One-Step Synthesis of Versatile Antimicrobial Nano-Architected Implant Coatings for Hard and Soft Tissue Healing.

Dental implant failure remains a prevalent problem around the globe. The integration of implants at the interface of soft and hard tissues is complex and susceptible to instability and infections. Modifications to the surface of titanium implants have been developed to improve the performance, yet insufficient integration and biofilm formation remain major problems. Introducing nanostructures on the surface to augment the implant-tissue contact holds promise for facilitated implant integration; however, current coating processes are limited in their versatility or costs. We present a highly modular single-step approach to produce multicomponent porous bioactive nanostructured coatings on implants. Inorganic nanoparticle building blocks with complex compositions and architectures are synthesized in situ and deposited on the implants in a single step using scalable liquid-feed flame spray pyrolysis. We present hybrid coatings based on ceria and bioglass, which render the implant surfaces superhydrophilic, promote cell adhesion, and exhibit antimicrobial properties. By modifications to the bioglass/ceria nanohybrid composition and architecture that prevent biomineralization, the coating can instead be tailored toward soft tissue healing. The one-step synthesis of nano-architected tissue-specific coatings has great potential in dental implantology and beyond.

Inorganic nanohybrids combat antibiotic-resistant bacteria hiding within human macrophages.

Bacterial infections are one of the main health concerns humanity faces today and bacterial resistances and protection mechanisms are set to aggravate the issue in the coming years. An increasing number of bacterial strains evades antibiotic treatment by hiding inside cells. Conventional antimicrobial agents are unable to penetrate or be retained in the infected mammalian cells. Recent approaches to overcome these limitations have focused on load-carrier systems, requiring a triggered discharge leading to complex release kinetics. The unison of potent antimicrobial activity with high mammalian cell compatibility is a prerequisite for intracellular activity, which is not well-met by otherwise well-established inorganic systems, such as silver-based nanoparticles. In this work, load and carrier are combined into one functional inorganic nanoparticle system, which unites antimicrobial activity with mammalian cell compatibility. These multicomponent nanohybrids based on cerium oxide are produced in one step, yet unite complex materials. The nanoparticles form suprastructures of similar size and surface charge as bacteria, therefore facilitating the uptake into the same subcellular compartments, where they unleash their antibacterial effect. Such intrinsically antibacterial nanohybrids significantly reduce bacterial survival inside macrophages without harming the latter. Furthermore, blocking of nanoparticle endocytosis and subcellular electron microscopy elucidate the mechanism of action. Taken together, this work presents the first demonstration of antibacterial activity of ceria-based nanoparticles inside of mammalian cells and offers a route to straightforward and robust intracellular antibacterial agents that do not depend on payload delivery or biological constituents.

Bi2O3 boosts brightness, biocompatibility and stability of Mn-doped Ba3(VO4)2 as NIR-II contrast agent.

Deep-tissue fluorescence imaging remains a major challenge as there is limited availability of bright biocompatible materials with high photo- and chemical stability. Contrast agents with emission wavelengths above 1000 nm are most favorable for deep tissue imaging, offering deeper penetration and less scattering than those operating at shorter wavelengths. Organic fluorophores suffer from low stability while inorganic nanomaterials (e.g. quantum dots) are based typically on heavy metals raising toxicity concerns. Here, we report scalable flame aerosol synthesis of water-dispersible Ba3(VO4)2 nanoparticles doped with Mn5+ which exhibit a narrow emission band at 1180 nm upon near-infrared excitation. Their co-synthesis with Bi2O3 results in even higher absorption and ten-fold increased emission intensity. The addition of Bi2O3 also improved both chemical stability and cytocompatibility by an order of magnitude enabling imaging deep within tissue. Taken together, these bright particles offer excellent photo-, chemical and colloidal stability in various media with cytocompatibility to HeLa cells superior to existing commercial contrast agents.

Correlative Cathodoluminescence Electron Microscopy: Immunolabeling Using Rare-Earth Element Doped Nanoparticles.

