Multivariate Silicification-Assisted Single Enzyme Structure Augmentation for Improved Enzymatic Activity-Stability Trade-Off.

The ability to finely tune/balance the structure and rigidity of enzymes to realize both high enzymatic activity and long-term stability is highly desired but highly challenging. In this work, we propose a new concept of silica-enzyme, referred to as "silicazyme", where solid inorganic silica was controlled hybridization with fragile enzyme under moderate condition at single-enzyme level, realizing simultaneous structure augmentation, long-term stability, and high enzymatic activity preservation. A multivariate silicification approach was utilized and occurred around individual enzymes to allow conformal coating. To realize a high activity-stability trade-off the structure flexibility/rigidity of silicazyme was optimized by a component-adjustment-ternary (CAT) plot method. Moreover, the multivariate organosilica frameworks bring great advantages including surface microenvironment adjustability, reversible modification capability, and functional extensibility through the rich chemistry of silica. Overall silicazymes represent a new class of enzymes that promise to broaden their utilization in catalysis, separations, and nanomedicine.

Synthetic Biohybrids of Red Blood Cells and Cascaded-Enzymes@ Metal-Organic Frameworks for Hyperuricemia Treatment.

Hyperuricemia, caused by an imbalance between the rates of production and excretion of uric acid (UA), may greatly increase the mortality rates in patients with cardiovascular and cerebrovascular diseases. Herein, for fast-acting and long-lasting hyperuricemia treatment, armored red blood cell (RBC) biohybrids, integrated RBCs with proximal, cascaded-enzymes of urate oxidase (UOX) and catalase (CAT) encapsulated within ZIF-8 framework-based nanoparticles, have been fabricated based on a super-assembly approach. Each component is crucial for hyperuricemia treatment: 1) RBCs significantly increase the circulation time of nanoparticles; 2) ZIF-8 nanoparticles-based superstructure greatly enhances RBCs resistance against external stressors while preserving native RBC properties (such as oxygen carrying capability); 3) the ZIF-8 scaffold protects the encapsulated enzymes from enzymatic degradation; 4) no physical barrier exists for urate diffusion, and thus allow fast degradation of UA in blood and neutralizes the toxic by-product H2 O2 . In vivo results demonstrate that the biohybrids can effectively normalize the UA level of an acute hyperuricemia mouse model within 2 h and possess a longer elimination half-life (49.7 ± 4.9 h). They anticipate that their simple and general method that combines functional nanomaterials with living cell carriers will be a starting point for the development of innovative drug delivery systems.

Future of Mesoporous Silica Nanoparticles in Nanomedicine: Protocol for Reproducible Synthesis, Characterization, Lipid Coating, and Loading of Therapeutics (Chemotherapeutic, Proteins, siRNA and mRNA).

Owing to their uniform and tunable particle size, pore size, and shape, along with their modular surface chemistry and biocompatibility, mesoporous silica nanoparticles (MSNs) have found extensive applications as nanocarriers to deliver therapeutic, diagnostic and combined "theranostic" cargos to cells and tissues. Although thoroughly investigated, MSN have garnered FDA approval for only one MSN system via oral administration. One possible reason is that there is no recognized, reproducible, and widely adopted MSN synthetic protocol, meaning not all MSNs are created equal in the laboratory nor in the eyes of the FDA. This manuscript provides the sol-gel and MSN research communities a reproducible, fully characterized synthetic protocol to synthesize MSNs and corresponding lipid-coated MSN delivery vehicles with predetermined particle size, pore size, and drug loading and release characteristics. By carefully articulating the step-by-step synthetic procedures and highlighting critical points and troubleshooting, augmented with videos and schematics, this Article will help researchers entering this rapidly expanding field to yield reliable results.

Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

Dimensional Reduction of Metal-Organic Frameworks for Enhanced Cryopreservation of Red Blood Cells.

