the Surface Enhanced Resonance Raman Scattering (SERRS) technique is being proposed as alternative to fluorescent methods in detection of biomarkers, because SERRS nanostructures are bright as fluorescent tags but more stable and clearly detectable using the narrow Raman "fingerprints" of a suitable reporter. Here we show that biocompatible SERRS active gold nanostructures, functionalized with an engineered PreS1 peptide (AuNP@PEG-PreS1), detect the presence of the SerpinB3 antigen overexpressed on liver tumor cells, a biomarker of the onset of liver cell carcinomatous transformation. A proper engineering of the targeting unit, linked to the nanostructure by a polymer chain, affords a sensitivity and specificity larger than 80%, at subnanomolar concentrations. Taking into account the high sensitivity of SERRS and that SB3 overexpression is an early event in liver cell carcinomatous transformation, AuNP@PEG-PreS1 nanostructures could be used in routine diagnostic activities, to improve the accuracy of HCC detection in particular in patients with chronic liver diseases.

Light-Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein.

Light-induced pulsed EPR dipolar spectroscopic methods allow the determination of nanometer distances between paramagnetic sites. Here we employ orthogonal spin labels, a chromophore triplet state and a stable radical, to carry out distance measurements in singly nitroxide-labeled human neuroglobin. We demonstrate that Zn-substitution of neuroglobin, to populate the Zn(II) protoporphyrin IX triplet state, makes it possible to perform light-induced pulsed dipolar experiments on hemeproteins, extending the use of light-induced dipolar spectroscopy to this large class of metalloproteins. The versatility of the method is ensured by the employment of different techniques: relaxation-induced dipolar modulation enhancement (RIDME) is applied for the first time to the photoexcited triplet state. In addition, an alternative pulse scheme for laser-induced magnetic dipole (LaserIMD) spectroscopy, based on the refocused-echo detection sequence, is proposed for accurate zero-time determination and reliable distance analysis.

Melatonin and Docosahexaenoic Acid Decrease Proliferation of PNT1A Prostate Benign Cells via Modulation of Mitochondrial Bioenergetics and ROS Production.

Prostate cancer development has been associated with changes in mitochondrial activity and reactive oxygen species (ROS) production. Melatonin (MLT) and docosahexaenoic acid (DHA) have properties to modulate both, but their protective role, mainly at early stages of prostate cancer, remains unclear. In this study, the effects of MLT and DHA, combined or not, on PNT1A cells with regard to mitochondria bioenergetics, ROS production, and proliferation-related pathways were examined. Based on dose response and lipid accumulation assays, DHA at 100 µM and MLT at 1 µM for 48 h were chosen. DHA doubled and MLT reduced (40%) superoxide anion production, but coincubation (DM) did not normalize to control. Hydrogen peroxide production decreased after MLT incubation only (p < 0.01). These alterations affected the area and perimeter of mitochondria, since DHA increased whereas MLT decreased, but such hormone has no effect on coincubation. DHA isolated did not change the oxidative phosphorylation rate (OXPHOS), but decreased (p < 0.001) the mitochondrial bioenergetic reserve capacity (MBRC) which is closely related to cell responsiveness to stress conditions. MLT, regardless of DHA, ameliorated OXPHOS and recovered MBRC after coincubation. All incubations decreased AKT phosphorylation; however, only MLT alone inhibited p-mTOR. MLT increased p-ERK1/2 and, when combined to DHA, increased GSTP1 expression (p < 0.01). DHA did not change the testosterone levels in the medium, whereas MLT alone or coincubated decreased by about 20%; however, any incubation affected AR expression. Moreover, incubation with luzindole revealed that MLT effects were MTR1/2-independent. In conclusion, DHA increased ROS production and impaired mitochondrial function which was probably related to AKT inactivation; MLT improved OXPHOS and decreased ROS which was related to AKT/mTOR dephosphorylation, and when coincubated, the antiproliferative action was related to mitochondrial bioenergetic modulation associated to AKT and ERK1/2 regulation. Together, these findings point to the potential application of DHA and MLT towards the prevention of proliferative prostate diseases.

A surface enhanced Raman scattering based colloid nanosensor for developing therapeutic drug monitoring.

Competitive reactions, on the surface of plasmonic nanostructures, allow exploiting SERS signals for quantitative Therapeutic Drug Monitoring. As an example, the concentration of Erlotinib, an anti-EGFR small molecule, used for the treatment of non-small cell lung and pancreatic cancer, is determined. The numerous side effects and the variability of patient responses make Erlotinib a good candidate for monitoring. The new SERS based sensor can estimate Erlotinib down to nanomolar concentration and is based on the chemical reaction of the drug and of a competitor SERS reporter on the surface of gold nanostructures. Colloid solutions of naked gold nanoparticles obtained by laser ablation in solution were used for obtaining nanostructures with very efficient hot spots for SERS and with a clean surface for chemistry. Detection of the drug in the nanomolar concentration range is shown to be possible also in spiked plasma samples.

Understanding the good and poor cell targeting activity of gold nanostructures functionalized with molecular units for the epidermal growth factor receptor.

