Nanodiamonds in biomedical research: Therapeutic applications and beyond.

Nanodiamonds (NDs) comprise a family of carbon-based nanomaterials (i.e. diameter <100 nm) with the same sp3 lattice structure that gives natural diamonds their exceptional hardness and electrical insulating properties. Among all carbon nanomaterials-e.g. carbon nanotubes, nanodots, and fullerenes-NDs are of particular interest for biomedical applications because they offer high biocompatibility, stability in vivo, and a dynamic surface chemistry that can be manipulated to perform a seemingly limitless variety of ultra-specific tasks. NDs are already deepening our understanding of basic biological processes, while numerous laboratories continue studying these nanomaterials with an aim of making seismic improvements in the prevention, diagnosis, and treatment of human diseases. This review surveys approximately 2,000 the most recent articles published in the last 5 years and includes references to more than 150 of the most relevant publications on the biomedical applications of NDs. The findings are categorized by contemporary lines of investigation based on potential applications, namely: genetics and gene editing, drug delivery systems, neural interfacing, biomedical sensors, synthetic biology, and organ and tissue regeneration. This review also includes a brief background of NDs and the methods currently developed for their synthesis and preparation. Finally, recommendations for future investigations are offered.

Bacterial therapies at the interface of synthetic biology and nanomedicine.

Bacteria are emerging as living drugs to treat a broad range of disease indications. However, the inherent advantages of these replicating and immunostimulatory therapies also carry the potential for toxicity. Advances in synthetic biology and the integration of nanomedicine can address this challenge through the engineering of controllable systems that regulate spatial and temporal activation for improved safety and efficacy. Here, we review recent progress in nanobiotechnology-driven engineering of bacteria-based therapies, highlighting limitations and opportunities that will facilitate clinical translation.

Multiscale engineering of brain organoids for disease modeling.

Brain organoids hold great potential for modeling human brain development and pathogenesis. They recapitulate certain aspects of the transcriptional trajectory, cellular diversity, tissue architecture and functions of the developing brain. In this review, we explore the engineering strategies to control the molecular-, cellular- and tissue-level inputs to achieve high-fidelity brain organoids. We review the application of brain organoids in neural disorder modeling and emerging bioengineering methods to improve data collection and feature extraction at multiscale. The integration of multiscale engineering strategies and analytical methods has significant potential to advance insight into neurological disorders and accelerate drug development.

Tackling Severe Neutrophilic Inflammation in Airway Disorders with Functionalized Nanosheets.

Severe airway inflammatory disorders impose a significant societal burden, and the available treatments are unsatisfactory. High levels of neutrophil extracellular trap (NET) and cell-free DNA (cfDNA) were detected in the inflammatory microenvironment of these diseases, which are closely associated with persistent uncontrolled neutrophilic inflammation. Although DNase has proven to be effective in mitigating neutrophilic airway inflammation in mice by reducing cfDNA and NET levels, its clinical use is hindered by severe side effects. Here, we synthesized polyglycerol-amine (PGA) with a series of hydroxyl/amine ratios and covered them with black phosphorus (BP) nanosheets. The BP nanosheets functionalized with polyglycerol-50% amine (BP-PGA50) efficiently lowered cfDNA levels, suppressed toll-like receptor 9 (TLR9) activation and inhibited NET formation in vitro. Importantly, BP-PGA50 nanosheets demonstrated substantial accumulation in inflamed airway tissues, excellent biocompatibility, and potent inflammation modulation ability in model mice. The 2D sheet-like structure of BP-PGA50 was identified as a crucial factor for the therapeutic efficacy, and the hydroxyl/amine ratio was revealed as a significant parameter to regulate the protein resistance, cfDNA-binding efficacy, and cytotoxicity. This study shows the promise of the BP-PGA50 nanosheet for tackling uncontrolled airway inflammation, which is also significant for the treatment of other neutrophilic inflammatory diseases. In addition, our work also highlights the importance of proper surface functionalization, such as hydroxyl/amine ratio, in therapeutic nanoplatform construction for inflammation modulation.

