Sphingomyelinase-responsive nanomicelles for targeting atherosclerosis.

Atherosclerosis, a leading cause of cardiovascular diseases requires approaches to enhance disease monitoring and treatment. Nanoparticles offer promising potential in this area by being customisable to target components or molecular processes within plaques, while carrying diagnostic and therapeutic agents. However, the number of biomarkers available to target this disease is limited. This study investigated the use of sphingomyelin-based nanomicelles triggered by sphingomyelinase (SMase) in atherosclerotic plaques. Accumulation of iron oxide-based nanomicelles in the plaque was demonstrated by fluorescence, MR imaging and electron microscopy. These findings demonstrate the possibility of utilising SMase as a mechanism to retain nanoprobes within plaques, thus opening up possibilities for future therapeutic interventions.

A Comparative Study of Ultrasmall Calcium Carbonate Nanoparticles for Targeting and Imaging Atherosclerotic Plaque.

Atherosclerosis is a complex disease that can lead to life-threatening events, such as myocardial infarction and ischemic stroke. Despite the severity of this disease, diagnosing plaque vulnerability remains challenging due to the lack of effective diagnostic tools. Conventional diagnostic protocols lack specificity and fail to predict the type of atherosclerotic lesion and the risk of plaque rupture. To address this issue, technologies are emerging, such as noninvasive medical imaging of atherosclerotic plaque with customized nanotechnological solutions. Modulating the biological interactions and contrast of nanoparticles in various imaging techniques, including magnetic resonance imaging, is possible through the careful design of their physicochemical properties. However, few examples of comparative studies between nanoparticles targeting different hallmarks of atherosclerosis exist to provide information about the plaque development stage. Our work demonstrates that Gd (III)-doped amorphous calcium carbonate nanoparticles are an effective tool for these comparative studies due to their high magnetic resonance contrast and physicochemical properties. In an animal model of atherosclerosis, we compare the imaging performance of three types of nanoparticles: bare amorphous calcium carbonate and those functionalized with the ligands alendronate (for microcalcification targeting) and trimannose (for inflammation targeting). Our study provides useful insights into ligand-mediated targeted imaging of atherosclerosis through a combination of in vivo imaging, ex vivo tissue analysis, and in vitro targeting experiments.

Corrigendum: Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

[This corrects the article DOI: 10.3389/fphar.2023.1021535.].

Biomimetic and Functional Nanomaterials for Molecular Imaging.

Welcome to this Special Issue of the journal Pharmaceutics entitled "Biomimetic and Functional Nanomaterials for Molecular Imaging," which focuses on the exciting advancements in molecular imaging facilitated by biomaterials and nanotechnology [...].

γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

Impact of a TAK-1 inhibitor as a single or as an add-on therapy to riociguat on the metabolic reprograming and pulmonary hypertension in the SUGEN5416/hypoxia rat model.

Background: Despite increasing evidence suggesting that pulmonary arterial hypertension (PAH) is a complex disease involving vasoconstriction, thrombosis, inflammation, metabolic dysregulation and vascular proliferation, all the drugs approved for PAH mainly act as vasodilating agents. Since excessive TGF-ß signaling is believed to be a critical factor in pulmonary vascular remodeling, we hypothesized that blocking TGFß-activated kinase 1 (TAK-1), alone or in combination with a vasodilator therapy (i.e., riociguat) could achieve a greater therapeutic benefit. Methods: PAH was induced in male Wistar rats by a single injection of the VEGF receptor antagonist SU5416 (20 mg/kg) followed by exposure to hypoxia (10%O2) for 21 days. Two weeks after SU5416 administration, vehicle, riociguat (3 mg/kg/day), the TAK-1 inhibitor 5Z-7-oxozeaenol (OXO, 3 mg/kg/day), or both drugs combined were administered for 7 days. Metabolic profiling of right ventricle (RV), lung tissues and PA smooth muscle cells (PASMCs) extracts were performed by magnetic resonance spectroscopy, and the differences between groups analyzed by multivariate statistical methods. Results: In vitro, riociguat induced potent vasodilator effects in isolated pulmonary arteries (PA) with negligible antiproliferative effects and metabolic changes in PASMCs. In contrast, 5Z-7-oxozeaenol effectively inhibited the proliferation of PASMCs characterized by a broad metabolic reprogramming but had no acute vasodilator effects. In vivo, treatment with riociguat partially reduced the increase in pulmonary arterial pressure (PAP), RV hypertrophy (RVH), and pulmonary vascular remodeling, attenuated the dysregulation of inosine, glucose, creatine and phosphocholine (PC) in RV and fully abolished the increase in lung IL-1ß expression. By contrast, 5Z-7-oxozeaenol significantly reduced pulmonary vascular remodeling and attenuated the metabolic shifts of glucose and PC in RV but had no effects on PAP or RVH. Importantly, combined therapy had an additive effect on pulmonary vascular remodeling and induced a significant metabolic effect over taurine, amino acids, glycolysis, and TCA cycle metabolism via glycine-serine-threonine metabolism. However, it did not improve the effects induced by riociguat alone on pulmonary pressure or RV remodeling. None of the treatments attenuated pulmonary endothelial dysfunction and hyperresponsiveness to serotonin in isolated PA. Conclusion: Our results suggest that inhibition of TAK-1 induces antiproliferative effects and its addition to short-term vasodilator therapy enhances the beneficial effects on pulmonary vascular remodeling and RV metabolic reprogramming in experimental PAH.

