Versatile Design of NO-Generating Proteolipid Nanovesicles for Alleviating Vascular Injury.

Vascular injury is central to the pathogenesis and progression of cardiovascular diseases, however, fostering alternative strategies to alleviate vascular injury remains a persisting challenge. Given the central role of cell-derived nitric oxide (NO) in modulating the endogenous repair of vascular injury, NO-generating proteolipid nanovesicles (PLV-NO) are designed that recapitulate the cell-mimicking functions for vascular repair and replacement. Specifically, the proteolipid nanovesicles (PLV) are versatilely fabricated using membrane proteins derived from different types of cells, followed by the incorporation of NO-generating nanozymes capable of catalyzing endogenous donors to produce NO. Taking two vascular injury models, two types of PLV-NO are tailored to meet the individual requirements of targeted diseases using platelet membrane proteins and endothelial membrane proteins, respectively. The platelet-based PLV-NO (pPLV-NO) demonstrates its efficacy in targeted repair of a vascular endothelium injury model through systemic delivery. On the other hand, the endothelial cell (EC)-based PLV-NO (ePLV-NO) exhibits suppression of thrombosis when modified onto a locally transplanted small-diameter vascular graft (SDVG). The versatile design of PLV-NO may enable a promising therapeutic option for various vascular injury-evoked cardiovascular diseases.

Data-Driven Design of Triple-Targeted Protein Nanoprobes for Multiplexed Imaging of Cancer Lymphatic Metastasis.

Targeted imaging of cancer lymphatic metastasis remains challenging due to its highly heterogeneous molecular and phenotypic diversity. Herein, triple-targeted protein nanoprobes capable of specifically binding to three targets for imaging cancer lymphatic metastasis, through a data-driven design approach combined with a synthetic biology-based assembly strategy, are introduced. Specifically, to address the diversity of metastatic lymph nodes (LNs), a combination of three targets, including C-X-C motif chemokine receptor 4 (CXCR4), transferrin receptor protein 1 (TfR1), and vascular endothelial growth factor receptor 3 (VEGFR3) is identified, leveraging machine leaning-based bioinformatics analysis and examination of LN tissues from patients with gastric cancer. Using this identified target combination, ferritin nanocage-based nanoprobes capable of specifically binding to all three targets are designed through the self-assembly of genetically engineered ferritin subunits using a synthetic biology approach. Using these nanoprobes, multiplexed imaging of heterogeneous metastatic LNs is successfully achieved in a polyclonal lymphatic metastasis animal model. In 19 freshly resected human gastric specimens, the signal from the triple-targeted nanoprobes significantly differentiates metastatic LNs from benign LNs. This study not only provides an effective nanoprobe for imaging highly heterogeneous lymphatic metastasis but also proposes a potential strategy for guiding the design of targeted nanomedicines for cancer lymphatic metastasis.

Greatly isolated heterogeneous circulating tumor cells using hybrid engineered cell membrane-camouflaged magnetic nanoparticles.

BACKGROUND: Circulating tumor cells (CTCs) are considered as a useful biomarker for early cancer diagnosis, which play a crucial role in metastatic process. Unfortunately, the tumor heterogeneity and extremely rare occurrence rate of CTCs among billions of interfering leukocytes seriously hamper the sensitivity and purity of CTCs isolation. METHODS: To address these, we firstly used microfluidic chips to detect the broad-spectrum of triple target combination biomarkers in CTCs of 10 types of cancer patients, including EpCAM, EGFR and Her2. Then, we constructed hybrid engineered cell membrane-camouflaged magnetic nanoparticles (HE-CM-MNs) for efficient capture of heterogeneous CTCs with high-purity, which was enabled by inheriting the recognition ability of HE-CM for various CTCs and reducing homologous cell interaction with leukocytes. Compared with single E-CM-MNs, HE-CM-MNs showed a significant improvement in the capture efficiency for a cell mixture, with an efficiency of 90%. And the capture efficiency of HE-CM-MNs toward 12 subpopulations of tumor cells was ranged from 70 to 85%. Furthermore, by using HE-CM-MNs, we successfully isolated heterogeneous CTCs with high purity from clinical blood samples. Finally, the captured CTCs by HE-CM-MNs could be used for gene mutation analysis. CONCLUSIONS: This study demonstrated the promising potential of HE-CM-MNs for heterogeneous CTCs detection and downstream analysis.

