PEGylation-Dependent Metabolic Rewiring of Macrophages with Silk Fibroin Nanoparticles

Silk fibroin nanoparticles are emerging as promising nanomedicines, but their full therapeutic potential is yet to be realized. These nanoparticles can be readily PEGylated to improve colloidal stability and to tune degradation and drug release profiles; however, the relationship between silk fibroin nanoparticle PEGylation and macrophage activation still requires elucidation. Here, we used in vitro assays and nuclear magnetic resonance based metabolomics to examine the inflammatory phenotype and metabolic profiles of macrophages following their exposure to unmodified or PEGylated silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles, but they showed different phenotypic and metabolic responses to each nanoparticle type. Unmodified silk fibroin nanoparticles induced the upregulation of several processes, including production of proinflammatory mediators (e.g., cytokines), release of nitric oxide, and promotion of antioxidant activity. These responses were accompanied by changes in the macrophage metabolomic profiles that were consistent with a proinflammatory state and that indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. By contrast, PEGylated silk fibroin nanoparticles induced milder changes to both inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is PEGylation-dependent. Overall, PEGylation of silk fibroin nanoparticles reduced the inflammatory and metabolic responses initiated by macrophages, and this observation could be used to guide the therapeutic applications of these nanoparticles. © 2019 American Chemical Society

Metabolic reprogramming of macrophages exposed to silk, poly(lactic-co-glycolic acid) and silica nanoparticles

Monitoring macrophage metabolism in response to nanoparticle exposure provides new insights into biological outcomes, such as inflammation or toxicity, and supports the design of tailored nanomedicines. We describe the metabolic signature of macrophages exposed to nanoparticles ranging in diameter from 100 to 125 nm and made from silk, poly(lactic-co-glycolic acid) or silica. Nanoparticles of this size and type are currently at various stages of pre-clinical and clinical development for drug delivery applications. We used 1H NMR analysis of cell extracts and culture media to quantify the changes in the intracellular and extracellular metabolomes of macrophages in response to nanoparticle exposure. Increased glycolytic activity, an altered tricarboxylic acid cycle and reduced ATP generation were consistent with a pro-inflammatory phenotype. Furthermore, amino acids possibly arising from autophagy, the creatine kinase/phosphocreatine system and a few osmolytes and antioxidants emerged as important players in the metabolic reprogramming of macrophages exposed to nanoparticles. This metabolic signature was a common response to all nanoparticles tested; however, the direction and magnitude of some variations were clearly nanoparticle specific, indicating material-induced biological specificity. Overall, metabolic reprogramming of macrophages can be achieved with nanoparticle treatments, modulated through the choice of the material, and monitored using 1H NMR metabolomics

Comparative Metabolomics Study of the Impact of Articaine and Lidocaine on the Metabolism of SH-SY5Y Neuronal Cells

Articaine (ATC) and lidocaine (LDC) are the local anesthetics (LAs) currently most employed in dentistry. Cases of paresthesia, reported more frequently for ATC, have raised concerns about their potential neurotoxicity, calling for further investigation of their biological effects in neuronal cells. In this work, the impact of ATC and LDC on the metabolism of SH-SY5Y cells was investigated through 1H NMR metabolomics. For each LA, in vitro cultured cells were exposed to concentrations causing 10 and 50% reductions in cell viability, and their metabolic intracellular and extracellular profiles were characterized. Most effects were common to ATC and LDC, although with varying magnitudes. The metabolic variations elicited by the two LAs suggested (i) downregulation of glycolysis and of glucose-dependent pathways (e.g., one-carbon metabolism and hexosamine biosynthetic pathway), (ii) disturbance of branched chain amino acids (BCAA) catabolism, (iii) downregulation of TCA cycle anaplerotic fueling and activation of alternative energy producing pathways, (iv) interference with choline metabolism and (v) lipid droplet build-up. Interestingly, LDC had a greater impact on membrane phospholipid turnover, as suggested by higher phosphatidylcholine to phosphocholine conversion. Moreover, LDC elicited an increase in triglycerides, whereas cholesteryl esters accumulated in ATC-exposed cells, suggesting a different composition and handling of lipid droplets.publishe

Silk Hydrogel Substrate Stress Relaxation Primes Mesenchymal Stem Cell Behavior in 2D

Tissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1β, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1β signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels.

Odd–even effect in the formation and extraction performance of Ionic-Liquid-Based aqueous biphasic systems

Ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been extensively investigated in the separation of high-value biomolecules. However, the understanding of the molecular-level mechanisms ruling phase separation and extraction performance of these systems is crucial to successfully design effective separation processes. In this work, IL-based ABS composed of K2HPO4 and cholinium carboxylate ILs ([Ch][CnCO2] with n = 1 to 7, comprising anions with odd and even alkyl chain length) were investigated. The respective ternary phase diagrams, including binodal curves, tie-lengths, tie-line lengths and critical points, as well as the Setschenow salting-out coefficients (ks) that is a quantitative measure of the two-phase formation ability, were determined at 298 K. The extraction performance of these systems was then evaluated for four amino acids (L-tryptophan, L-phenylalanine, L-tyrosine, L-3,4-dihydroxyphenylalanine/L-dopa). It was found that ILs composed of anions with even alkyl chains display slightly higher ks values, meaning that these ILs are more easily salted-out or more easily phase separate to form ABS. On the other hand, ABS formed by ILs with anions comprising odd alkyl chains lead to slightly higher partition coefficients of amino acids. Beyond the neat ILs odd-even effect resulting from their nanostructuration, being this a well-known phenomenon occurring in a series of their thermophysical properties, it is here shown the existence of an odd-even effect displayed by the IL anion aliphatic moiety in aqueous solution, visible both in the two-phase formation ability and extraction performance of ABS. These findings contribute to elucidate the molecular-level mechanisms governing ABS formation and partitioning of biomolecules, ultimately allowing the design of effective separation platforms.publishe

On the Relevance of Soft Tissue Sarcomas Metabolic Landscape Mapping

Soft tissue sarcomas (STS) prognosis is disappointing, with current treatment strategies being based on a "fit for all" principle and not taking distinct sarcoma subtypes specificities and genetic/metabolic differences into consideration. The paucity of precision therapies in STS reflects the shortage of studies that seek to decipher the sarcomagenesis mechanisms. There is an urge to improve STS diagnosis precision, refine STS classification criteria, and increase the capability of identifying STS prognostic biomarkers. Single-omics and multi-omics studies may play a key role on decodifying sarcomagenesis. Metabolomics provides a singular insight, either as a single-omics approach or as part of a multi-omics strategy, into the metabolic adaptations that support sarcomagenesis. Although STS metabolome is scarcely characterized, untargeted and targeted metabolomics approaches employing different data acquisition methods such as mass spectrometry (MS), MS imaging, and nuclear magnetic resonance (NMR) spectroscopy provided important information, warranting further studies. New chromatographic, MS, NMR-based, and flow cytometry-based methods will offer opportunities to therapeutically target metabolic pathways and to monitorize the response to such metabolic targeting therapies. Here we provide a comprehensive review of STS omics applications, comprising a detailed analysis of studies focused on the metabolic landscape of these tumors.publishe

Endo- and exometabolome crosstalk in mesenchymal stem cells undergoing osteogenic differentiation

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.publishe