Design and fabrication of a microfluidic system with embedded circular channels for rotary cell culture.

The development of functional blood vessels is today a fundamental pillar in the evaluation of new therapies and diagnostic agents. This article describes the manufacture and subsequent functionalization, by means of cell culture, of a microfluidic device with a circular section. Its purpose is to simulate a blood vessel in order to test new treatments for pulmonary arterial hypertension. The manufacture was carried out using a process in which a wire with a circular section determines the dimensions of the channel. To fabricate the blood vessel, cells were seeded under rotary cell culture to obtain a homogeneous cell seeding in the inner wall of the devices. This is a simple and reproducible method that allows the generation of blood vessel models in vitro.

Machine Learning-Assisted High-Throughput SERS Classification of Cell Secretomes.

During the response to different stress conditions, damaged cells react in multiple ways, including the release of a diverse cocktail of metabolites. Moreover, secretomes from dying cells can contribute to the effectiveness of anticancer therapies and can be exploited as predictive biomarkers. The nature of the stress and the resulting intracellular responses are key determinants of the secretome composition, but monitoring such processes remains technically arduous. Hence, there is growing interest in developing tools for noninvasive secretome screening. In this regard, it has been previously shown that the relative concentrations of relevant metabolites can be traced by surface-enhanced Raman scattering (SERS), thereby allowing label-free biofluid interrogation. However, conventional SERS approaches are insufficient to tackle the requirements imposed by high-throughput modalities, namely fast data acquisition and automatized analysis. Therefore, machine learning methods were implemented to identify cell secretome variations while extracting standard features for cell death classification. To this end, ad hoc microfluidic chips were devised, to readily conduct SERS measurements through a prototype relying on capillary pumps made of filter paper, which eventually would function as the SERS substrates. The developed strategy may pave the way toward a faster implementation of SERS into cell secretome classification, which can be extended even to laboratories lacking highly specialized facilities.

Luminescent Carbon Nanodots Doped with Gadolinium (III): Purification Criteria, Chemical and Biological Characterization of a New Dual Fluorescence/MR Imaging Agent.

Carbon Dots (CDs) are luminescent quasi-spherical nanoparticles, possessing water solubility, high biocompatibility, and tunable chemical and physical properties for a wide range of applications, including nanomedicine and theranostics. The evaluation of new purification criteria, useful to achieve more reliable CDs, free from the interference of artifacts, is currently an object of debate in the field. Here, new CDs doped with gadolinium (Gd (III)), named Gd@CNDs, are presented as multifunctional probes for Magnetic Resonance Imaging (MRI). This new system is a case of study, to evaluate and/or combine different purification strategies, as a crucial approach to generate CDs with a better performance. Indeed, these new amorphous Gd@CNDs display good homogeneity, and they are free from emissive side products. Gd@CNDs (7-10 nm) contain 7% of Gd (III) w/w, display suitable and stable longitudinal relaxivity (r1 ) and with emissive behavior, therefore potentially useful for both MR and fluorescence imaging. They show good biocompatibility in both cellular and in vivo studies, cell permeability, and the ability to generate contrast in cellular pellets. Finally, MRI recording T1 -weighted images on mice after intravenous injection of Gd@CNDs, show signal enhancement in the liver, spleen, and kidney 30 min postinjection.

