Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.

Multinucleation resets human macrophages for specialized functions at the expense of their identity.

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7-H3 (CD276) and can form granuloma-like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell-cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality.

Modulation of engineered nanomaterial interactions with organ barriers for enhanced drug transport.

The biomedical use of nanoparticles (NPs) has been the focus of intense research for over a decade. As most NPs are explored as carriers to alter the biodistribution, pharmacokinetics and bioavailability of associated drugs, the delivery of these NPs to the tissues of interest remains an important topic. To date, the majority of NP delivery studies have used tumor models as their tool of interest, and the limitations concerning tumor targeting of systemically administered NPs have been well studied. In recent years, the focus has also shifted to other organs, each presenting their own unique delivery challenges to overcome. In this review, we discuss the recent advances in leveraging NPs to overcome four major biological barriers including the lung mucus, the gastrointestinal mucus, the placental barrier, and the blood-brain barrier. We define the specific properties of these biological barriers, discuss the challenges related to NP transport across them, and provide an overview of recent advances in the field. We discuss the strengths and shortcomings of different strategies to facilitate NP transport across the barriers and highlight some key findings that can stimulate further advances in this field.

Human Autosomal Recessive DNA Polymerase Delta 3 Deficiency Presenting as Omenn Syndrome.

The DNA polymerase δ complex (PolD), comprising catalytic subunit POLD1 and accessory subunits POLD2, POLD3, and POLD4, is essential for DNA synthesis and is central to genome integrity. We identified, by whole exome sequencing, a homozygous missense mutation (c.1118A > C; p.K373T) in POLD3 in a patient with Omenn syndrome. The patient exhibited severely decreased numbers of naïve T cells associated with a restricted T-cell receptor repertoire and a defect in the early stages of TCR recombination. The patient received hematopoietic stem cell transplantation at age 6 months. He manifested progressive neurological regression and ultimately died at age 4 years. We performed molecular and functional analysis of the mutant POLD3 and assessed cell cycle progression as well as replication-associated DNA damage. Patient fibroblasts showed a marked defect in S-phase entry and an enhanced number of double-stranded DNA break-associated foci despite normal expression levels of PolD components. The cell cycle defect was rescued by transduction with WT POLD3. This study validates autosomal recessive POLD3 deficiency as a novel cause of profound T-cell deficiency and Omenn syndrome.

The Use of Alternative Strategies for Enhanced Nanoparticle Delivery to Solid Tumors.

Nanomaterial (NM) delivery to solid tumors has been the focus of intense research for over a decade. Classically, scientists have tried to improve NM delivery by employing passive or active targeting strategies, making use of the so-called enhanced permeability and retention (EPR) effect. This phenomenon is made possible due to the leaky tumor vasculature through which NMs can leave the bloodstream, traverse through the gaps in the endothelial lining of the vessels, and enter the tumor. Recent studies have shown that despite many efforts to employ the EPR effect, this process remains very poor. Furthermore, the role of the EPR effect has been called into question, where it has been suggested that NMs enter the tumor via active mechanisms and not through the endothelial gaps. In this review, we provide a short overview of the EPR and mechanisms to enhance it, after which we focus on alternative delivery strategies that do not solely rely on EPR in itself but can offer interesting pharmacological, physical, and biological solutions for enhanced delivery. We discuss the strengths and shortcomings of these different strategies and suggest combinatorial approaches as the ideal path forward.

Cu-doped TiO2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies.

Nanoparticle-mediated cancer immunotherapy holds great promise, but more efforts are needed to obtain nanoformulations that result in a full scale activation of innate and adaptive immune components that specifically target the tumors. We generated a series of copper-doped TiO2 nanoparticles in order to tune the kinetics and full extent of Cu2+ ion release from the remnant TiO2 nanocrystals. Fine-tuning nanoparticle properties resulted in a formulation of 33% Cu-doped TiO2 which enabled short-lived hyperactivation of dendritic cells and hereby promoted immunotherapy. The nanoparticles result in highly efficient activation of dendritic cells ex vivo, which upon transplantation in tumor bearing mice, exceeded the therapeutic outcomes obtained with classically stimulated dendritic cells. Efficacious but simple nanomaterials that can promote dendritic cancer cell vaccination strategies open up new avenues for improved immunotherapy and human health.

Gold nanostructures: synthesis, properties, and neurological applications.

