X-ray radio-enhancement by Ti3C2Tx MXenes in soft tissue sarcoma.

Radiotherapy is a cornerstone of cancer treatment. However, due to the low tissue specificity of ionizing radiation, damage to the surrounding healthy tissue of the tumor remains a significant challenge. In recent years, radio-enhancers based on inorganic nanomaterials have gained considerable interest. Beyond the widely explored metal and metal oxide nanoparticles, 2D materials, such as MXenes, could present potential benefits because of their inherently large specific surface area. In this study, we highlight the promising radio-enhancement properties of Ti3C2Tx MXenes. We demonstrate that atomically thin layers of titanium carbides (Ti3C2Tx MXenes) are efficiently internalized and well-tolerated by mammalian cells. Contrary to MXenes suspended in aqueous buffers, which fully oxidize within days, yielding rice-grain shaped rutile nanoparticles, the MXenes internalized by cells oxidize at a slower rate. This is consistent with cell-free experiments that have shown slower oxidation rates in cell media and lysosomal buffers compared to dispersants without antioxidants. Importantly, the MXenes exhibit robust radio-enhancement properties, with dose enhancement factors reaching up to 2.5 in human soft tissue sarcoma cells, while showing no toxicity to healthy human fibroblasts. When compared to oxidized MXenes and commercial titanium dioxide nanoparticles, the intact 2D titanium carbide flakes display superior radio-enhancement properties. In summary, our findings offer evidence for the potent radio-enhancement capabilities of Ti3C2Tx MXenes, marking them as a promising candidate for enhancing radiotherapy.

Multiscale multimodal characterization and simulation of structural alterations in failed bioprosthetic heart valves.

Calcific degeneration is the most frequent type of heart valve failure, with rising incidence due to the ageing population. The gold standard treatment to date is valve replacement. Unfortunately, calcification oftentimes re-occurs in bioprosthetic substitutes, with the governing processes remaining poorly understood. Here, we present a multiscale, multimodal analysis of disturbances and extensive mineralisation of the collagen network in failed bioprosthetic bovine pericardium valve explants with full histoanatomical context. In addition to highly abundant mineralized collagen fibres and fibrils, calcified micron-sized particles previously discovered in native valves were also prevalent on the aortic as well as the ventricular surface of bioprosthetic valves. The two mineral types (fibres and particles) were detectable even in early-stage mineralisation, prior to any macroscopic calcification. Based on multiscale multimodal characterisation and high-fidelity simulations, we demonstrate that mineral occurrence coincides with regions exposed to high haemodynamic and biomechanical indicators. These insights obtained by multiscale analysis of failed bioprosthetic valves serve as groundwork for the evidence-based development of more durable alternatives. STATEMENT OF SIGNIFICANCE: Bioprosthetic valve calcification is a well-known clinically significant phenomenon, leading to valve failure. The nanoanalytical characterisation of bioprosthetic valves gives insights into the highly abundant, extensive calcification and disorganization of the collagen network and the presence of calcium phosphate particles previously reported in native cardiovascular tissues. While the collagen matrix mineralisation can be primarily attributed to a combination of chemical and mechanical alterations, the calcified particles are likely of host cellular origin. This work presents a straightforward route to mineral identification and characterization at high resolution and sensitivity, and with full histoanatomical context and correlation to hemodynamic and biomechanical indicators, hence providing design cues for improved bioprosthetic valve alternatives.

Metal-Organic Framework Mediated Radio-Enhancement Assessed in High-Throughput-Compatible 3D Tumor Spheroid Co-Cultures.

Inorganic nanomaterials have gained increasing attention in radiation oncology, owing to their radiation therapy enhancing properties. To accelerate candidate material selection and overcome the disconnect between conventional 2D cell culture and in vivo findings, screening platforms unifying high-throughput with physiologically relevant endpoint analysis based on 3D in vitro models are promising. Here, a 3D tumor spheroid co-culture model based on cancerous and healthy human cells is presented for the concurrent assessment of radio-enhancement efficacy, toxicity, and intratissural biodistribution with full ultrastructural context of radioenhancer candidate materials. Its potential for rapid candidate materials screening is showcased based on the example of nano-sized metal-organic frameworks (nMOFs) and direct benchmarking against gold nanoparticles (the current "gold standard"). Dose enhancement factors (DEFs) ranging between 1.4 and 1.8 are measured for Hf-, Ti-, TiZr-, and Au-based materials in 3D tissues and are overall lower than in 2D cell cultures, where DEF values exceeding 2 are found. In summary, the presented co-cultured tumor spheroid-healthy fibroblast model with tissue-like characteristics may serve as high-throughput platform enabling rapid, cell line-specific endpoint analysis for therapeutic efficacy and toxicity assessment, as well as accelerated radio-enhancer candidate screening.