The understanding of living systems and their building blocks relies on the assessment of structure-function relationships at the nanoscale. Although electron microscopy (EM) gives access to ultrastructural imaging with nanometric resolution, the unambiguous localization of specific molecules is challenging. An EM approach capable of localizing biomolecules with respect to the cellular ultrastructure will offer a direct route to the molecular blueprints of biological systems. In an approach departing from conventional correlative imaging, an electron beam may be used as excitation source to generate optical emission with nanometric resolution, that is, cathodoluminescence (CL). Once suitable luminescent labels become available, CL may be harnessed to enable identification of biomolecule labels based on spectral signatures rather than electron density and size. This work presents CL-enabled immunolabeling based on rare-earth element doped nanoparticle-labels allowing specific molecules to be visualized at nanoscale resolution in the context of the cellular ultrastructure. Folic acid decorated nanoparticles exhibiting single particle CL emission are employed to specifically label receptors and identify characteristic receptor clustering on the surface of cancer cells. This demonstration of CL immunotargeting gives access to protein localization in the context of the cellular ultrastructure and paves the way for immunolabeling of multiple proteins in EM.

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.

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context.

The understanding of living systems and their building blocks relies heavily on the assessment of structure-function relationships at the nanoscale. Ever since the development of the first optical microscope, the reliance of scientists across disciplines on microscopy has increased. The development of the first electron microscope and with it the access to information at the nanoscale has prompted numerous disruptive discoveries. While fluorescence imaging allows identification of specific entities based on the labelling with fluorophores, the unlabelled constituents of the samples remain invisible. In electron microscopy on the other hand, structures can be comprehensively visualized based on their distinct electron density and geometry. Although electron microscopy is a powerful tool, it does not implicitly provide information on the location and activity of specific organic molecules. While correlative light and electron microscopy techniques have attempted to unify the two modalities, the resolution mismatch between the two data sets poses major challenges. Recent developments in optical super resolution microscopy enable high resolution correlative light and electron microscopy, however, with considerable constraints due to sample preparation requirements. Labelling of specific structures directly for electron microscopy using small gold nanoparticles (i.e. immunogold) has been used extensively. However, identification of specific entities solely based on electron contrast, and the differentiation from endogenous dense granules, remains challenging. Recently, the use of correlative cathodoluminescence electron microscopy (CCLEM) imaging based on luminescent inorganic nanocrystals has been proposed. While nanometric resolution can be reached for both the electron and the optical signal, high energy electron beams are potentially damaging to the sample. In this review, we discuss the opportunities of (volumetric) multi-color single protein labelling based on correlative cathodoluminescence electron microscopy, and its prospective impact on biomedical research in general. We elaborate on the potential challenges of correlative cathodoluminescence electron microscopy-based bioimaging and benchmark CCLEM against alternative high-resolution correlative imaging techniques.

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.

Uptake, distribution and radio-enhancement effects of gold nanoparticles in tumor microtissues.

Radiotherapy is an integral and highly effective part of cancer therapy, applicable in over 50% of patients affected by cancer. Due to the low specificity of the X-ray irradiation, the maximal radiation dose is greatly limited in order to avoid damage to surrounding healthy tissue. The limitations in applicable dose oftentimes result in the survival of a subpopulation of radio-resistant cells that then cause cancer reoccurence. Approaches based on tumor-targeted high atomic number inorganic nanoparticles have been proposed to locally increase the photoelectric absorption cross-section of tumors relative to healthy tissue. However, the complex interplay between the nanoparticle radio-enhancers and the tumor tissue has led to poor translation of in vitro findings to (pre)clinics. Here, we report the development of a tumor microtissue model along with analytical imaging for the quantitative assessment of nanoparticle-based radio-enhancement as a function of nanoparticle size, uptake and intratissural distribution. The advanced in vitro model exhibits key features of cancerous tissues, including diminished susceptibility to drugs and attenuated response to nanoparticle treatment compared to corresponding conventional 2D cell cultures. Whereas radio-enhancement effects between 2D and 3D cell cultures were comparable for 5 nm gold particles, the limited penetration of 50 nm gold nanoparticles into 3D microtissues led to a significantly reduced radio-enhancement effect in 3D compared to 2D. Taken together, tumor microtissues, which in stark contrast to 2D cell culture exhibit tissue-like features, may provide a valuable high-throughput intermediate pre-selection step in the preclinical translation of nanoparticle-based radio-enhancement therapy designs.