Assessment of Regional Lung Ventilation with Positron Emission Tomography Using the Radiofluorinated Gas [18F]SF6: Application to an Animal Model of Impaired Ventilation.

PURPOSE: Clinical ventilation studies are primarily performed with computerized tomography (CT) and more often with single-photon emission Computerized tomography (SPECT) using radiolabelled aerosols, both presenting certain limitations. Here, we investigate the use of the radiofluorinated gas [18F]SF6 as a positron emission tomography (PET) ventilation marker in an animal model of impaired lung ventilation. PROCEDURES: Sprague-Dawley rats (n = 15) were randomly assigned to spontaneous ventilation (sham group), endotracheal administration of phosphate-buffered saline (PBS group), or endotracheal administration of lipopolysaccharide (LPS group). PET-[18F]SF6 images (10-min acquisition) were acquired at t = 48 h after LPS or PBS administration under mechanical ventilation. CT images were acquired after each PET session. Volumes of interest were manually delineated in the lungs on CT images, and voxel-by-voxel analysis was carried out on PET images to obtain the corresponding histograms. After the imaging sessions, lungs were harvested to conduct histological analysis. RESULTS: Ventilation studies in sham animals showed uniform distribution of [18F]SF6 and fast elimination of the radioactivity after discontinuation of the administration. For PBS- and LPS-treated rats, ventilation defects were observed on PET images in some animals, identified as regions with low presence of the radiolabelled gas. Hypoventilated areas co-localized with regions with higher x-ray attenuation than healthy lungs on the CT images, suggesting the presence of oedema and, in some cases, atelectasis. Histograms obtained from PET images showed quasi-Gaussian distributions for control animals, while PBS- and LPS-treated animals demonstrated the presence of hypoventilated voxels. Deviation of the histograms from Gaussian distribution correlated with histological score was obtained by ex vivo histological analysis. CONCLUSIONS: [18F]SF6 is an appropriate marker of regional lung ventilation and may find application in the early diagnose of acute lung disease.

A Comprehensive Introduction to Magnetic Resonance Imaging Relaxometry and Contrast Agents.

The development of imaging technologies allowing noninvasive observation through solid bodies has represented a breakthrough in medical diagnosis, facilitating the comprehension of biomolecular events underlying disease and the development of more efficient therapeutic approaches. Some of the traditional limitations of these techniques are nowadays fading away thanks to the combination of imaging with nanotechnology, allowing the development of novel functional biomaterials that increase the sensitivity of detection, enable sensitivity to specific targets, and facilitate the combination of therapeutic and diagnostic functions (theragnosis) with molecular platforms functioning simultaneously as imaging probes and drug delivery carriers. The design of such functional biomaterials requires a comprehensive understanding of the principles that govern the generation of signal and modulation of contrast for a given imaging modality to exploit its capabilities to the maximal level. In this sense, magnetic resonance imaging (MRI) is a technique that presents a complex relationship between the detected signal and the physical-chemical properties of its sourcing matter, allowing the generation of multiple image contrasts. Thus, while magnetic resonance imaging is a highly versatile imaging modality, it requires specific knowledge of its physical principles to take advantage of all of its possibilities. This work reviews the origin of the image signal and contrast in MRI and the concepts of relaxometry and MRI contrast agents.

Delayed alveolar clearance of nanoparticles through control of coating composition and interaction with lung surfactant protein A.

