An artificial metabzyme for tumour-cell-specific metabolic therapy.

Metabolic dysregulation constitutes a pivotal feature of cancer progression. Enzymes with multiple metal active sites play a major role in this process. Here we report the first metabolic-enzyme-like FeMoO4 nanocatalyst, dubbed 'artificial metabzyme'. It showcases dual active centres, namely, Fe2+ and tetrahedral Mo4+, that mirror the characteristic architecture of the archetypal metabolic enzyme xanthine oxidoreductase. Employing spatially dynamic metabolomics in conjunction with the assessments of tumour-associated metabolites, we demonstrate that FeMoO4 metabzyme catalyses the metabolic conversion of tumour-abundant xanthine into uric acid. Subsequent metabolic adjustments orchestrate crosstalk with immune cells, suggesting a potential therapeutic pathway for cancer. Our study introduces an innovative paradigm in cancer therapy, where tumour cells are metabolically reprogrammed to autonomously modulate and directly interface with immune cells through the intervention of an artificial metabzyme, for tumour-cell-specific metabolic therapy.

Electrochemical Biosensor for Point-of-Care Testing of Low-Abundance Biomarkers of Neurological Diseases.

The neurofilament protein light chain (NEFL) is a potential biomarker of neurodegenerative diseases, and interleukin-6 (IL-6) is also closely related to neuroinflammation. Especially, NEFL and IL-6 are the two most low-abundance known protein markers of neurological diseases, making their detection very important for the early diagnosis and prognosis prediction of such kinds of diseases. Nevertheless, quantitative detection of low concentrations of NEFL and IL-6 in serum remains quite difficult, especially in the point-of-care test (POCT). Herein, we developed a portable, sensitive electrochemical biosensor combined with smartphones that can be applied to multiple scenarios for the quantitative detection of NEFL and IL-6, meeting the need of the POCT. We used a double-antibody sandwich configuration combined with polyenzyme-catalyzed signal amplification to improve the sensitivity of the biosensor for the detection of NEFL and IL-6 in sera. We could detect NEFL as low as 5.22 pg/mL and IL-6 as low as 3.69 pg/mL of 6 µL of serum within 2 h, demonstrating that this electrochemical biosensor worked well with serum systems. Results also showed its superior detection capabilities over those of high-sensitivity ELISA for serum samples. Importantly, by detecting NEFL and IL-6 in sera, the biosensor showed its potential for the POCT model detection of all known biomarkers of neurological diseases, making it possible for the mass screening of patients with neurodegenerative diseases.

A DNA origami device spatially controls CD95 signalling to induce immune tolerance in rheumatoid arthritis.

DNA origami is capable of spatially organizing molecules into sophisticated geometric patterns with nanometric precision. Here we describe a reconfigurable, two-dimensional DNA origami with geometrically patterned CD95 ligands that regulates immune cell signalling to alleviate rheumatoid arthritis. In response to pH changes, the device reversibly transforms from a closed to an open configuration, displaying a hexagonal pattern of CD95 ligands with ~10 nm intermolecular spacing, precisely mirroring the spatial arrangement of CD95 receptor clusters on the surface of immune cells. In a collagen-induced arthritis mouse model, DNA origami elicits robust and selective activation of CD95 death-inducing signalling in activated immune cells located in inflamed synovial tissues. Such localized immune tolerance ameliorates joint damage with no noticeable side effects. This device allows for the precise spatial control of cellular signalling, expanding our understanding of ligand-receptor interactions and is a promising platform for the development of pharmacological interventions targeting these interactions.

Enhanced Detection of Novel Low-Frequency Gene Fusions via High-Yield Ligation and Multiplexed Enrichment Sequencing.

Panel-based methods are commonly employed for the analysis of novel gene fusions in precision diagnostics and new drug development in cancer. However, these methods are constrained by limitations in ligation yield and the enrichment of novel gene fusions with low variant allele frequencies. In this study, we conducted a pioneering investigation into the stability of double-stranded adapter DNA, resulting in improved ligation yield and enhanced conversion efficiency. Additionally, we implemented blocker displacement amplification, achieving a remarkable 7-fold enrichment of novel gene fusions. Leveraging the pre-enrichment achieved with this approach, we successfully applied it to Nanopore sequencing, enabling ultra-fast analysis of novel gene fusions within one hour with high sensitivity. This method offers a robust and remarkably sensitive mean of analyzing novel gene fusions, promising the discovery of pivotal biomarkers that can significantly improve cancer diagnostics and the development of new therapeutic strategies.

