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To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report the development of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.
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The use of precious metal electrocatalysts in clean electrochemical energy conversion and storage applications is widespread, but the sustainability of these materials, in terms of their availability and cost, is constrained. In this research, iron triad-based bimetallic nitrogen-doped carbon (M-N-C) materials were investigated as potential bifunctional electrocatalysts for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). The synthesis of bimetallic FeCo-N-C, CoNi-N-C, and FeNi-N-C catalysts involved a precisely optimized carbonization process of their respective metal-organic precursors. Comprehensive structural analysis was undertaken to elucidate the morphology of the prepared M-N-C materials, while their electrocatalytic performance was assessed through cyclic voltammetry and rotating disk electrode measurements in a 0.1 M KOH solution. All bimetallic catalyst materials demonstrated impressive bifunctional electrocatalytic performance in both the ORR and the OER. However, the FeNi-N-C catalyst proved notably more stable, particularly in the OER conditions. Employed as a bifunctional catalyst for ORR/OER within a customized zinc-air battery, FeNi-N-C exhibited a remarkable discharge-charge voltage gap of only 0.86 V, alongside a peak power density of 60 mW cm-2. The outstanding stability of FeNi-N-C, operational for about 55 h at 2 mA cm-2, highlights its robustness for prolonged application.
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In vivo phage display is widely used for identification of organ- or disease-specific homing peptides. However, the current in vivo phage biopanning approaches fail to assess biodistribution of specific peptide phages across tissues during the screen, thus necessitating laborious and time-consuming post-screening validation studies on individual peptide phages. Here, we adopted bioinformatics tools used for RNA sequencing for analysis of high-throughput sequencing (HTS) data to estimate the representation of individual peptides during biopanning in vivo. The data from in vivo phage screen were analyzed using differential binding-relative representation of each peptide in the target organ versus in a panel of control organs. Application of this approach in a model study using low-diversity peptide T7 phage library with spiked-in brain homing phage demonstrated brain-specific differential binding of brain homing phage and resulted in identification of novel lung- and brain-specific homing peptides. Our study provides a broadly applicable approach to streamline in vivo peptide phage biopanning and to increase its reproducibility and success rate.
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Técnicas de Visualização da Superfície Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Peptídeos/metabolismo , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
Melanocortin-4 receptors (MC4 R) are unique among G-protein-coupled receptors (GPCRs) as they have endogenous ligands that can exhibit inverse agonistic properties in the case of elevated basal activity. It is known that the constitutive activity of GPCRs strongly affects the ligand-dependent physiological responses, but little is known about these regulatory mechanisms. Since several metal ions have been shown to be important modulators of the signal transduction of GPCRs, we hypothesized that metal ions regulate the basal activity of MC4 Rs. Implementation of a fluorescence anisotropy assay and novel redshifted fluorescent peptides enabled kinetic characterization of ligand binding to MC4 R expressed on budded baculoviruses. We show that Ca2+ is required for high-affinity ligand binding, but Zn2+ and Cu2+ in the presence of Ca2+ behave as negative allosteric modulators of ligand binding to MC4 R. FRET-based cAMP biosensor was used to measure the activation of MC4 R stably expressed in CHO-K1 cells. At low micromolar concentrations, Zn2+ caused MC4 R-dependent activation of the cAMP pathway, whereas Cu2+ reduced the activity of MC4 R even below the basal level. These findings indicate that at physiologically relevant concentrations can Zn2+ and Cu2+ function as MC4 R agonists or inverse agonists, respectively. This means that depending on the level of constitutive activity induced by Zn2+ ions, the pharmacological effect of orthosteric ligands of MC4 R can be switched from a partial to an inverse agonist. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Science badges can be found at https://cos.io/our-services/open-science-badges/.
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Cobre/metabolismo , AMP Cíclico/metabolismo , Receptor Tipo 4 de Melanocortina/agonistas , Receptor Tipo 4 de Melanocortina/metabolismo , Transdução de Sinais/fisiologia , Zinco/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Células CHO , Cobre/farmacologia , Cricetinae , Cricetulus , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Receptor Tipo 4 de Melanocortina/química , Células Sf9 , Transdução de Sinais/efeitos dos fármacos , Zinco/farmacologiaRESUMO
Oncofetal fibronectin (FN-EDB) and tenascin-C C domain (TNC-C) are nearly absent in extracellular matrix of normal adult tissues but upregulated in malignant tissues. Both FN-EDB and TNC-C are developed as targets of antibody-based therapies. Here we used peptide phage biopanning to identify a novel targeting peptide (PL1, sequence: PPRRGLIKLKTS) that interacts with both FN-EDB and TNC-C. Systemic PL1-functionalized model nanoscale payloads [iron oxide nanoworms (NWs) and metallic silver nanoparticles] homed to glioblastoma (GBM) and prostate carcinoma xenografts, and to non-malignant angiogenic neovessels induced by VEGF-overexpression. Antibody blockage experiments demonstrated that PL1 tumor homing involved interactions with both receptor proteins. Treatment of GBM mice with PL1-targeted model therapeutic nanocarrier (NWs loaded with a proapoptotic peptide) resulted in reduced tumor growth and increased survival, whereas treatment with untargeted particles had no effect. PL1 peptide may have applications as an affinity ligand for delivery of diagnostic and therapeutic compounds to microenvironment of solid tumors.