To increase the red blood cell (RBC) cryopreservation efficiency by metal-organic frameworks (MOFs), a dimensional reduction approach has been proposed. Namely, 3D MOF nanoparticles are progressively reduced to 2D ultra-thin metal-organic layers (MOLs). We found that 2D MOLs are beneficial for enhanced interactions of the interfacial hydrogen-bonded water network and increased utilization of inner ordered structures, due to the higher surface-to-volume ratio. Specifically, a series of hafnium (Hf)-based 2D MOLs with different thicknesses (monolayer to stacked multilayers) and densities of hydrogen bonding sites have been synthesized. Both ice recrystallization inhibition activity (IRI) and RBCs cryopreservation assay confirm the pronounced better IRI activity and excellent cell recovery efficiency (up to ≈63 % at a very low concentration of 0.7 mg mL-1 ) of thin-layered Hf-MOLs compared to their 3D counterparts, thereby verifying the dimensional reduction strategy to improved cryoprotectant behaviors.

CTB-targeted protocells enhance ability of lanthionine ketenamine analogs to induce autophagy in motor neuron-like cells.

Impaired autophagy, a cellular digestion process that eliminates proteins and damaged organelles, has been implicated in neurodegenerative diseases, including motor neuron disorders. Motor neuron targeted upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue, activates autophagy in neuronal cell lines. We hypothesized that analogs of LK can be targeted to motor neurons using nanoparticles to improve autophagy flux. Using a mouse motor neuron-like hybrid cell line (NSC-34), we tested the effect of three different LK analogs on autophagy modulation, either alone or loaded in nanoparticles. For fluorescence visualization of autophagy flux, we used a mCherry-GFP-LC3 plasmid reporter. We also evaluated protein expression changes in LC3-II/LC3-I ratio obtained by western blot, as well as presence of autophagic vacuoles per cell obtained by electron microscopy. Delivering LK analogs with targeted nanoparticles significantly enhanced autophagy flux in differentiated motor neuron-like cells compared to LK analogs alone, suggesting the need of a delivery vehicle to enhance their efficacy. In conclusion, LK analogs loaded in nanoparticles targeting motor neurons constitute a promising treatment option to induce autophagy flux, which may serve to mitigate motor neuron degeneration/loss and preserve motor function in motor neuron disease.

Analysis and Biomedical Applications of Functional Cargo in Extracellular Vesicles.

Extracellular vesicles (EVs) can facilitate essential communication among cells in a range of pathophysiological conditions including cancer metastasis and progression, immune regulation, and neuronal communication. EVs are membrane-enclosed vesicles generated through endocytic origin and contain many cellular components, including proteins, lipids, nucleic acids, and metabolites. Over the past few years, the intravesicular content of EVs has proven to be a valuable biomarker for disease diagnostics, involving cancer, cardiovascular diseases, and central nervous system diseases. This review aims to provide insight into EV biogenesis, composition, function, and isolation, present a comprehensive overview of emerging techniques for EV cargo analysis, highlighting their major technical features and limitations, and summarize the potential role of EV cargos as biomarkers in disease diagnostics. Further, progress and remaining challenges will be discussed for clinical diagnostic outlooks.

Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies.

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo. The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo, enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Dual-Stage Irradiation of Size-Switchable Albumin Nanocluster for Cascaded Tumor Enhanced Penetration and Photothermal Therapy.