Tailoring the Colloidal Stability, Magnetic Separability, and Cytocompatibility of High-Capacity Magnetic Anion Exchangers.

Extracorporeal blood purification has been applied to artificially support kidney or liver function. However, convection and diffusion based blood purification systems have limited removal rates for high molecular weight and hydrophobic molecules. This limitation is due to the finite volume of infusion and limited membrane permeability, respectively. Adsorption provides an attractive alternative for the removal of higher molecular weight compounds. The use of adsorption resins containing ion exchanging groups to capture specific molecules has become well-established. Instead of stationary adsorption resins, however, ion exchanging polymers may be immobilized on magnetic particles and serve as freely diffusing, mobile, high capacity solid phase of ion exchange chromatography. While small beads with high surface area are attractive in terms of mass transfer and binding, unifying high capturing capacity with rapid and quantitative bead recovery remains an issue. Therefore, most of the current magnetic ion exchangers are based on micron-sized beads or require long times to separate. In addition to unfavorable magnetic recovery rates, the usually poor cytocompatibility limits their applicability in biomedicine. Here, we report on the synthesis and performance of polycationic polymer coated magnetic nanoflowers (MNF) for highly efficacious anion capturing. We demonstrate accurate control over the polymer content and composition on the beads and show its direct influence on colloidal stability, capturing capacity and magnetic separability. We present the removal of clinically relevant targets by capturing bilirubin with capacities 2-fold higher than previous work as well as quantitative heparin removal. Additionally, we illustrate how copolymerization of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) with poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) leads to improved cytocompatibility of the polymer-coated MNF capturing agents while retaining high capturing capacities. Taken together, we present a nanoparticle/polymer material, which upon future in vivo validation, unifies high binding capacities and magnetic separability for rapid toxin capturing and hence fulfills key requirements of clinical utility.

Ultrabright and Stable Luminescent Labels for Correlative Cathodoluminescence Electron Microscopy Bioimaging.

The mechanistic understanding of structure-function relationships in biological systems heavily relies on imaging. While fluorescence microscopy allows the study of specific proteins following their labeling with fluorophores, electron microscopy enables holistic ultrastructural analysis based on differences in electron density. To identify specific proteins in electron microscopy, immunogold labeling has become the method of choice. However, the distinction of immunogold-based protein labels from naturally occurring electron dense granules and the identification of several different proteins in the same sample remain challenging. Correlative cathodoluminescence electron microscopy (CCLEM) bioimaging has recently been suggested to provide an attractive alternative based on labels emitting characteristic light. While luminescence excitation by an electron beam enables subdiffraction imaging, structural damage to the sample by high-energy electrons has been identified as a potential obstacle. Here, we investigate the feasibility of various commonly used luminescent labels for CCLEM bioimaging. We demonstrate that organic fluorophores and semiconductor quantum dots suffer from a considerable loss of emission intensity, even when using moderate beam voltages (2 kV) and currents (0.4 nA). Rare-earth element-doped nanocrystals, in particular Y2O3:Tb3+ and YVO4:Bi3+,Eu3+ nanoparticles with green and orange-red emission, respectively, feature remarkably high brightness and stability in the CCLEM bioimaging setting. We further illustrate how these nanocrystals can be readily differentiated from morphologically similar naturally occurring dense granules based on optical emission, making them attractive nanoparticle core materials for molecular labeling and (multi)color CCLEM.

Lanthanide-Doped Hafnia Nanoparticles for Multimodal Theranostics: Tailoring the Physicochemical Properties and Interactions with Biological Entities.