Genetically engineered membrane-based nanoengagers for immunotherapy of pancreatic cancer.

Modulating macrophages presents a promising avenue in tumor immunotherapy. However, tumor cells have evolved mechanisms to evade macrophage activation and phagocytosis. Herein, we introduced a bispecific antibody-based nanoengager to facilitate the recognition and phagocytosis of tumor cells by macrophages. Specifically, we genetically engineered two single chain variable fragments (scFv) onto cell membrane: anti-CD40 scFv for engaging with macrophages and anti-Claudin18.2 (CLDN18.2) scFv for interacting with tumor cells. These nanoengagers were further constructed by coating scFv-anchored membrane into PLGA nanoparticle core. Our developed nanoengagers significantly boosted immune responses, including increased recognition and phagocytosis of tumor cells by macrophages, enhanced activation and antigen presentation, and elevated cytotoxic T lymphocyte activity. These combined benefits resulted in enhancing antitumor efficacy against highly aggressive "cold" pancreatic cancer. Overall, this study offers a versatile nanoengager design for immunotherapy, achieved through genetically engineering to incorporate antibody-anchored membrane.

Self-Assembly of Heterogeneous Ferritin Nanocages for Tumor Uptake and Penetration.

Well-defined nanostructures are crucial for precisely understanding nano-bio interactions. However, nanoparticles (NPs) fabricated through conventional synthesis approaches often lack poor controllability and reproducibility. Herein, a synthetic biology-based strategy is introduced to fabricate uniformly reproducible protein-based NPs, achieving precise control over heterogeneous components of the NPs. Specifically, a ferritin assembly toolbox system is developed that enables intracellular assembly of ferritin subunits/variants in Escherichia coli. Using this strategy, a proof-of-concept study is provided to explore the interplay between ligand density of NPs and their tumor targets/penetration. Various ferritin hybrid nanocages (FHn) containing human ferritin heavy chains (FH) and light chains are accurately assembled, leveraging their intrinsic binding with tumor cells and prolonged circulation time in blood, respectively. Further studies reveal that tumor cell uptake is FH density-dependent through active binding with transferrin receptor 1, whereas in vivo tumor accumulation and tissue penetration are found to be correlated to heterogeneous assembly of FHn and vascular permeability of tumors. Densities of 3.7 FH/100 nm2 on the nanoparticle surface exhibit the highest degree of tumor accumulation and penetration, particularly in tumors with high permeability compared to those with low permeability. This study underscores the significance of nanoparticle heterogeneity in determining particle fate in biological systems.

Machine-Learning-Assisted Nanozyme Design: Lessons from Materials and Engineered Enzymes.

Nanozymes are nanomaterials that exhibit enzyme-like biomimicry. In combination with intrinsic characteristics of nanomaterials, nanozymes have broad applicability in materials science, chemical engineering, bioengineering, biochemistry, and disease theranostics. Recently, the heterogeneity of published results has highlighted the complexity and diversity of nanozymes in terms of consistency of catalytic capacity. Machine learning (ML) shows promising potential for discovering new materials, yet it remains challenging for the design of new nanozymes based on ML approaches. Alternatively, ML is employed to promote optimization of intelligent design and application of catalytic materials and engineered enzymes. Incorporation of the successful ML algorithms used in the intelligent design of catalytic materials and engineered enzymes can concomitantly facilitate the guided development of next-generation nanozymes with desirable properties. Here, recent progress in ML, its utilization in the design of catalytic materials and enzymes, and how emergent ML applications serve as promising strategies to circumvent challenges associated with time-expensive and laborious testing in nanozyme research and development are summarized. The potential applications of successful examples of ML-aided catalytic materials and engineered enzymes in nanozyme design are also highlighted, with special focus on the unified aims in enhancing design and recapitulation of substrate selectivity and catalytic activity.

Ferritin nanocages: a versatile platform for nanozyme design.