Recent advances in technology are expected to increase our current understanding of neuroscience. Nanotechnology and nanomaterials can alter and control neural functionality in both in vitro and in vivo experimental setups. The intersection between neuroscience and nanoscience may generate long-term neural interfaces adapted at the molecular level. Owing to their intrinsic physicochemical characteristics, gold nanostructures (GNSs) have received much attention in neuroscience, especially for combined diagnostic and therapeutic (theragnostic) purposes. GNSs have been successfully employed to stimulate and monitor neurophysiological signals. Hence, GNSs could provide a promising solution for the regeneration and recovery of neural tissue, novel neuroprotective strategies, and integrated implantable materials. This review covers the broad range of neurological applications of GNS-based materials to improve clinical diagnosis and therapy. Sub-topics include neurotoxicity, targeted delivery of therapeutics to the central nervous system (CNS), neurochemical sensing, neuromodulation, neuroimaging, neurotherapy, tissue engineering, and neural regeneration. It focuses on core concepts of GNSs in neurology, to circumvent the limitations and significant obstacles of innovative approaches in neurobiology and neurochemistry, including theragnostics. We will discuss recent advances in the use of GNSs to overcome current bottlenecks and tackle technical and conceptual challenges.

Multicompartmental Microcapsules for Enzymatic Cascade Reactions Prepared through Gas Shearing and Surface Gelation.

Inspired by the structure of eukaryotic cells, multicompartmental microcapsules have gained increasing attention. However, challenges remain in the fabrication of "all-aqueous" (i.e., oil-free) microcapsules composed of accurately adjustable hierarchical compartments. This study reports on multicompartmental microcapsules with an innovative architecture. While multicompartmental cores of the microcapsules were fabricated through gas shearing, a shell was applied on the cores through surface gelation of alginate. Different from traditional multicompartmental microcapsules, thus obtained microcapsules have well-segregated compartments while the universal nature of the surface-gelation method allows us to finely tune the shell thicknesses of the microcapsules. The microcapsules are highly stable and cytocompatible and allow repeated enzymatic cascade reactions, which might make them of interest for complex biocatalysis or for mimicking physiological processes.

Gold nanoparticle delivery to solid tumors: a multiparametric study on particle size and the tumor microenvironment.

Nanoparticle (NP) delivery to solid tumors remains an actively studied field, where several recent studies have shed new insights into the underlying mechanisms and the still overall poor efficacy. In the present study, Au NPs of different sizes were used as model systems to address this topic, where delivery of the systemically administered NPs to the tumor as a whole or to tumor cells specifically was examined in view of a broad range of tumor-associated parameters. Using non-invasive imaging combined with histology, immunohistochemistry, single-cell spatial RNA expression and image-based single cell cytometry revealed a size-dependent complex interaction of multiple parameters that promoted tumor and tumor-cell specific NP delivery. Interestingly, the data show that most NPs are sequestered by tumor-associated macrophages and cancer-associated fibroblasts, while only few NPs reach the actual tumor cells. While perfusion is important, leaky blood vessels were found not to promote NP delivery, but rather that delivery efficacy correlated with the maturity level of tumor-associated blood vessels. In line with recent studies, we found that the presence of specialized endothelial cells, expressing high levels of CD276 and Plvap promoted both tumor delivery and tumor cell-specific delivery of NPs. This study identifies several parameters that can be used to determine the suitability of NP delivery to the tumor region or to tumor cells specifically, and enables personalized approaches for maximal delivery of nanoformulations to the targeted tumor.

Modularity of RBC hitchhiking with polymeric nanoparticles: testing the limits of non-covalent adsorption.

Red blood cell (RBC) hitchhiking has great potential in enhancing drug therapy, by improving targeting and reducing rapid clearance of nanoparticles (NPs). However, to improve the potential for clinical translation of RBC hitchhiking, a more thorough understanding of the RBC-NP interface is needed. Here, we evaluate the effects of NP surface parameters on the success and biocompatibility of NP adsorption to extracted RBCs from various species. Major differences in RBC characteristics between rabbit, mouse and human were proven to significantly impact NP adsorption outcomes. Additionally, the effects of NP design parameters, including NP hydrophobicity, zeta potential, surfactant concentration and drug encapsulation, on RBC hitchhiking are investigated. Our studies demonstrate the importance of electrostatic interactions in balancing NP adsorption success and biocompatibility. We further investigated the effect of varying the anti-coagulant used for blood storage. The results presented here offer new insights into the parameters that impact NP adsorption on RBCs that will assist researchers in experimental design choices for using RBC hitchhiking as drug delivery strategy.

Bio-Nanohybrid Gelatin/Quantum Dots for Cellular Imaging and Biosensing Applications.

The bio-nanohybrid gelatin protein/cadmium sulfide (Gel/CdS) quantum dots (QDs) have been designed via a facile one-pot strategy. The amino acids group of gelatin chelate Cd2+ and grow CdS QDs without any agglomeration. The 1H NMR spectra indicate that during the above process there are no alterations of the gelatin protein structure conformation and chemical functionalities. The prepared Gel/CdS QDs were characterized and their potential as a system for cellular imaging and the electrochemical sensor for hydrogen peroxide (H2O2) detection applications were investigated. The obtained results demonstrate that the developed Gel/CdS QDs system could offer a simple and convenient operating strategy both for the class of contrast agents for cell labeling and electrochemical sensors purposes.