High-Z metal oxide nanoparticles hold promise as imaging probes and radio-enhancers. Hafnium dioxide nanoparticles have recently entered clinical evaluation. Despite promising early clinical findings, the potential of HfO2 as a matrix for multimodal theranostics is yet to be developed. Here, we investigate the physicochemical properties and the potential of HfO2-based nanoparticles for multimodal theranostic imaging. Undoped and lanthanide (Eu3+, Tb3+, and Gd3+)-doped HfO2 nanoparticles were synthesized and functionalized with various moieties including poly(vinylpyrrolidone) (PVP), (3-aminopropyl)triethoxysilane (APTES), and folic acid (FA). We show that different synthesis routes, including direct precipitation, microwave-assisted synthesis, and sol-gel chemistry, allow preparation of hafnium dioxide particles with distinct physicochemical properties. Sol-gel based synthesis allows preparation of uniform nanoparticles with dopant incorporation efficiencies superior to the other two methods. Both luminescence and contrast properties can be tweaked by lanthanide doping. We show that MRI contrast can be unified with radio-enhancement by incorporating lanthanide dopants in the HfO2 matrix. Importantly, ion leaching from the HfO2 host matrix in lysosomal-like conditions was minimal. For Gd:HfO2 nanoparticles, leaching was reduced >10× compared to Gd2O3, and no relevant cytotoxic effects have been observed in monocyte-derived macrophages for nanoparticle concentrations up to 250 µg/mL. Chemical surface modification allows further tailoring of the cyto- and hemocompatibility and enables functionalization with molecular targeting entities, which lead to enhanced cellular uptake. Taken together, the present study illustrates the manifold properties of HfO2-based nanomaterials with prospective clinical utility beyond radio-enhancement.

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.

An advanced human in vitro co-culture model for translocation studies across the placental barrier.

Although various drugs, environmental pollutants and nanoparticles (NP) can cross the human placental barrier and may harm the developing fetus, knowledge on predictive placental transfer rates and the underlying transport pathways is mostly lacking. Current available in vitro placental transfer models are often inappropriate for translocation studies of macromolecules or NPs and do not consider barrier function of placental endothelial cells (EC). Therefore, we developed a human placental in vitro co-culture transfer model with tight layers of trophoblasts (BeWo b30) and placental microvascular ECs (HPEC-A2) on a low-absorbing, 3 µm porous membrane. Translocation studies with four model substances and two polystyrene (PS) NPs across the individual and co-culture layers revealed that for most of these compounds, the trophoblast and the EC layer both demonstrate similar, but not additive, retention capacity. Only the paracellular marker Na-F was substantially more retained by the BeWo layer. Furthermore, simple shaking, which is often applied to mimic placental perfusion, did not alter translocation kinetics compared to static exposure. In conclusion, we developed a novel placental co-culture model, which provides predictive values for translocation of a broad variety of molecules and NPs and enables valuable mechanistic investigations on cell type-specific placental barrier function.

Reduced Magnetic Coupling in Ultrasmall Iron Oxide T1 MRI Contrast Agents.

Contrast agents for magnetic resonance imaging (MRI) are essential for evidential visualization of soft tissues pathologies. Contrast-enhanced MRI can be carried out with T1- and T2-weighted sequences that require as contrast agents paramagnetic and superparamagnetic materials, respectively. The T1-weighted imaging is frequently preferred over T2-, as it induces a bright contrast for sharper image analysis and allows more rapid image acquisition. Commonly used and FDA-approved T1 contrast agents, however, were shown to be associated with nephrogenic systematic fibrosis due to Gd3+ release from the injected complexes. Here, ultrasmall iron oxide nanocrystals are produced by scalable flame aerosol technology and investigated as T1 MRI contrast agents by focusing on structure-function relationships and cytocompatibility. The optimized nanocrystals are shown to be a promising cytocompatible alternative to commercial Gd-complexes as they attain comparable relaxivities with no apparent cytotoxicity at clinically relevant concentrations tested in vitro against four different cell types (PC3, HepG2, THP-1, and red blood cells). By using SiO2 as a spacing material, the contrast enhancement could be finely tuned by decreasing the effective magnetic size of iron oxide resulting in significant T1 contrast enhancement due to reduced magnetic coupling.