Nanozymes are a class of nanomaterials with enzyme-like activities and have attracted increasing attention due to their potential applications in biomedicine. However, nanozyme design incorporating the desired properties remains challenging. Natural or genetically engineered protein scaffolds, such as ferritin nanocages, have emerged as a promising platform for nanozyme design due to their unique protein structure, natural biomineralization capacity, self-assembly properties, and high biocompatibility. In this review, we highlight the intrinsic properties of ferritin nanocages, especially for nanozyme design. We also discuss the advantages of genetically engineered ferritin in the versatile design of nanozymes over natural ferritin. Additionally, we summarize the bioapplications of ferritin-based nanozymes based on their enzyme-mimicking activities. In this perspective, we mainly provide potential insights into the utilization of ferritin nanocages for nanozyme design.

Machine-learning-assisted single-vessel analysis of nanoparticle permeability in tumour vasculatures.

The central dogma that nanoparticle delivery to tumours requires enhanced leakiness of vasculatures is a topic of debate. To address this, we propose a single-vessel quantitative analysis method by taking advantage of protein-based nanoprobes and image-segmentation-based machine learning (nano-ISML). Using nano-ISML, >67,000 individual blood vessels from 32 tumour models were quantified, revealing highly heterogenous vascular permeability of protein-based nanoparticles. There was a >13-fold difference in the percentage of high-permeability vessels in different tumours and >100-fold penetration ability in vessels with the highest permeability compared with vessels with the lowest permeability. Our data suggest passive extravasation and transendothelial transport were the dominant mechanisms for high- and low-permeability tumour vessels, respectively. To exemplify the nano-ISML-assisted rational design of nanomedicines, genetically tailored protein nanoparticles with improved transendothelial transport in low-permeability tumours were developed. Our study delineates the heterogeneity of tumour vascular permeability and defines a direction for the rational design of next-generation anticancer nanomedicines.

Tumor Cell Nanovaccines Based on Genetically Engineered Antibody-Anchored Membrane.

Despite the promise in whole-tumor cell vaccines, a key challenge is to overcome the lack of costimulatory signals. Here, agonistic-antibody-boosted tumor cell nanovaccines are reported by genetically engineered antibody-anchored membrane (AAM) technology, capable of effectively activating costimulatory pathways. Specifically, the AAM can be stably constructed following genetic engineering of tumor cell membranes with anti-CD40 single chain variable fragment (scFv), an agonistic antibody to induce costimulatory signals. The nanovaccines are versatilely designed and obtained based on the anti-CD40 scFv-anchored membrane and nanotechnology. Following vaccination, the anti-CD40 scFv-anchored membrane nanovaccine (Nano-AAM/CD40) significantly facilitates dendritic cell maturation in CD40-humanized transgenic mice and subsequent adaptive immune responses. Compared to membrane-based nanovaccines alone, the enhanced antitumor efficacy in both "hot" and "cold" tumor models of the Nano-AAM/CD40 demonstrates the importance of agonistic antibodies in development of tumor-cell-based vaccines. To expand the design of nanovaccines, further incorporation of cell lysates into the Nano-AAM/CD40 to conceptually construct tumor cell-like nanovaccines results in boosted immune responses and improved antitumor efficacy against malignant tumors inoculated into CD40-humanized transgenic mice. Overall, this genetically engineered AAM technology provides a versatile design of nanovaccines by incorporation of tumor-cell-based components and agonistic antibodies of costimulatory immune checkpoints.

Targeted delivery of nanomedicines for promoting vascular regeneration in ischemic diseases.

Ischemic diseases, the leading cause of disability and death, are caused by the restriction or blockage of blood flow in specific tissues, including ischemic cardiac, ischemic cerebrovascular and ischemic peripheral vascular diseases. The regeneration of functional vasculature network in ischemic tissues is essential for treatment of ischemic diseases. Direct delivery of pro-angiogenesis factors, such as VEGF, has demonstrated the effectiveness in ischemic disease therapy but suffering from several obstacles, such as low delivery efficacy in disease sites and uncontrolled modulation. In this review, we summarize the molecular mechanisms of inducing vascular regeneration, providing the guidance for designing the desired nanomedicines. We also introduce the delivery of various nanomedicines to ischemic tissues by passive or active targeting manner. To achieve the efficient delivery of nanomedicines in various ischemic diseases, we highlight targeted delivery of nanomedicines and controllable modulation of disease microenvironment using nanomedicines.

Neutrophil-mediated delivery of the combination of colistin and azithromycin for the treatment of bacterial infection.