Nanostructures can strongly interact with cells or other biological structures; furthermore when they are functionalized with targeting units, they are of great interest for a variety of applications in the biotechnology field like those for efficient imaging, diagnosis and therapy and in particular for cancer theranostics. Obtaining targeting with good specificity and sensitivity is a key necessity, which, however, is affected by the complexity of the interactions between the nanostructures and the biological components. In this work we report the study of specificity and sensitivity of gold nanoparticles functionalized with the peptide GE11 for the targeting of the epidermal growth factor receptor, expressed on many cells and, in particular, on many types of cancer cells. We show how a combination of spectroscopic measurements and molecular dynamics simulations allows the comprehension of the targeting activity of peptides linked to the surface of gold nanostructures and how the targeting is tuned by the presence of polyethylene glycol chains.

A SERRS/MRI multimodal contrast agent based on naked Au nanoparticles functionalized with a Gd(iii) loaded PEG polymer for tumor imaging and localized hyperthermia.

Multimodal contrast agents offer new interesting diagnostic possibilities, summing the benefits of multiple imaging techniques. Magnetic resonance and optical imaging are complementary techniques. The first allows total body screening, even though it suffers from low spatial resolution and needs high loadings, whereas the second shows lower penetration, but bright signals, and a higher spatial resolution and needs lower loadings. We present a plasmonic nanosystem as a MRI (magnetic resonance imaging) and SERRS (surface enhanced resonance Raman scattering) multimodal contrast agent. Naked gold nanoparticles, obtained by laser ablation synthesis in solution, are organized as a highly efficient SERRS substrate with a naphthalocyanine reporter and functionalized with a MRI contrast agent with a newly synthesized 3DOTA-PEG polymer, with a high GdIII loading. As a proof of concept, in vivo and ex vivo MRI and SERRS experiments are also performed. The plasmonic property of the nanosystem is then exploited to show its usefulness for localized hyperthermia.

Pathological lesions and global DNA methylation in rat prostate under streptozotocin-induced diabetes and melatonin supplementation.

Chronic hyperglycemia increases production of reactive oxygen species, which favors carcinogenesis. The association between diabetes and prostate cancer is controversial. Melatonin has antioxidant, anti-inflammatory, and antiproliferative properties. We investigated whether low doses of melatonin prevent the tissue alterations caused by diabetes and alter prostate histology of healthy rats. We also investigated whether experimental diabetes promoted the development of pathological lesions in the ventral prostate of rats. Melatonin was provided in drinking water (10 µg/kg/day) from age 5 weeks until the end of experiment. Diabetes was induced at 13 weeks by administration of streptozotocin (40 mg/kg, ip). Rats were euthanized at 14 or 21 weeks. Histological and stereological analyses were carried out and the incidence and density of malignant and pre-malignant lesions were assessed. Immunohistochemical assays of α-actin, cell proliferation (PCNA), Bcl-2, glutathione S-transferase (GSTPI), and DNA methylation (5-methylcytidine) were performed. Melatonin did not elicit conspicuous changes in the prostate of healthy animals; in diabetic animals there was a higher incidence of atrophy (93%), microinvasive carcinoma (10%), proliferative inflammatory atrophy, PIA (13%), prostatitis (26%), and prostate intraepithelial neoplasia, PIN (20%) associated with an increase of 40% in global DNA methylation. Melatonin attenuated epithelial and smooth muscle cell (smc) atrophy, especially at short-term diabetes-and normalized incidence of PIN (11%), inflammatory cells infiltrates, prostatitis (0%) and PIA (0%) at long-term diabetes. MLT was effective in preventing inflammatory disorders and PIN under diabetic condition. Although MLT has antioxidant action, it did not influence DNA methylation and not avoid carcinogenesis at low doses.

Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide.

Plasmonic nanostructures show important properties for biotechnological applications, but they have to be guided on the target for exploiting their potentialities. Antibodies are the natural molecules for targeting. However, their possible adverse immunogenic activity and their cost have suggested finding other valid substitutes. Small molecules like peptides can be an alternative source of targeting agents, even if, as single molecules, their binding affinity is usually not very good. GE11 is a small dodecapeptide with specific binding to the epidermal growth factor receptor (EGFR) and low immunogenicity. The present work shows that thousands of polyethylene glycol (PEG) chains modified with lysines and functionalized with GE11 on clusters of naked gold nanoparticles, obtained by laser ablation in water, achieves a better targeting activity than that recorded with nanoparticles decorated with the specific anti-EGFR antibody Cetuximab (C225). The insertion of the cationic spacer between the polymeric part of the ligand and the targeting peptide allows for a proper presentation of GE11 on the surface of the nanosystems. Surface enhanced resonance Raman scattering signals of the plasmonic gold nanoparticles are used for quantifying the targeting activity. Molecular dynamic calculations suggest that subtle differences in the exposition of the peptide on the PEG sea are important for the targeting activity.

Light-Induced Porphyrin-Based Spectroscopic Ruler for Nanometer Distance Measurements.

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin-labeling method based on the photoexcited triplet state (S=1). Four-pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N-terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8-8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR-derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)-porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.