Enabling continuous immune cell recirculation on a microfluidic array to study immunotherapeutic interactions in a recapitulated tumour microenvironment.

The effects of immunotherapeutics on interactions between immune and cancer cells are modulated by multiple components in the tumour microenvironment (TME), including endothelium and tumour stroma, which provide both a physical barrier and immunosuppressive stimuli. Herein, we report a recirculating chip to enable continuous immune cell recirculation through a microfluidic cell array to include these crucial players. This system consists of a three-layered cell array (µFCA) spatially emulating the TME, with tailored fluidic circuits establishing T cell recirculation. This platform enables the study of dynamics among the TME, immune cells in a circulatory system and cancer cell responses thereof. Through this system, we found that tumour endothelium hindered T cell infiltration into the reconstructed breast cancer tumour compartment. This negative effect was alleviated when treated with anti-human PD-L1 (programmed cell death ligand 1) antibody. Another key stromal component - cancer associated fibroblasts - attenuated T cell infiltration, compared against normal fibroblasts, and led to reduced apoptotic activity in cancer cells. These results confirm the capability of our tumour-on-a-chip system in identifying some key axes to target in overcoming barriers to immunotherapy by recapitulating immune cell interactions with the reconstructed TME. Our results also attest to the feasibility of scaling up this system for high-throughput cancer immunotherapeutic screening.

Inhaled immunoantimicrobials for the treatment of chronic obstructive pulmonary disease.

Effective therapeutic modalities and drug administration strategies for the treatment of chronic obstructive pulmonary disease (COPD) exacerbations are lacking. Here, mucus and biofilm dual-penetrating immunoantimicrobials (IMAMs) are developed for bridging antibacterial therapy and pro-resolving immunotherapy of COPD. IMAMs are constructed from ceftazidime (CAZ)-encapsulated hollow mesoporous silica nanoparticles (HMSNs) gated with a charge/conformation-transformable polypeptide. The polypeptide adopts a negatively charged, random-coiled conformation, masking the pores of HMSNs to prevent antibiotic leakage and allowing the nebulized IMAMs to efficiently penetrate the bronchial mucus and biofilm. Inside the acidic biofilm, the polypeptide transforms into a cationic and rigid α helix, enhancing biofilm retention and unmasking the pores to release CAZ. Meanwhile, the polypeptide is conditionally activated to disrupt bacterial membranes and scavenge bacterial DNA, functioning as an adjuvant of CAZ to eradicate lung-colonizing bacteria and inhibiting Toll-like receptor 9 activation to foster inflammation resolution. This immunoantibacterial strategy may shift the current paradigm of COPD management.

Chronic Opioid Treatment Arrests Neurodevelopment and Alters Synaptic Activity in Human Midbrain Organoids.

Understanding the impact of long-term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency. However, this has been limited by human brain inaccessibility and cross-species differences in animal models. Here, a human midbrain model is established that uses hiPSC-derived midbrain organoids to assess cell-type-specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single-cell mRNA sequencing of 25,510 cells from organoids in different treatment groups reveals that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single-cell level.

Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery.

Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown PCSK9 and ANGPTL3 expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Strep. pyogenes Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications.

An Engineered Nanoplatform with Tropism Toward Irradiated Glioblastoma Augments Its Radioimmunotherapy Efficacy.