Novel treatment strategies are in urgent need to deal with the rapid development of antibiotic-resistant superbugs. Combination therapies and targeted drug delivery have been exploited to promote treatment efficacies. In this study, we loaded neutrophils with azithromycin and colistin to combine the advantages of antibiotic combinations, targeted delivery, and immunomodulatory effect of azithromycin to treat infections caused by Gram-negative pathogens. Delivery of colistin into neutrophils was mediated by fusogenic liposome, while azithromycin was directly taken up by neutrophils. Neutrophils loaded with the drugs maintained the abilitity to generate reactive oxygen species and migrate. In vitro assays demonstrated enhanced bactericidal activity against multidrug-resistant pathogens and reduced inflammatory cytokine production by the drug-loaded neutrophils. A single intravenous administration of the drug-loaded neutrophils effectively protected mice from Pseudomonas aeruginosa infection in an acute pneumonia model. This study provides a potential effective therapeutic approach for the treatment of bacterial infections.

Lineage Tracing and Molecular Real-Time Imaging of Cancer Stem Cells.

The cancer stem cells (CSC) are the roots of cancer. The CSC hypothesis may provide a model to explain the tumor cell heterogeneity. Understand the biological mechanism of CSC will help the early detection and cure of cancer. The discovery of the dynamic changes in CSC will be possible by the using of bio-engineering techniques-lineage tracing. However, it is difficult to obtain real-time, continuous, and dynamic live-imaging information using the traditional approaches that take snapshots of time points from different animals. The goal of molecular imaging is to monitor the in situ, continuous molecular changes of cells in vivo. Therefore, the most advanced bioengineering lineage tracing approach, while using a variety of molecular detection methods, will maximize the presentation of CSC. In this review, we first introduce the method of lineage tracing, and then introduce the various components of molecular images to dynamic detect the CSC. Finally, we analyze the current situation and look forward the future of CSC detection.

Screening of Protein-Based Ultrasmall Nanozymes for Building Cell-Mimicking Catalytic Vesicles.

Enzymes are an important component for bottom-up building of synthetic/artificial cells. Nanozymes are nanomaterials with intrinsic enzyme-like properties, however, the construction of synthetic cells using nanozymes is difficult owing to their high surface energy or large size. Herein, the authors show a protein-based general platform that biomimetically integrates various ultrasmall metal nanozymes into protein shells. Specifically, eight metal-based ultrasmall nano-particles/clusters are in situ incorporated into ferritin nanocages that are self-assembled by 24 subunits of ferritin heavy chain. As a nanozyme generator, such a platform is suitable for screening the desired enzyme-like activities, including peroxidase (POD), oxidase (OXD), catalase (CAT) and superoxide dismutase (SOD). After screening, it is found that Ru intrinsically possesses the highest POD-like and CAT-like activities, while Mn and Pt show the highest OXD-like and SOD-like activities, respectively. Additionally, the inducers/inhibitors of various nanozymes are screened from more than 50 compounds to improve or inhibit their enzyme-like activities. Based on the screened nanozymes and their inhibitors, a proof-of-conceptually constructs cell-mimicking catalytic vesicles to mimic or modulate the events of redox homeostasis in living cells. This study offers a type of artificial metalloenzyme based on nanotechnology and shows a choice for bottom-up enzyme-based synthetic cell systems in a fully synthetic manner.

Mitochondria-targeted nanozymes eliminate oxidative damage in retinal neovascularization disease.

Retinal neovascularization is typically accompanied by hypoxia-induced oxidative injury in the vascular system. This study developed an ultrasmall (6-8 nm) platinum (Pt) nanozyme loaded mitochondria-targeted liposome (Pt@MitoLipo) to alleviate hypoxia and eliminate excess reactive oxygen species (ROS) for effective retinal neovascularization disease therapy. Pt nanozymes possess superoxide dismutase (SOD) and catalase (CAT) cascade enzyme-like activities, which convert cytotoxic O2•- and H2O2 into nontoxic H2O and O2. Triphenylphosphonium (TPP)-conjugated liposomes were coated on the surface of Pt nanozymes to increase their biocompatibility and help them penetrate the cell membrane, escape from the lysosomal barrier, and target mitochondria, thus achieving precise scavenging of mitochondrial O2•- and relief from hypoxia. Using an oxygen-induced retinopathy (OIR) mouse model, we demonstrated that Pt@MitoLipo nanozymes significantly suppressed hypoxia-induced abnormal neovascularization and facilitated avascular normalization of the retina in vivo without any noticeable toxicity. This study provides a promising way to break through cellular barriers and target scavenging mitochondrial O2•- and illustrates the potential of ROS-scavenging and hypoxia relief in retinal neovascularization disease therapy.