Upconversion dual-photosensitizer-expressing bacteria for near-infrared monochromatically excitable synergistic phototherapy.

Synergistic phototherapy stands for superior treatment prospects than a single phototherapeutic modality. However, the combined photosensitizers often suffer from incompatible excitation mode, limited irradiation penetration depth, and lack of specificity. We describe the development of upconversion dual-photosensitizer-expressing bacteria (UDPB) for near-infrared monochromatically excitable combination phototherapy. UDPB are prepared by integrating genetic engineering and surface modification, in which bacteria are encoded to simultaneously express photothermal melanin and phototoxic KillerRed protein and the surface primary amino groups are derived to free thiols for biorthogonal conjugation of upconversion nanoparticles. UDPB exhibit a near-infrared monochromatic irradiation-mediated dual-activation characteristic as the photothermal conversion of melanin can be initiated directly, while the photodynamic effect of KillerRed can be stimulated indirectly by upconverted visible light emission. UDPB also show living features to colonize hypoxic lesion sites and inhibit pathogens via bacterial community competition. In two murine models of solid tumor and skin wound infection, UDPB separately induce robust antitumor response and a rapid wound healing effect.

Robotic Actuation-Mediated Quantitative Mechanogenetics for Noninvasive and On-Demand Cancer Therapy.

Cell mechanotransduction signals are important targets for physical therapy. However, current physiotherapy heavily relies on ultrasound, which is generated by high-power equipment or amplified by auxiliary drugs, potentially causing undesired side effects. To address current limitations, a robotic actuation-mediated therapy is developed that utilizes gentle mechanical loads to activate mechanosensitive ion channels. The resulting calcium influx precisely regulated the expression of recombinant tumor suppressor protein and death-associated protein kinase, leading to programmed apoptosis of cancer cell line through caspase-dependent pathway. In stark contrast to traditional gene therapy, the complete elimination of early- and middle-stage tumors (volume ≤ 100 mm3) and significant growth inhibition of late-stage tumor (500 mm3) are realized in tumor-bearing mice by transfecting mechanogenetic circuits and treating daily with quantitative robotic actuation in a form of 5 min treatment over the course of 14 days. Thus, this massage-derived therapy represents a quantitative strategy for cancer treatment.

Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

Programmed Remodeling of the Tumor Milieu to Enhance NK Cell Immunotherapy Combined with Chemotherapy for Pancreatic Cancer.

Natural killer (NK) cell-based adoptive immunotherapy has demonstrated encouraging therapeutic effects in clinical trials for hematological cancers. However, the effectiveness of treatment for solid tumors remains a challenge due to insufficient recruitment and infiltration of NK cells into tumor tissues. Herein, a programmed nanoremodeler (DAS@P/H/pp) is designed to remodel dense physical stromal barriers and for dysregulation of the chemokine of the tumor environment to enhance the recruitment and infiltration of NK cells in tumors. The DAS@P/H/pp is triggered by the acidic tumor environment, resulting in charge reversal and subsequent hyaluronidase (HAase) release. HAase effectively degrades the extracellular matrix, promoting the delivery of immunoregulatory molecules and chemotherapy drugs into deep tumor tissues. In mouse models of pancreatic cancer, this nanomediated strategy for the programmed remodeling of the tumor microenvironment significantly boosts the recruitment of NK92 cells and their tumor cell-killing capabilities under the supervision of multiplexed near-infrared-II fluorescence.

DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance.

Biomarker discovery is essential for the understanding, diagnosis, targeted therapy and prognosis assessment of malignant diseases. However, it remains a huge challenge due to the lack of sensitive methods to identify disease-specific rare molecules. Here we present MORAC, molecular recognition based on affinity and catalysis, which enables the effective identification of candidate biomarkers with low abundance. MORAC relies on a class of DNAzymes, each cleaving a sole RNA linkage embedded in their DNA chain upon specifically sensing a complex system with no prior knowledge of the system's molecular content. We show that signal amplification from catalysis ensures the DNAzymes high sensitivity (for target probing); meanwhile, a simple RNA-to-DNA mutation can shut down their RNA cleavage ability and turn them into a pure affinity tool (for target pulldown). Using MORAC, we identify previously unknown, low-abundance candidate biomarkers with clear clinical value, including apolipoprotein L6 in breast cancer and seryl-tRNA synthetase 1 in polyps preceding colon cancer.

Atomically precise photothermal nanomachines.