Multiscale Multimodal Investigation of the Intratissural Biodistribution of Iron Nanotherapeutics with Single Cell Resolution Reveals Co-Localization with Endogenous Iron in Splenic Macrophages.

Imaging of iron-based nanoparticles (NPs) remains challenging because of the presence of endogenous iron in tissues that is difficult to distinguish from exogenous iron originating from the NPs. Here, an analytical cascade for characterizing the biodistribution of biomedically relevant iron-based NPs from the organ scale to the cellular and subcellular scales is introduced. The biodistribution on an organ level is assessed by elemental analysis and quantification of magnetic iron by electron paramagnetic resonance, which allowed differentiation of exogenous and endogenous iron. Complementary to these bulk analysis techniques, correlative whole-slide optical and electron microscopy provided spatially resolved insight into the biodistribution of endo- and exogenous iron accumulation in macrophages, with single-cell and single-particle resolution, revealing coaccumulation of iron NPs with endogenous iron in splenic macrophages. Subsequent transmission electron microscopy revealed two types of morphologically distinct iron-containing structures (exogenous nanoparticles and endogenous ferritin) within membrane-bound vesicles in the cytoplasm, hinting at an attempt of splenic macrophages to extract and recycle iron from exogenous nanoparticles. Overall, this strategy enables the distinction of endo- and exogenous iron across scales (from cm to nm, based on the analysis of thousands of cells) and illustrates distribution on organ, cell, and organelle levels.

Modular stimuli-responsive hydrogel sealants for early gastrointestinal leak detection and containment.

Millions of patients every year undergo gastrointestinal surgery. While often lifesaving, sutured and stapled reconnections leak in around 10% of cases. Currently, surgeons rely on the monitoring of surrogate markers and clinical symptoms, which often lack sensitivity and specificity, hence only offering late-stage detection of fully developed leaks. Here, we present a holistic solution in the form of a modular, intelligent suture support sealant patch capable of containing and detecting leaks early. The pH and/or enzyme-responsive triggerable sensing elements can be read out by point-of-need ultrasound imaging. We demonstrate reliable detection of the breaching of sutures, in as little as 3 hours in intestinal leak scenarios and 15 minutes in gastric leak conditions. This technology paves the way for next-generation suture support materials that seal and offer disambiguation in cases of anastomotic leaks based on point-of-need monitoring, without reliance on complex electronics or bulky (bio)electronic implantables.

The Role of Inorganics in Preeclampsia Assessed by Multiscale Multimodal Characterization of Placentae.

Preeclampsia is one of the most dangerous diseases in pregnancy. Because of the hypertensive nature of preeclampsia, placental calcifications are believed to be a predictor for its occurrence, analogous to their role in cardiovascular diseases. However, the prevalence and the relevance of calcifications for the clinical outcome with respect to preeclampsia remains controversial. In addition, the role of other inorganic components present in the placental tissue in the development of preeclampsia has rarely been investigated. In this work, we therefore characterized inorganic constituents in placental tissue in groups of both normotensive and preeclamptic patients (N = 20 each) using a multi-scale and multi-modal approach. Examinations included elemental analysis (metallomics), sonography, computed tomography (CT), histology, scanning electron microscopy, X-ray fluorescence and energy dispersive X-ray spectroscopy. Our data show that tissue contents of several heavy metals (Al, Cd, Ni, Co, Mn, Pb, and As) were elevated whereas the Rb content was decreased in preeclamptic compared to normotensive placentae. However, the median mineral content (Ca, P, Mg, Na, K) was remarkably comparable between the two groups and CT showed lower calcified volumes and fewer crystalline deposits in preeclamptic placentae. Electron microscopy investigations revealed four distinct types of calcifications, all predominantly composed of calcium, phosphorus and oxygen with variable contents of magnesium in tissues of both maternal and fetal origin in both preeclamptic and normotensive placentae. In conclusion our study suggests that heavy metals, combined with other factors, can be associated with the development of preeclampsia, however, with no obvious correlation between calcifications and preeclampsia.