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Sistemas de Liberação de Medicamentos , Fibronectinas/metabolismo , Neoplasias/tratamento farmacológico , Peptídeos/uso terapêutico , Tenascina/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Linhagem Celular Tumoral , Compostos Férricos/química , Glioblastoma/tratamento farmacológico , Humanos , Imageamento por Ressonância Magnética , Nanopartículas Metálicas/química , Camundongos Nus , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Prata/químicaRESUMO
Tumor-selective drug conjugates can potentially improve the prognosis for patients affected by glioblastoma (GBM) - the most common and malignant type of brain cancer with no effective cure. Here we evaluated a novel tumor penetrating peptide that targets cell surface p32, LinTT1 (AKRGARSTA), as a GBM targeting ligand for systemically-administered nanoparticles. LinTT1-functionalization increased tumor homing of iron oxide nanoworms (NWs) across a panel of five GBM models ranging from infiltratively-disseminating to angiogenic phenotypes. LinTT1-NWs homed to CD31-positive tumor blood vessels, including to transdifferentiated endothelial cells, and showed co-localization with tumor macrophages and lymphatic vessels. LinTT1 functionalization also resulted in increased GBM delivery of other types of systemically-administered nanoparticles: silver nanoparticles and albumin-paclitaxel nanoparticles. Finally, LinTT1-guided proapoptotic NWs exerted strong anti-glioma activity in two models of GBM, including doubling the lifespan of the mice in an aggressive orthotopic stem cell-like GBM that recapitulates the histological hallmarks of human GBM. Our study suggests that LinTT1 targeting strategy can be used to increase GBM uptake of systemic nanoparticles for improved imaging and therapy.
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Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Glioblastoma/tratamento farmacológico , Nanopartículas , Peptídeos/administração & dosagem , Albuminas/administração & dosagem , Albuminas/farmacocinética , Animais , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Feminino , Compostos Férricos/química , Glioblastoma/patologia , Humanos , Masculino , Nanopartículas Metálicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Peptídeos/química , Prata/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Power generation and other industries using solid fossil fuels like coal, lignite, oil shale and peat are responsible for producing large quantities of solid residues that are often chemically reactive and/or unstable and are disposed in holding ponds and deposition sites. Stability and long-term behaviour of such deposits are typically studied in short-term laboratory experiments that cannot describe nor predict long-term changes taking place in these materials. Here, we study long-term (>40â¯years) transformations, in highly alkaline conditions, of the Ca-rich ash deposit in Estonia composed of oil shale processing residues from the Eesti power plant. Detailed mineralogical, chemical and micromorphological analyses using X-ray diffraction, X-ray fluorescence, 29Si nuclear magnetic resonance, scanning electron microscopy and other methods were applied in order to identify the composition of the waste with a focus on formation and transformation of semicrystalline phases in the deposit. The results show progressive formation of calcium-silicate-hydrate (C-S-H) type phase at the expense of silicate minerals and amorphous glass phases with increasing depth and age of the sediments, from about 25% in the upper part of the depository to over 60% in the oldest-deepest part. This demonstrates that over time the high alkalinity of the ash is responsible for initiating natural alkali-activation. The formation of C-S-H-type phases increases the mechanical strength of the sediment and ensures long-term stability of waste deposits. These findings may encourage the use of these ashes in binder or other construction material production or as construction aggregates.
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Attaching affinity ligands to nanoparticles (NPs) increases selectivity for targeting cells and tissues, and can result in improved sensitivity and reduced off-target toxicity in diagnostic and therapeutic systems. The decision over key features - NP size, shape, coating strategies and targeting ligands for clinical translation is often hampered by a lack of quantitative in vivo NP homing assays. Sensitive, internally controlled assays are needed which allow for quantitative comparisons (auditions) among various formulations of targeted NPs. We recently reported the development of peptide-functionalized, isotopically-barcoded silver NPs (AgNPs) for ultrasensitive studies centered on measuring relative ratios of NP internalization into cultured cells. Here we evaluated the application of this technology for NP homing studies in live mice using peptides with previously described tissue tropism; one peptide that favors vascular beds of the normal lungs (RPARPAR; receptor neuropilin-1, or NRP-1) and another that is selective for central nervous system vessels (CAGALCY). Equimolar mixtures of the peptide-targeted Ag107-NPs and Ag109 control particles were mixed and injected intravenously. Distribution profiles of Ag107 and Ag109 in tissue extracts were determined simultaneously through inductively coupled plasma mass spectrometry (ICP-MS). Compared to non-targeted particles up to â¼9-fold increased lung accumulation was seen for RPARPAR-OH AgNPs (but not for AgNPs functionalized with RPARPAR-NH2, which does not bind to NRP-1). Similarly, AgNPs functionalized with the brain-homing CAGALCY peptide were overrepresented in brain extracts. Spatial distribution (mapping) analysis by laser ablation ICP-MS (LA-ICP-MS) was used to determine the ratio Ag107/Ag109 in tissue cryosections. The mapping demonstrated preferential accumulation of the RPARPAR-AgNPs in the perivascular areas around pulmonary veins, and CAGALCY AgNPs accumulated in discrete areas of the brain (e.g. in the vessels of cerebellar fibrillary tracts). Based on these results, the internally controlled ratiometric AgNP system is suitable for quantitative studies of the effect of targeting ligands on NP biodistribution, at average tissue concentration and distribution at the microscopic level. The platform might be particularly relevant for target sites with high local variability in uptake, such as tumors.
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Nanopartículas Metálicas , Terapia de Alvo Molecular , Prata/farmacocinética , Animais , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Peptídeos/administração & dosagem , Distribuição TecidualRESUMO
Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 ± 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 ± 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.