Protein corona-coated immunomagnetic nanoparticles with enhanced isolation of circulating tumor cells.

Immunomagnetic nanoparticles (IMNs) have been widely developed as a detection tool to isolate rare circulating tumor cells (CTCs) from whole blood as a potential method for early cancer diagnosis, metastasis examination, and treatment guidance. However, a spontaneous interaction between nanoparticles and proteins results in the formation of a protein corona that reduces the performance of IMNs when they enter body fluids. To address this issue, the protein corona was precoated onto magnetic nanoparticles (C-MNs), and then their surfaces were conjugated with an immuno-antibody. The adsorption of proteins on C-MNs was decreased 6-fold and non-specific cell binding was reduced 5-fold, compared with magnetic nanoparticles (MNs). Furthermore, the immuno-antibody functionalized C-MNs (IC-MNs) maintained highly specific CTC capture performance when exposed to blood plasma. By using artificial spiked blood samples, IC-MNs exhibited 90.2% CTC isolation efficiency, compared with 60.3% by using IMNs. IC-MNs also successfully captured CTCs with high purity in 24 out of 26 female breast cancer patient blood samples. This work demonstrated that a novel preformed protein corona strategy can provide a useful clinically applicable diagnostic tool.

Dynamic O-GlcNAcylation coordinates ferritinophagy and mitophagy to activate ferroptosis.

Ferroptosis is a regulated iron-dependent cell death characterized by the accumulation of lipid peroxidation. A myriad of facets linking amino acid, lipid, redox, and iron metabolisms were found to drive or to suppress the execution of ferroptosis. However, how the cells decipher the diverse pro-ferroptotic stress to activate ferroptosis remains elusive. Here, we report that protein O-GlcNAcylation, the primary nutrient sensor of glucose flux, orchestrates both ferritinophagy and mitophagy for ferroptosis. Following the treatment of ferroptosis stimuli such as RSL3, a commonly used ferroptosis inducer, there exists a biphasic change of protein O-GlcNAcylation to modulate ferroptosis. Pharmacological or genetic inhibition of O-GlcNAcylation promoted ferritinophagy, resulting in the accumulation of labile iron towards mitochondria. Inhibition of O-GlcNAcylation resulted in mitochondria fragmentation and enhanced mitophagy, providing an additional source of labile iron and rendering the cell more sensitive to ferroptosis. Mechanistically, we found that de-O-GlcNAcylation of the ferritin heavy chain at S179 promoted its interaction with NCOA4, the ferritinophagy receptor, thereby accumulating labile iron for ferroptosis. Our findings reveal a previously uncharacterized link of dynamic O-GlcNAcylation with iron metabolism and decision-making for ferroptosis, thus offering potential therapeutic intervention for fighting disease.

Prediction and Design of Nanozymes using Explainable Machine Learning.

An abundant number of nanomaterials have been discovered to possess enzyme-like catalytic activity, termed nanozymes. It is identified that a variety of internal and external factors influence the catalytic activity of nanozymes. However, there is a lack of essential methodologies to uncover the hidden mechanisms between nanozyme features and enzyme-like activity. Here, a data-driven approach is demonstrated that utilizes machine-learning algorithms to understand particle-property relationships, allowing for classification and quantitative predictions of enzyme-like activity exhibited by nanozymes. High consistency between predicted outputs and the observations is confirmed by accuracy (90.6%) and R2 (up to 0.80). Furthermore, sensitive analysis of the models reveals the central roles of transition metals in determining nanozyme activity. As an example, the models are successfully applied to predict or design desirable nanozymes by uncovering the hidden relationship between different periods of transition metals and their enzyme-like performance. This study offers a promising strategy to develop nanozymes with desirable catalytic activity and demonstrates the potential of machine learning within the field of material science.

Combining Doxorubicin-Conjugated Polymeric Nanoparticles and 5-Aminolevulinic Acid for Enhancing Radiotherapy against Lung Cancer.