Combining radiotherapy with immune checkpoint blockade therapy offers a promising approach to treat glioblastoma multiforme (GBM), yet challenges such as limited effectiveness and immune-related adverse events (irAEs) persist. These issues are largely due to the failure in targeting immunomodulators directly to the tumor microenvironment. To address this, a biomimetic nanoplatform that combines a genetically modified mesenchymal stem cell (MSC) membrane with a bioactive nanoparticle core for chemokine-directed radioimmunotherapy of GBM is developed. The CC chemokine receptor 2 (CCR2)-overexpressing MSC membrane acts as a tactical tentacle to achieve radiation-induced tropism toward the abundant chemokine (CC motif) ligand 2 (CCL2) in irradiated gliomas. The nanoparticle core, comprising diselenide-bridged mesoporous silica nanoparticles (MSNs) and PD-L1 antibodies (αPD-L1), enables X-ray-responsive drug release and radiosensitization. In two murine models with orthotopic GBM tumors, this nanoplatform reinvigorated immunogenic cell death, and augmented the efficacy and specificity of GBM radioimmunotherapy, with reduced occurrence of irAEs. This study suggests a promising radiation-induced tropism strategy for targeted drug delivery, and presents a potent nanoplatform that enhances the efficacy and safety of radio-immunotherapy.

Modeling the Effects of Protracted Cosmic Radiation in a Human Organ-on-Chip Platform.

Galactic cosmic radiation (GCR) is one of the most serious risks posed to astronauts during missions to the Moon and Mars. Experimental models capable of recapitulating human physiology are critical to understanding the effects of radiation on human organs and developing radioprotective measures against space travel exposures. The effects of systemic radiation are studied using a multi-organ-on-a-chip (multi-OoC) platform containing engineered tissue models of human bone marrow (site of hematopoiesis and acute radiation damage), cardiac muscle (site of chronic radiation damage) and liver (site of metabolism), linked by vascular circulation with an endothelial barrier separating individual tissue chambers from the vascular perfusate. Following protracted neutron radiation, the most damaging radiation component in deep space, a greater deviation of tissue function is observed as compared to the same cumulative dose delivered acutely. Further, by characterizing engineered bone marrow (eBM)-derived immune cells in circulation, 58 unique genes specific to the effects of protracted neutron dosing are identified, as compared to acutely irradiated and healthy tissues. It propose that this bioengineered platform allows studies of human responses to extended radiation exposure in an "astronaut-on-a-chip" model that can inform measures for mitigating cosmic radiation injury.

Human vascular organoids with a mosaic AKT1 mutation recapitulate Proteus syndrome.

Vascular malformation, a key clinical phenotype of Proteus syndrome, lacks effective models for pathophysiological study and drug development due to limited patient sample access. To bridge this gap, we built a human vascular organoid model replicating Proteus syndrome's vasculature. Using CRISPR/Cas9 genome editing and gene overexpression, we created induced pluripotent stem cells (iPSCs) embodying the Proteus syndrome-specific AKTE17K point mutation for organoid generation. Our findings revealed that AKT overactivation in these organoids resulted in smaller sizes yet increased vascular connectivity, although with less stable connections. This could be due to the significant vasculogenesis induced by AKT overactivation. This phenomenon likely stems from boosted vasculogenesis triggered by AKT overactivation, leading to increased vascular sprouting. Additionally, a notable increase in dysfunctional PDGFRß+ mural cells, impaired in matrix secretion, was observed in these AKT-overactivated organoids. The application of AKT inhibitors (ARQ092, AZD5363, or GDC0068) reversed the vascular malformations; the inhibitors' effectiveness was directly linked to reduced connectivity in the organoids. In summary, our study introduces an innovative in vitro model combining organoid technology and gene editing to explore vascular pathophysiology in Proteus syndrome. This model not only simulates Proteus syndrome vasculature but also holds potential for mimicking vasculatures of other genetically driven diseases. It represents an advance in drug development for rare diseases, historically plagued by slow progress.

Isotropic multi-scale neuronal reconstruction from high-ratio expansion microscopy with contrastive unsupervised deep generative models.