The silanol content and in vitro cytolytic activity of flame-made silica.

HYPOTHESIS: The surface chemistry of synthetic amorphous silicas is essential for their applicational performance and for understanding their interactions with biological matter. Synthesis of silica by flame spray pyrolysis (FSP) allows to control the content and type of hydroxyl groups which also affects the cytolytic activity. EXPERIMENTS: By controlling the FSP process variables, silica nanoparticles with the same specific surface area but different surface chemistry and content of internal silanols are prepared by combustion of hexamethyldisiloxane sprays, as characterized by Raman and infrared spectroscopy, thermogravimetric analysis, and titration with lithium alanate. Cytolytic activity is assessed in terms of membrane damage in human blood monocytes in vitro. FINDINGS: Unlike commercial fumed silica, FSP-made silicas contain a significant amount of internal silanol groups and a high surface hydroxyl density, up to ∼8OH/nm2, similar to silicas made by wet-chemistry. Increasing the residence time of particles at high temperature during their synthesis reduces the internal and surface hydroxyl content and increases the relative amount of isolated silanols. This suggests incomplete oxidation of the silica matrix especially in short and "cold" flames and indicates that the silica particle formation pathway involves Si(OH)4. The surface chemistry differences translate into lower cytolytic activity for "cold-" than "hot-flame" silicas.

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.

An energy transfer kinetic probe for OH-quenchers in the Nd(3+):YPO4 nanocrystals suitable for imaging in the biological tissue transparency window.

Tetragonal xenotime-type yttrium orthophosphate (YPO4) Nd(3+) doped nanoparticles suitable for biomedical applications were prepared by microwave-hydrothermal treatment. We applied the energy transfer probing based on the analysis of kinetics of impurity quenching to determine the presence and spatial position of -OH fluorescence quenching acceptors in the impurity-containing nanoparticles. We show that the impurity quenching kinetics of the 0.1 at% Nd(3+) doped YPO4 nanoparticles is a two stage (ordered and disordered) static kinetics, determined by a direct energy transfer to the -OH acceptors. Analyzing the ordered stage, we assume that the origin of the -OH groups is the protonation of the phosphate groups, while analyzing the disordered stage, we assume the presence of water molecules in the mesopores. We determine the dimension of the space of the -OH acceptors as d = 3 and quantify their absolute concentration using the disordered Förster stage of kinetics. We use the late stage of kinetics of fluorescence hopping (CDD ≫ CDA) quenching (the fluctuation asymptotics) at 1 at% Nd(3+) concentration as an energy transfer probe to quantify the relative concentration of -OH molecular groups compared to an optically active rare-earth dopant in the volume of NPs, when energy migration over Nd(3+) donors to the -OH acceptors accelerates fluorescence quenching. In doing so we use just one parameter α = γ(A)/γ(D) = n(A)√[C(DA)]/n(D)√[C(DD)], defined by the relation of concentration of the -OH acceptors to the concentration of an optically active dopant. The higher is the α, the higher is the relative concentration of -OH acceptors in the volume of nanoparticles. We find α = 2.95 for the 1 at% Nd(3+):YPO4 NPs that, according to the equation for α, and the results obtained for the values of the microparameters CDD(Nd-Nd) = 24.6 nm(6) ms(-1) and CDA(Nd-OH) = 0.6 nm(6) ms(-1), suggests twenty times higher concentration for acceptors other than donors. As the main result we have established that the majority of -OH acceptors is located not on the surface of the Nd(3+):YPO4 nanoparticles, as many researchers assumed, but in their volume, and can be either associated with crystal structure defects or located in the mesopores.