The coating composition of nanomedicines is one of the main features in determining the medicines' fate, clearance, and immunoresponse in the body. To highlight the coatings' impact in pulmonary administration, two micellar superparamagnetic iron oxide nanoparticles (SPION) were compared. These nanoparticles are similar in size and charge but have different coatings: either phosphatidylcholine (PC-SPION) or bovine serum albumin (BSA-SPION). The aim of the study was to increase the understanding of the nano-bio interaction with the cellular and non-cellular components of the lung and underline valuable coatings either for local lung-targeted drug delivery in theranostic application or patient-friendly route systemic administration. PC-SPION and BSA-SPION were deposited in the alveoli by in vivo instillation and, despite the complexity of imaging the lung, SPION were macroscopically visualized by MRI. Impressively, PC-SPION were retained within the lungs for at least a week, while BSA-SPION were cleared more rapidly. The different lung residence times were confirmed by histological analysis and supported by a flow cytometry analysis of the SPION interactions with different myeloid cell populations. To further comprehend the way in which these nanoformulations interact with lung components at the molecular level, we used fluorescence spectroscopy, turbidity measurements, and dynamic light scattering to evaluate the interactions of the two SPION with surfactant protein A (SP-A), a key protein in setting up the nanoparticle behavior in the alveolar fluid. We found that SP-A induced aggregation of PC-SPION, but not BSA-SPION, which likely caused PC-SPION retention in the lung without inducing inflammation. In conclusion, the two SPION show different outcomes from interaction with SP-A leading to distinctive fate in the lung. PC-SPION hold great promise as imaging and theranostic agents when prolonged pulmonary drug delivery is required.

2 deoxy-D-glucose augments the mitochondrial respiratory chain in heart.

2-Deoxy-D-glucose (2DG) has recently received emergency approval for the treatment of COVID-19 in India, after a successful clinical trial. SARS-CoV-2 infection of cultured cells is accompanied by elevated glycolysis and decreased mitochondrial function, whereas 2DG represses glycolysis and stimulates respiration, and restricts viral replication. While 2DG has pleiotropic effects on cell metabolism in cultured cells it is not known which of these manifests in vivo. On the other hand, it is known that 2DG given continuously can have severe detrimental effects on the rodent heart. Here, we show that the principal effect of an extended, intermittent 2DG treatment on mice is to augment the mitochondrial respiratory chain proteome in the heart; importantly, this occurs without vacuolization, hypertrophy or fibrosis. The increase in the heart respiratory chain proteome suggests an increase in mitochondrial oxidative capacity, which could compensate for the energy deficit caused by the inhibition of glycolysis. Thus, 2DG in the murine heart appears to induce a metabolic configuration that is the opposite of SARS-CoV-2 infected cells, which could explain the compound's ability to restrict the propagation of the virus to the benefit of patients with COVID-19 disease.

Ultrasmall Manganese Ferrites for In Vivo Catalase Mimicking Activity and Multimodal Bioimaging.

Manganese ferrite nanoparticles display interesting features in bioimaging and catalytic therapies. They have been recently used in theranostics as contrast agents in magnetic resonance imaging (MRI), and as catalase-mimicking nanozymes for hypoxia alleviation. These promising applications encourage the development of novel synthetic procedures to enhance the bioimaging and catalytic properties of these nanomaterials simultaneously. Herein, a cost-efficient synthetic microwave method is developed to manufacture ultrasmall manganese ferrite nanoparticles as advanced multimodal contrast agents in MRI and positron emission tomography (PET), and improved nanozymes. Such a synthetic method allows doping ferrites with Mn in a wide stoichiometric range (Mnx Fe3-x O4 , 0.1 ≤ x ≤ 2.4), affording a library of nanoparticles with different magnetic relaxivities and catalytic properties. These tuned magnetic properties give rise to either positive or dual-mode MRI contrast agents. On the other hand, higher levels of Mn doping enhance the catalytic efficiency of the resulting nanozymes. Finally, through their intracellular catalase-mimicking activity, these ultrasmall manganese ferrite nanoparticles induce an unprecedented tumor growth inhibition in a breast cancer murine model. All of these results show the robust characteristics of these nanoparticles for nanobiotechnological applications.

Heteroplasmy of Wild-Type Mitochondrial DNA Variants in Mice Causes Metabolic Heart Disease With Pulmonary Hypertension and Frailty.

BACKGROUND: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is transmitted uniparentally and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of >1 mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent nonpathologic mtDNA heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. METHODS: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiologic, and phenotyping techniques. We focused on in vivo imaging techniques for noninvasive assessment of cardiac and pulmonary energy metabolism. RESULTS: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. CONCLUSIONS: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.

Benchtop nuclear magnetic resonance-based metabolomic approach for the diagnosis of bovine tuberculosis.