Radiation therapy (RT) concurrent with chemotherapy improves local lung cancer control but may cause systemic toxicity. There is an unmet clinical need of treatments that can selectively sensitize cancer cells to RT. Herein, we explored a radiosensitizing strategy that combines doxorubicin (DOX)-encapsulated polyaspartamide nanoparticles and 5-aminolevulinic acid (5-ALA). The DOX-polyaspartamide nanoparticles were coupled with NTSmut, a ligand specific to neurotensin receptor type 1 (NTSR1), for lung cancer targeting. DOX was coupled to the polymer backbone through a pH-sensitive hydrazone linker, which allows for controlled release of the drug in an acidic tumor micromovement. Meanwhile, 5-ALA accumulates in the cancer cell's mitochondria, forming protoporphyrin (PpIX) that amplifies RT-induced oxidative stress. When tested in vitro in H1299 cells, DOX-encapsulated nanoparticles in conjugation with 5-ALA enhanced cancer cell killing owing to the complementary radiosensitizing effects of DOX and 5-ALA. In vivo studies confirmed that the combination improved tumor suppression relative to RT alone without causing toxicity to normal tissues. Overall, our study suggests an effective and selective radiosensitizing approach.

Biomimetic Design of Artificial Hybrid Nanocells for Boosted Vascular Regeneration in Ischemic Tissues.

Restoration of sufficient blood supply for the treatment of ischemia remains a significant scientific and clinical challenge. Here, a cell-like nanoparticle delivery technology is introduced that is capable of recapitulating multiple cell functions for the spatiotemporal triggering of vascular regeneration. Specifically, a copper-containing protein is successfully prepared using a recombinant protein scaffold based on a de novo design strategy, which facilitates the timely release of nitric oxide and improved accumulation of particles within ischemic tissues. Through closely mimicking physiological cues, the authors demonstrate the benefits of bioactive factors secreted from hypoxic stem cells on promoting angiogenesis. Following this cell-mimicking manner, artificial hybrid nanosized cells (Hynocell) are constructed by integrating the hypoxic stem cell secretome into nanoparticles with surface coatings of cell membranes fused with copper-containing protein. The Hynocell, hybridized with different cell-derived components, provides synergistic effects on targeting ischemic tissues and promoting vascular regeneration in acute hindlimb ischemia and acute myocardial infarction models. This study offers new insights into the utilization of nanotechnology to potentiate the development of cell-free therapeutics.

Bioorthogonal catalytic nanozyme-mediated lysosomal membrane leakage for targeted drug delivery.

Rationale: Employing in situ bioorthogonal catalysis within subcellular organelles, such as lysosomes, remains a challenge. Lysosomal membranes pose an intracellular barrier for drug sequestration, thereby greatly limiting drug accumulation and concentrations at intended targets. Here, we provide a proof-of-concept report of a nanozyme-based strategy that mediates in situ bioorthogonal uncaging reactions within lysosomes, followed by lysosomal escape and the release of uncaged drugs into the cytoplasm. Methods: A model system composed of a protein-based nanozyme platform (based on the transition metals Co, Fe, Mn, Rh, Ir, Pt, Au, Ru and Pd) and caged compound fluorophores was designed to screen for nanozyme/protecting group pairings. The optimized nanozyme/protecting group pairing was then selected for utilization in the design of anti-cancer pro-drugs and drug delivery systems. Results: Our screening system identified Pd nanozymes that mimic mutant P450BM3 activity and specifically cleave propargylic ether groups. We found that the intrinsic peroxidase-like activity of Pd nanozymes induced the production of free radicals under acid conditions, resulting in lysosomal membrane leakage of uncaged molecules into the cytoplasm. Using a multienzyme synergistic approach, our Pd nanozymes achieved in situ bioorthogonal catalysis and nanozyme-mediated lysosomal membrane leakage, which were successfully applied to the design of model pro-drugs for anti-cancer therapy. The extension of our nanozyme system to the construction of a liposome-based "all-in-one" delivery system offers promise for realizing efficacious in vivo tumor-targeted therapies. Conclusions: This strategy shows a promising new direction by utilizing nanotechnology for drug development through in situ catalyzing bioorthogonal chemistry within specific subcellular organelles.