A Multiparametric Evaluation of Quantum Dot Size and Surface-Grafted Peptide Density on Cellular Uptake and Cytotoxicity.

Despite the progress in nanotechnology for biomedical applications, great efforts are still being employed in optimizing nanoparticle (NP) design parameters to improve functionality and minimize bionanotoxicity. In this study, we developed CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) that are compact ligand-coated and surface-functionalized with an HIV-1-derived TAT cell-penetrating peptide (CPP) analog to improve both biocompatibility and cellular uptake. Multiparametric studies were performed in different mammalian and murine cell lines to compare the effects of varying QD size and number of surface CPPs on cellular uptake, viability, generation of reactive oxygen species, mitochondrial health, cell area, and autophagy. Our results showed that the number of cell-associated NPs and their respective toxicity are higher for the larger QDs. Meanwhile, increasing the number of surface CPPs also enhanced cellular uptake and induced cytotoxicity through the generation of mitoROS and autophagy. Thus, here we report the optimal size and surface CPP combinations for improved QD cellular uptake.

Model-Based Nanoengineered Pharmacokinetics of Iron-Doped Copper Oxide for Nanomedical Applications.

The progress in nanomedicine (NM) using nanoparticles (NPs) is mainly based on drug carriers for the delivery of classical chemotherapeutics. As low NM delivery rates limit therapeutic efficacy, an entirely different approach was investigated. A homologous series of engineered CuO NPs was designed for dual purposes (carrier and drug) with a direct chemical composition-biological functionality relationship. Model-based dissolution kinetics of CuO NPs in the cellular interior at post-exposure conditions were controlled through Fe-doping for intra/extra cellular Cu2+ and biological outcome. Through controlled ion release and reactions taking place in the cellular interior, tumors could be treated selectively, in vitro and in vivo. Locally administered NPs enabled tumor cells apoptosis and stimulated systemic anti-cancer immune responses. We clearly show therapeutic effects without tumor cells relapse post-treatment with 6 % Fe-doped CuO NPs combined with myeloid-derived suppressor cell silencing.

A Magnetic Chameleon: Biocompatible Lanthanide Fluoride Nanoparticles with Magnetic Field Dependent Tunable Contrast Properties as a Versatile Contrast Agent for Low to Ultrahigh Field MRI and Optical Imaging in Biological Window.

A novel type of multimodal, magnetic resonance imaging/optical imaging (MRI/OI) contrast agent was developed, based on core-shell lanthanide fluoride nanoparticles composed of a ß-NaHoF4 core plus a ß-NaGdF4:Yb3+ , Tm3+ shell with an average size of ∼24 nm. The biocompatibility of the particles was ensured by a surface modification with poly acrylic acid (PAA) and further functionalization with an affinity ligand, folic acid (FA). When excited using 980 nm near infrared (NIR) radiation, the contrast agent (CA) shows intense emission at 802 nm with lifetime of 791±3 µs, due to the transition 3 H4 →3 H6 of Tm3+ . Proton nuclear magnetic relaxation dispersion (1 H-NMRD) studies and magnetic resonance (MR) phantom imaging showed that the newly synthesized nanoparticles, decorated with poly(acrylic acid) and folic acid on the surface (NP-PAA-FA), can act mainly as a T1 -weighted contrast agent below 1.5 T, a T1 /T2 dual-weighted contrast agent at 3 T, and as highly efficient T2 -weighted contrast agent at ultrahigh fields. In addition, NP-PAA-FA showed very low cytotoxicity and no detectable cellular damage up to a dose of 500 µg mL-1 .

The impact of nanoparticle-driven lysosomal alkalinization on cellular functionality.

BACKGROUND: The biomedical use of nanosized materials is rapidly gaining interest, which drives the quest to elucidate the behavior of nanoparticles (NPs) in a biological environment. Apart from causing direct cell death, NPs can affect cellular wellbeing through a wide range of more subtle processes that are often overlooked. Here, we aimed to study the effect of two biomedically interesting NP types on cellular wellbeing. RESULTS: In the present work, gold and SiO2 NPs of similar size and surface charge are used and their interactions with cultured cells is studied. Initial screening shows that at subcytotoxic conditions gold NPs induces cytoskeletal aberrations while SiO2 NPs do not. However, these transformations are only transient. In-depth investigation reveals that Au NPs reduce lysosomal activity by alkalinization of the lysosomal lumen. This leads to an accumulation of autophagosomes, resulting in a reduced cellular degradative capacity and less efficient clearance of damaged mitochondria. The autophagosome accumulation induces Rac and Cdc42 activity, and at a later stage activates RhoA. These transient cellular changes also affect cell functionality, where Au NP-labelled cells display significantly impeded cell migration and invasion. CONCLUSIONS: These data highlight the importance of in-depth understanding of bio-nano interactions to elucidate how one biological parameter (impact on cellular degradation) can induce a cascade of different effects that may have significant implications on the further use of labeled cells.