Nuclear and cellular, micro and nano calcification in Alzheimer's disease patients and correlation to phosphorylated Tau.

Brain calcification (calcium phosphate mineral formation) has been reported in the past 100 years in the brains of Alzheimer's disease (AD) patients. However, the association between calcification and AD, the triggers for calcification, and its role within the disease are not clear. On the other hand, hyperphosphorylated Tau protein (pTau) tangles have been widely studied and recognized as an essential factor in developing AD. In this work, calcification in the brains of AD patients is characterized by advanced electron microscopy and fluorescence microscopy. Results are then compared to samples from cognitively healthy, age-matched donors, and the colocalization of calcification and pTau is investigated. Here, we show that AD patients' brains present microcalcification associated with the neural cell nuclei and cell projections, and that these are strongly related to the presence of pTau. The link between microcalcification and pTau suggests a potential mechanism of brain cell damage. Together with the formation of amyloid plaques and neurofibrillary tangles, microcalcification in neuronal cells adds to a better understanding of the pathology of AD. Finally, the presence of microcalcification in the neuronal cells of AD patients may assist in AD diagnosis, and may open avenues for developing intervention strategies based on inhibition of calcification. STATEMENT OF SIGNIFICANCE: Brain calcification has been reported in the past 100 years in the brains of Alzheimer's disease (AD) patients. However, the association between calcification and AD is not clear. Hyperphosphorylated Tau protein (pTau) has been studied and recognized as a key factor in developing AD. We show here that AD patients' brains present microcalcification associated with the neuronal cell nuclei and cell projections, and that these are related to the presence of pTau. The study of calcification in brain cells can contribute to a better understanding of the biochemical mechanisms associated with AD and might also reveal that calcification is part of the full disease mechanism. Moreover, this work opens the possibility for using calcification as a biomarker to identify AD.

Annexin A1-dependent tethering promotes extracellular vesicle aggregation revealed with single-extracellular vesicle analysis.

Extracellular vesicles (EVs) including plasma membrane-derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)-dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Multiscale Analysis of Metal Oxide Nanoparticles in Tissue: Insights into Biodistribution and Biotransformation.

Metal oxide nanoparticles have emerged as exceptionally potent biomedical sensors and actuators due to their unique physicochemical features. Despite fascinating achievements, the current limited understanding of the molecular interplay between nanoparticles and the surrounding tissue remains a major obstacle in the rationalized development of nanomedicines, which is reflected in their poor clinical approval rate. This work reports on the nanoscopic characterization of inorganic nanoparticles in tissue by the example of complex metal oxide nanoparticle hybrids consisting of crystalline cerium oxide and the biodegradable ceramic bioglass. A validated analytical method based on semiquantitative X-ray fluorescence and inductively coupled plasma spectrometry is used to assess nanoparticle biodistribution following intravenous and topical application. Then, a correlative multiscale analytical cascade based on a combination of microscopy and spectroscopy techniques shows that the topically applied hybrid nanoparticles remain at the initial site and are preferentially taken up into macrophages, form apatite on their surface, and lead to increased accumulation of lipids in their surroundings. Taken together, this work displays how modern analytical techniques can be harnessed to gain unprecedented insights into the biodistribution and biotransformation of complex inorganic nanoparticles. Such nanoscopic characterization is imperative for the rationalized engineering of safe and efficacious nanoparticle-based systems.

The multiscale hierarchical structure of Heloderma suspectum osteoderms and their mechanical properties.