BACKGROUND AND OBJECTIVE: Current methods for imaging reconstruction from high-ratio expansion microscopy (ExM) data are limited by anisotropic optical resolution and the requirement for extensive manual annotation, creating a significant bottleneck in the analysis of complex neuronal structures. METHODS: We devised an innovative approach called the IsoGAN model, which utilizes a contrastive unsupervised generative adversarial network to sidestep these constraints. This model leverages multi-scale and isotropic neuron/protein/blood vessel morphology data to generate high-fidelity 3D representations of these structures, eliminating the need for rigorous manual annotation and supervision. The IsoGAN model introduces simplified structures with idealized morphologies as shape priors to ensure high consistency in the generated neuronal profiles across all points in space and scalability for arbitrarily large volumes. RESULTS: The efficacy of the IsoGAN model in accurately reconstructing complex neuronal structures was quantitatively assessed by examining the consistency between the axial and lateral views and identifying a reduction in erroneous imaging artifacts. The IsoGAN model accurately reconstructed complex neuronal structures, as evidenced by the consistency between the axial and lateral views and a reduction in erroneous imaging artifacts, and can be further applied to various biological samples. CONCLUSION: With its ability to generate detailed 3D neurons/proteins/blood vessel structures using significantly fewer axial view images, IsoGAN can streamline the process of imaging reconstruction while maintaining the necessary detail, offering a transformative solution to the existing limitations in high-throughput morphology analysis across different structures.

Starfysh integrates spatial transcriptomic and histologic data to reveal heterogeneous tumor-immune hubs.

Spatially resolved gene expression profiling provides insight into tissue organization and cell-cell crosstalk; however, sequencing-based spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for rigorous interpretation of cell states, mostly do not use associated histology images and are not capable of inferring shared neighborhoods across multiple tissues. Here we present Starfysh, a computational toolbox using a deep generative model that incorporates archetypal analysis and any known cell type markers to characterize known or new tissue-specific cell states without a single-cell reference. Starfysh improves the characterization of spatial dynamics in complex tissues using histology images and enables the comparison of niches as spatial hubs across tissues. Integrative analysis of primary estrogen receptor (ER)-positive breast cancer, triple-negative breast cancer (TNBC) and metaplastic breast cancer (MBC) tissues led to the identification of spatial hubs with patient- and disease-specific cell type compositions and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC.

Comparative Analysis of Nucleic Acid-Binding Polymers as Potential Anti-Inflammatory Nanocarriers.

Conventionally, nanocarriers are used to regulate the controlled release of therapeutic payloads. Increasingly, they can also be designed to have an intrinsic therapeutic effect. For example, a positively charged nanocarrier can bind damage-associated molecular patterns, inhibiting toll-like receptor (TLR) pathway activation and thus modulating inflammation. These nucleic acid-binding nanomaterials (NABNs), which scavenge pro-inflammatory stimuli, exist in diverse forms, ranging from soluble polymers to nanoparticles and 2D nanosheets. Unlike conventional drugs that primarily address inflammation symptoms, these NABPs target the upstream inflammation initiation pathway by removing the agonists responsible for inflammation. Many NABNs have demonstrated effectiveness in murine models of inflammatory diseases. However, these scavengers have not been systematically studied and compared within a single setting. Herein, we screen a subset of the most potent NABNs to define their relative efficiency in scavenging cell-free nucleic acids and inhibiting various TLR pathways. This study helps interpret existing in vivo results and provides insights into the future design of anti-inflammatory nanocarriers.

Degradation-resistant implanted biomaterials establish an immunosuppressive microenvironment that induces T cell exhaustion by recruiting myeloid cells.

Implanted biomaterials have transformed healthcare and the treatment of injury and disease, but their influence on the local immune landscape remains unclear. Here we discovered that degradation-resistant titanium-based implants establish an immunosuppressive microenvironment by recruiting myeloid cells, including monocytes, macrophages, neutrophils, and myeloid-lineage dendritic cells. Unlike normal tissues, the tissues nearby implants exhibit an chronic inflamed and immunosuppressive status characterised by myeloid-rich, T cell-exhaustion gene signature by single-cell RNA sequencing. Vitamin C treatment provides an effective strategy to rescue the immunosuppressive microenvironment, which can be used as a regular supplement to reduce the risk of malignant cell survival around the implants.