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non-tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high-field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low-field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB-vaccinated healthy control (n = 10) and healthy PTB-unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77-1). In summary, plasma fingerprinting using HF and LF-NMR differentiated TB subjects from uninfected animals, and PTB and PTB-vaccinated subjects who may provide a TB-false positive, highlighting the use of LF-NMR-based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods.

Metabolomic diferences between COVID-19 and H1N1 influenza induced ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a type of respiratory failure characterized by lung inflammation and pulmonary edema. Coronavirus disease 2019 (COVID-19) is associated with ARDS in the more severe cases. This study aimed to compare the specificity of the metabolic alterations induced by COVID-19 or Influenza A pneumonia (IAP) in ARDS. METHODS: Eighteen patients with ARDS due to COVID-19 and twenty patients with ARDS due to IAP, admitted to the intensive care unit. ARDS was defined as in the American-European Consensus Conference. As compared with patients with COVID-19, patients with IAP were younger and received more often noradrenaline to maintain a mean arterial pressure > 65 mm Hg. Serum samples were analyzed by Nuclear Magnetic Resonance Spectroscopy. Multivariate Statistical Analyses were used to identify metabolic differences between groups. Metabolic pathway analysis was performed to identify the most relevant pathways involved in ARDS development. RESULTS: ARDS due to COVID-19 or to IAP induces a different regulation of amino acids metabolism, lipid metabolism, glycolysis, and anaplerotic metabolism. COVID-19 causes a significant energy supply deficit that induces supplementary energy-generating pathways. In contrast, IAP patients suffer more marked inflammatory and oxidative stress responses. The classificatory model discriminated against the cause of pneumonia with a success rate of 100%. CONCLUSIONS: Our findings support the concept that ARDS is associated with a characteristic metabolomic profile that may discriminate patients with ARDS of different etiologies, being a potential biomarker for the diagnosis, prognosis, and management of this condition.

HAP-Multitag, a PET and Positive MRI Contrast Nanotracer for the Longitudinal Characterization of Vascular Calcifications in Atherosclerosis.

Vascular microcalcifications are associated with atherosclerosis plaque instability and, therefore, to increased mortality. Because of this key role, several imaging probes have been developed for their in vivo identification. Among them, [18F]FNa is the gold standard, showing a large uptake in the whole skeleton by positron emission tomography. Here, we push the field toward the combined anatomical and functional early characterization of atherosclerosis. For this, we have developed hydroxyapatite (HAP)-multitag, a bisphosphonate-functionalized 68Ga core-doped magnetic nanoparticle showing high affinity toward most common calcium salts present in microcalcifications, particularly HAP. We characterized this interaction in vitro and in vivo, showing a massive uptake in the atherosclerotic lesion identified by positron emission tomography (PET) and positive contrast magnetic resonance imaging (MRI). In addition, this accumulation was found to be dependent on the calcification progression, with a maximum uptake in the microcalcification stage. These results confirmed the ability of HAP-multitag to identify vascular calcifications by PET/(T1)MRI during the vulnerable stages of the plaque progression.

Assessing the Potential of Molecular Imaging for Myelin Quantification in Organotypic Cultures.

Ex vivo models for the noninvasive study of myelin-related diseases represent an essential tool to understand the mechanisms of diseases and develop therapies against them. Herein, we assessed the potential of multimodal imaging traceable myelin-targeting liposomes to quantify myelin in organotypic cultures. Methods: MRI testing was used to image mouse cerebellar tissue sections and organotypic cultures. Demyelination was induced by lysolecithin treatment. Myelin-targeting liposomes were synthetized and characterized, and their capacity to quantify myelin was tested by fluorescence imaging. Results: Imaging of freshly excised tissue sections ranging from 300 µm to 1 mm in thickness was achieved with good contrast between white (WM) and gray matter (GM) using T2w MRI. The typical loss of stiffness, WM structures, and thickness of organotypic cultures required the use of diffusion-weighted methods. Designed myelin-targeting liposomes allowed for semiquantitative detection by fluorescence, but the specificity for myelin was not consistent between assays due to the unspecific binding of liposomes. Conclusions: With respect to the sensitivity, imaging of brain tissue sections and organotypic cultures by MRI is feasible, and myelin-targeting nanosystems are a promising solution to quantify myelin ex vivo. With respect to specificity, fine tuning of the probe is required. Lipid-based systems may not be suitable for this goal, due to unspecific binding to tissues.