Osteoderms are hard tissues embedded in the dermis of vertebrates and have been suggested to be formed from several different mineralized regions. However, their nano architecture and micro mechanical properties had not been fully characterized. Here, using electron microscopy, µ-CT, atomic force microscopy and finite element simulation, an in-depth characterization of osteoderms from the lizard Heloderma suspectum, is presented. Results show that osteoderms are made of three different mineralized regions: a dense apex, a fibre-enforced region comprising the majority of the osteoderm, and a bone-like region surrounding the vasculature. The dense apex is stiff, the fibre-enforced region is flexible and the mechanical properties of the bone-like region fall somewhere between the other two regions. Our finite element analyses suggest that when combined into the osteoderm structure, the distinct tissue regions are able to shield the body of the animal by bearing the external forces. These findings reveal the structure-function relationship of the Heloderma suspectum osteoderm in unprecedented detail. STATEMENT OF SIGNIFICANCE: The structures of bone and teeth have been thoroughly investigated. They provide a basis not only for understanding the mechanical properties and functions of these hard tissues, but also for the de novo design of composite materials. Osteoderms, however, are hard tissues that must possess mechanical properties distinct from teeth and bone to function as a protective armour. Here we provide a detailed analysis of the nanostructure of vertebrate osteoderms from Heloderma suspectum, and show that their mechanical properties are determined by their multiscale hierarchical tissue. We believe this study contributes to advance the current knowledge of the structure-function relationship of the hierarchical structures in the Heloderma suspectum osteoderm. This knowledge might in turn provide a source of inspiration for the design of bioinspired and biomimetic materials.

Nano-analytical characterization of endogenous minerals in healthy placental tissue: mineral distribution, composition and ultrastructure.

Despite its crucial role, the placenta is the least understood human organ. Recent clinical studies indicate a direct association between placental calcification and maternal and offspring health. This study reveals distinct characteristics of minerals formed during gestational ageing using cutting-edge nano-analytical characterization and paves the way for investigations focused on the identification of potential markers for disease risks in a clinical setting based on atypical placental mineral fingerprints.

Pathological Mineralization: The Potential of Mineralomics.

Pathological mineralization has been reported countless times in the literature and is a well-known phenomenon in the medical field for its connections to a wide range of diseases, including cancer, cardiovascular, and neurodegenerative diseases. The minerals involved in calcification, however, have not been directly studied as extensively as the organic components of each of the pathologies. These have been studied in isolation and, for most of them, physicochemical properties are hitherto not fully known. In a parallel development, materials science methods such as electron microscopy, spectroscopy, thermal analysis, and others have been used in biology mainly for the study of hard tissues and biomaterials and have only recently been incorporated in the study of other biological systems. This review connects a range of soft tissue diseases, including breast cancer, age-related macular degeneration, aortic valve stenosis, kidney stone diseases, and Fahr's syndrome, all of which have been associated with mineralization processes. Furthermore, it describes how physicochemical material characterization methods have been used to provide new information on such pathologies. Here, we focus on diseases that are associated with calcium-composed minerals to discuss how understanding the properties of these minerals can provide new insights on their origins, considering that different conditions and biological features are required for each type of mineral to be formed. We show that mineralomics, or the study of the properties and roles of minerals, can provide information which will help to improve prevention methods against pathological mineral build-up, which in the cases of most of the diseases mentioned in this review, will ultimately lead to new prevention or treatment methods for the diseases. Importantly, this review aims to highlight that chemical composition alone cannot fully support conclusions drawn on the nature of these minerals.

Calcified nodules in retinal drusen are associated with disease progression in age-related macular degeneration.

Drusen are lipid-, mineral-, and protein-containing extracellular deposits that accumulate between the basal lamina of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM) of the human eye. They are a defining feature of age-related macular degeneration (AMD), a common sight-threatening disease of older adults. The appearance of heterogeneous internal reflectivity within drusen (HIRD) on optical coherence tomography (OCT) images has been suggested to indicate an increased risk of progression to advanced AMD. Here, in a cohort of patients with AMD and drusen, we show that HIRD indicated an increased risk of developing advanced AMD within 1 year. Using multimodal imaging in an independent cohort, we demonstrate that progression to AMD was associated with increasing degeneration of the RPE overlying HIRD. Morphological analysis of clinically imaged cadaveric human eye samples revealed that HIRD was formed by multilobular nodules. Nanoanalytical methods showed that nodules were composed of hydroxyapatite and that they differed from spherules and BrM plaques, other refractile features also found in the retinas of patients with AMD. These findings suggest that hydroxyapatite nodules may be indicators of progression to advanced AMD and that using multimodal clinical imaging to determine the composition of macular calcifications may help to direct therapeutic strategies and outcome measures in AMD.