Standard Cellular Testing Conditions Generate an Exaggerated Nanoparticle Cytotoxicity Profile.

Cellular internalization of nanoparticles (NPs) is key to many biomedical applications and serves as a model to investigate the potential toxicity of NPs on entire organisms. Large discrepancies between in vitro and in vivo nanotoxicity data however exist, suggesting that cellular systems may not be optimal for predictive in vivo toxicology. Here, we use validated multiparametric high-content imaging protocols to evaluate the impact of common cell culture conditions on NP cytotoxicity studies. The data show that high NP to cell ratios, typical for cellular studies, stress the cells by high endocytosis levels that overstimulate mitochondria, resulting in oxidative stress-mediated mitochondrial damage, which induces autophagy. Using proliferation-restricted models, we show that lowering endocytosis levels overcomes most toxicity while resulting in higher final cellular NP numbers. The data suggest that many common NP cytotoxicity mechanisms may partially be an artifact caused by overstimulated endocytosis.

The role of intracellular trafficking of CdSe/ZnS QDs on their consequent toxicity profile.

BACKGROUND: Nanoparticle interactions with cellular membranes and the kinetics of their transport and localization are important determinants of their functionality and their biological consequences. Understanding these phenomena is fundamental for the translation of such NPs from in vitro to in vivo systems for bioimaging and medical applications. Two CdSe/ZnS quantum dots (QD) with differing surface functionality (NH2 or COOH moieties) were used here for investigating the intracellular uptake and transport kinetics of these QDs. RESULTS: In water, the COOH- and NH2-QDs were negatively and positively charged, respectively, while in serum-containing medium the NH2-QDs were agglomerated, whereas the COOH-QDs remained dispersed. Though intracellular levels of NH2- and COOH-QDs were very similar after 24 h exposure, COOH-QDs appeared to be continuously internalised and transported by endosomes and lysosomes, while NH2-QDs mainly remained in the lysosomes. The results of (intra)cellular QD trafficking were correlated to their toxicity profiles investigating levels of reactive oxygen species (ROS), mitochondrial ROS, autophagy, changes to cellular morphology and alterations in genes involved in cellular stress, toxicity and cytoskeletal integrity. The continuous flux of COOH-QDs perhaps explains their higher toxicity compared to the NH2-QDs, mainly resulting in mitochondrial ROS and cytoskeletal remodelling which are phenomena that occur early during cellular exposure. CONCLUSIONS: Together, these data reveal that although cellular QD levels were similar after 24 h, differences in the nature and extent of their cellular trafficking resulted in differences in consequent gene alterations and toxicological effects.

Genotoxic capacity of Cd/Se semiconductor quantum dots with differing surface chemistries.

Quantum dots (QD) have unique electronic and optical properties promoting biotechnological advances. However, our understanding of the toxicological structure-activity relationships remains limited. This study aimed to determine the biological impact of varying nanomaterial surface chemistry by assessing the interaction of QD with either a negative (carboxyl), neutral (hexadecylamine; HDA) or positive (amine) polymer coating with human lymphoblastoid TK6 cells. Following QD physico-chemical characterisation, cellular uptake was quantified by optical and electron microscopy. Cytotoxicity was evaluated and genotoxicity was characterised using the micronucleus assay (gross chromosomal damage) and the HPRT forward mutation assay (point mutagenicity). Cellular damage mechanisms were also explored, focusing on oxidative stress and mitochondrial damage. Cell uptake, cytotoxicity and genotoxicity were found to be dependent on QD surface chemistry. Carboxyl-QD demonstrated the smallest agglomerate size and greatest cellular uptake, which correlated with a dose dependent increase in cytotoxicity and genotoxicity. Amine-QD induced minimal cellular damage, while HDA-QD promoted substantial induction of cell death and genotoxicity. However, HDA-QD were not internalised by the cells and the damage they caused was most likely due to free cadmium release caused by QD dissolution. Oxidative stress and induced mitochondrial reactive oxygen species were only partially associated with cytotoxicity and genotoxicity induced by the QD, hence were not the only mechanisms of importance. Colloidal stability, nanoparticle (NP) surface chemistry, cellular uptake levels and the intrinsic characteristics of the NPs are therefore critical parameters impacting genotoxicity induced by QD.