The triple-negative breast cancer (TNBC) microenvironment makes a feature of aberrant vasculature, high interstitial pressure, and compact extracellular matrix, which combine to reduce the delivery and penetration of therapeutic agents, bringing about incomplete elimination of cancer cells. Herein, employing the tumor penetration strategy of size-shrinkage combined with ligand modification, we constructed a photothermal nanocluster for cascaded deep penetration in tumor parenchyma and efficient eradication of TNBC cells. In our approach, the photothermal agent indocyanine green (ICG) is laded in human serum albumin (HSA), which is cross-linked by a thermally labile azo linker (VA057) and then further modified with a tumor homing/penetrating tLyP-1 peptide (HP), resulting in a TNBC-targeting photothermal-responsive size-switchable albumin nanocluster (ICG@HSA-Azo-HP). Aided by the enhanced permeability and retention effect and guidance of HP, the ca. 149 nm nanoclusters selectively accumulate in the tumor site and then, upon mild irradiation with the 808 nm laser, disintegrate into 11 nm albumin fractions that possess enhanced intratumoral diffusion ability. Meanwhile, HP initiates the CendR pathway among the nutrient-deficient tumor cells and facilitates the transcellular delivery of the nanocluster and its disintegrated fractions for subsequent therapy. By employing this size-shrinkage and peptide-initiated transcytosis strategy, ICG@HSA-Azo-HP possesses excellent penetration capabilities and shows extensive penetration depth in three-dimensional multicellular tumor spheroids after irradiation. Moreover, with a superior photothermal conversion effect, the tumor-penetrating nanocluster can implement effective photothermal therapy throughout the tumor tissue under a second robust irradiation. Both in vivo orthotopic and ectopic TNBC therapy confirmed the efficient tumor inhibition of ICG@HSA-Azo-HP after dual-stage irradiation. The synergistic penetration strategy of on-demanded size-shrinkage and ligand guidance accompanied by clinically feasible NIR irradiation provides a promising approach for deep-penetrating TNBC therapy.

Robust and Long-Term Cellular Protein and Enzymatic Activity Preservation in Biomineralized Mammalian Cells.

Preservation of evolved biological structure and function in robust engineering materials is of interest for storage of biological samples before diagnosis and development of vaccines, sensors, and enzymatic reactors and has the potential to avoid cryopreservation and its associated cold-chain issues. Here, we demonstrate that "freezing cells in amorphous silica" is a powerful technique for long-term preservation of whole mammalian cell proteomic structure and function at room temperature. Biomimetic silicification employs the crowded protein microenvironment of mammalian cells as a catalytic framework to proximally transform monomeric silicic acid into silicates forming a nanoscopic silica shell over all biomolecular interfaces. Silicification followed by dehydration preserves and passivates proteomic information within a nanoscale thin silica coating that exhibits size selective permeability (<3.6 nm), preventing protein leaching and protease degradation of cellular contents, while providing access of small molecular constituents for cellular enzymatic reaction. Exposure of dehydrated silicified cells to mild etchant or prolonged hydrolysis removes the silica, completely rerevealing biomolecular components and restoring their accessibility and functionality.

Uptake and Toxicity of Respirable Carbon-Rich Uranium-Bearing Particles: Insights into the Role of Particulates in Uranium Toxicity.

Particulate matter (PM) presents an environmental health risk for communities residing close to uranium (U) mine sites. However, the role of the particulate form of U on its cellular toxicity is still poorly understood. Here, we investigated the cellular uptake and toxicity of C-rich U-bearing particles as a model organic particulate containing uranyl citrate over a range of environmentally relevant concentrations of U (0-445 µM). The cytotoxicity of C-rich U-bearing particles in human epithelial cells (A549) was U-dose-dependent. No cytotoxic effects were detected with soluble U doses. Carbon-rich U-bearing particles with a wide size distribution (<10 µm) presented 2.7 times higher U uptake into cells than the particles with a narrow size distribution (<1 µm) at 100 µM U concentration. TEM-EDS analysis identified the intracellular translocation of clusters of C-rich U-bearing particles. The accumulation of C-rich U-bearing particles induced DNA damage and cytotoxicity as indicated by the increased phosphorylation of the histone H2AX and cell death, respectively. These findings reveal the toxicity of the particulate form of U under environmentally relevant heterogeneous size distributions. Our study opens new avenues for future investigations on the health impacts resulting from environmental exposures to the particulate form of U near mine sites.

Bioinspired Cell Silicification: From Extracellular to Intracellular.