An engineered, quantifiable in vitro model for analysing the effect of proteostasis-targeting drugs on tissue physical properties.

Cellular function depends on the maintenance of protein homeostasis (proteostasis) by regulated protein degradation. Chronic dysregulation of proteostasis is associated with neurodegenerative and age-related diseases, and drugs targeting components of the protein degradation apparatus are increasingly used in cancer therapies. However, as chronic imbalances rather than loss of function mediate their pathogenesis, research models that allow for the study of the complex effects of drugs on tissue properties in proteostasis-associated diseases are almost completely lacking. Here, to determine the functional effects of impaired proteostatic fine-tuning, we applied a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which we pharmacologically perturbed protein degradation. We show that low-level inhibition of VCP/p97 and the proteasome, two major components of the degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that failed to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the utility of quantitative materials science approaches for tackling long-standing questions in biology and medicine, and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. Our findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.

Scanning electron microscopy for blood micro-crystals in aortic stenosis patients.

BACKGROUND: Micro-crystals of calcium phosphate have been detected on the aortic valve of patients with aortic stenosis using scanning electron microscopy. It is not known whether crystalisation is specific to heart valve tissue or a general blood-derived process. METHODS: To this end we modified the method to determine whether calcium phosphate micro-crystals were present in the blood of patients with aortic stenosis. The method was first validated by adding synthetic calcium phosphate hydroxyapatite micro-crystals to healthy volunteer blood samples and determining the lower limit of detection. Then the method was used to examine the blood of 63 patients with echocardiographically confirmed aortic stenosis and 69 unaffected controls undergoing echocardiography for other reasons. Serum calcium and phosphate were measured and the calcium phosphate product compared in cases and controls. RESULTS: In the validation study, synthetic hydroxyapatite micro-crystals were identified down to a lower concentration limit of 0.008mg/mL. In the experimental study no particles were identified in any patient, with or without aortic stenosis, even though serum calcium phosphate was higher in cases compared with controls 2.6mmol/L (2.58-2.77) versus 2.47mmol/L (2.36-2.57), p = 0.005 for the difference. CONCLUSION: The results of our study confirm a positive association between serum calcium phosphate and aortic stenosis, but indicate that the calcium phosphate particles found in valve tissue do not precipitate freely in the blood.

Facile meltPEGylation of flame-made luminescent Tb3+-doped yttrium oxide particles: hemocompatibility, cellular uptake and comparison to silica.

Flame aerosol technology is a versatile method for scalable synthesis of nanoparticles. Since particles are produced and collected in a dry state, dispersibility and further functionalization could pose hurdles to their biomedical use. We report on a one-pot, scalable and robust procedure for the PEGylation of flame-made yttria and silica nanoparticles. We demonstrate improved colloidal stability, attenuated activation of blood coagulation and decreased uptake into phagocytic cells, all of which pave the way for facilitated biomedical use of flame-made oxide nanoparticles.

Removal of Cells from Body Fluids by Magnetic Separation in Batch and Continuous Mode: Influence of Bead Size, Concentration, and Contact Time.

The magnetic separation of pathogenic compounds from body fluids is an appealing therapeutic concept. Recently, removal of a diverse array of pathogens has been demonstrated using extracorporeal dialysis-type devices. The contact time between the fluid and the magnetic beads in such devices is limited to a few minutes. This poses challenges, particularly if large compounds such as bacteria or cells need to be removed. Here, we report on the feasibility to remove cells from body fluids in a continuous dialysis type of setting. We assessed tumor cell removal efficiencies from physiological fluids with or without white blood cells using a range of different magnetic bead sizes (50-4000 nm), concentrations, and contact times. We show that tumor cells can be quantitatively removed from body fluids within acceptable times (1-2 min) and bead concentrations (0.2 mg per mL). We further present a mathematical model to describe the minimal bead number concentration needed to remove a certain number of cells, in the presence of competing nonspecific uptake. The present study paves the way for investigational studies to assess the therapeutic potential of cell removal by magnetic blood purification in a dialysis-like setting.