In nature, biosilicification directs the formation of elaborate amorphous silica exoskeletons that provide diatoms mechanically strong, chemically inert, non-decomposable silica armor conferring chemical and thermal stability as well as resistance to microbial attack, without changing the optical transparency or adversely effecting nutrient and waste exchange required for growth. These extraordinary silica/cell biocomposites have inspired decades of biomimetic research aimed at replication of diatoms' hierarchically organized exoskeletons, immobilization of cells or living organisms within silica matrices and coatings to protect them against harmful external stresses, genetic re-programming of cellular functions by virtue of physico-chemical confinement within silica, cellular integration into devices, and endowment of cells with non-native, abiotic properties through facile silica functionalization. In this Perspective, we focus our discussions on the development and concomitant challenges of bioinspired cell silicification ranging from "cells encapsulated within 3D silica matrices" and "cells encapsulated within 2D silica shells" to extra- and intracellular silica replication, wherein all biomolecular interfaces are encased within nanoscopic layers of amorphous silica. We highlight notable examples of advances in the science and technology of biosilicification and consider challenges to advancing the field, where we propose cellular "mineralization" with arbitrary nanoparticle exoskeletons as a generalizable means to impart limitless abiotic properties and functions to cells, and, based on the interchangeability of water and silicic acid and analogies between amorphous ice and amorphous silica, we consider "freezing" cells within amorphous silica as an alternative to cryo-preservation.

Advanced Nanomaterials-Assisted Cell Cryopreservation: A Mini Review.

Cell cryopreservation is of vital significance both for transporting and storing cells before experimental/clinical use. Cryoprotectants (CPAs) are necessary additives in the preserving medium in cryopreservation, preventing cells from freeze-thaw injuries. Traditional organic solvents have been widely used in cell cryopreservation for decades. Given the obvious damage to cells due to their undesirable cytotoxicity and the burdensome post-thaw washing cycles before use, traditional CPAs are more and more likely to be replaced by modern ones with lower toxicity, less processing, and higher efficiency. As materials science thrives, nanomaterials are emerging to serve as potent vehicles for delivering nontoxic CPAs or inherent CPAs comparable to or even superior to conventional ones. This review will introduce some advanced nanomaterials (e.g., organic/inorganic nanoCPAs, nanodelivery systems) utilized for cell cryopreservation, providing broader insights into this developing field.

Synthesis of Polyhedral Metal-Organic Framework@Mesoporous Silica Hybrid Nanocomposites with Branched Shapes.

The structural modulation of multicompartment porous nanomaterials is one of the major challenges of nanoscience. Herein, by utilizing the polyhedral effects/characteristics of metal-organic frameworks (MOFs), we present a versatile approach to construct MOF-organosilica hybrid branched nanocomposites with MOF cores, SiO2 shells, and periodic mesoporous organosilica (PMO) branches. The morphology, structure, and functions of the obtained hybrid nanocomposites can be facilely modulated by varying particle size, shape, or crystalline structures of the MOF cores. Specifically, these branched multicompartment porous nanoparticles exhibit evasion behaviors in epithelial cells compared with macrophage cells, which may endow them great potential as a vehicle for immunotherapy.

Direct Transfer of Mesoporous Silica Nanoparticles between Macrophages and Cancer Cells.

Macrophages line the walls of microvasculature, extending processes into the blood flow to capture foreign invaders, including nano-scale materials. Using mesoporous silica nanoparticles (MSNs) as a model nano-scale system, we show the interplay between macrophages and MSNs from initial uptake to intercellular trafficking to neighboring cells along microtubules. The nature of cytoplasmic bridges between cells and their role in the cell-to-cell transfer of nano-scale materials is examined, as is the ability of macrophages to function as carriers of nanomaterials to cancer cells. Both direct administration of nanoparticles and adoptive transfer of nanoparticle-loaded splenocytes in mice resulted in abundant localization of nanomaterials within macrophages 24 h post-injection, predominately in the liver. While heterotypic, trans-species nanomaterial transfer from murine macrophages to human HeLa cervical cancer cells or A549 lung cancer cells was robust, transfer to syngeneic 4T1 breast cancer cells was not detected in vitro or in vivo. Cellular connections and nanomaterial transfer in vivo were rich among immune cells, facilitating coordinated immune responses.