Interfacing molecular machines to inorganic nanoparticles can, in principle, lead to hybrid nanomachines with extended functions. Here we demonstrate a ligand engineering approach to develop atomically precise hybrid nanomachines by interfacing gold nanoclusters with tetraphenylethylene molecular rotors. When gold nanoclusters are irradiated with near-infrared light, the rotation of surface-decorated tetraphenylethylene moieties actively dissipates the absorbed energy to sustain the photothermal nanomachine with an intact structure and steady efficiency. Solid-state nuclear magnetic resonance and femtosecond transient absorption spectroscopy reveal that the photogenerated hot electrons are rapidly cooled down within picoseconds via electron-phonon coupling in the nanomachine. We find that the nanomachine remains structurally and functionally intact in mammalian cells and in vivo. A single dose of near-infrared irradiation can effectively ablate tumours without recurrence in tumour-bearing mice, which shows promise in the development of nanomachine-based theranostics.

Programmable DNA Hydrogel Assisting Microcrystal Formulations for Sustained Locoregional Drug Delivery in Surgical Residual Tumor Lesions and Lymph Node Metastasis.

Surgical residual tumor lesions (R1 resection of surgical procedures (e.g., liver cancer infiltrating the diaphragm, surgical residual breast cancer, postoperative residual ovarian cancer) or boundary residual after ablation) and lymph node metastasis that cannot be surgically resected (retroperitoneal lymph nodes) significantly affect postoperative survival of tumor patients. This clinical conundrum poses three challenges for local drug delivery systems: stable and continuous delivery, good biocompatibility, and the ability to package new targeted drugs that can synergize with other treatments. Here, a drug-laden hydrogel generated from pure DNA strands and highly programmable in adjusting its mesh size is reported. Meanwhile, the DNA hydrogel can assist the microcrystallization of novel radiosensitizing drugs, ataxia telangiectasia and rad3-related protein (ATR) inhibitor (Elimusertib), further facilitating its long-term release. When applied to the tumor site, the hydrogel system demonstrates significant antitumor activity, minimized systemic toxicity, and has a modulatory effect on the tumor-immune cell interface. This drug-loaded DNA-hydrogel platform represents a novel modality for adjuvant therapy in patients with surgical residual tumor lesions and lymph node metastasis.

Triggerable Prodrug Nanocoating Enables On-Demand Activation of Microbial and Small-Molecular Therapeutics for Combination Treatment.

The synergy of living microbial and small-molecular therapeutics has been widely explored for treating a variety of diseases, while current combination strategies often suffer from low bioavailability, heterogeneous spatiotemporal distribution, and premature drug release. Here, the use of a triggerable prodrug nanocoating is reported to enable the on-demand activation of microbial and small-molecular therapeutics for combination treatment. As a proof-of-concept study, a reactive oxygen species-responsive aromatic thioacetal linker is employed to prepare cationic chitosan-drug conjugates, which can form a nanocoating on the surface of living bacteria via electrostatic interaction. Following administration, the wrapped bacteria can be prevented from in vivo insults by the shielding effect of the nanocoating and be co-delivered with the conjugated drug in a spatiotemporally synchronous manner. Upon reaching the lesion site, the upgraded reactive oxygen species trigger in situ cleavage of the thioacetal linker, resulting in the release of the conjugated drug and a linker-derived therapeutic cinnamaldehyde. Meanwhile, a charge reversal achieved by the generation of negatively charged thiolated chitosan induces the dissociation of the nanocoating, leading to synchronous release of the living bacteria. The adequate activation of the combined therapeutics at the lesion site exhibits superior synergistic treatment efficacy, as demonstrated by an in vivo assessment using a mouse model of colitis. This work presents an appealing approach to combine living microbial and small-molecular therapeutics for advanced therapy of diseases.

Reshaping Intratumoral Mononuclear Phagocytes with Antibody-Opsonized Immunometabolic Nanoparticles.

Mononuclear phagocytes (MPs) are vital components of host immune defenses against cancer. However, tumor-infiltrating MPs often present tolerogenic and pro-tumorigenic phenotypes via metabolic switching triggered by excessive lipid accumulation in solid tumors. Inspired by viral infection-mediated MP modulation, here enveloped immunometabolic nanoparticles (immeNPs) are designed to co-deliver a viral RNA analog and a fatty acid oxidation regulator for synergistic reshaping of intratumoral MPs. These immeNPs are camouflaged with cancer cell membranes for tumor homing and opsonized with anti-CD163 antibodies for specific MP recognition and uptake. It is found that internalized immeNPs coordinate lipid metabolic reprogramming with innate immune stimulation, inducing M2-to-M1 macrophage repolarization and tolerogenic-to-immunogenic dendritic cell differentiation for cytotoxic T cell infiltration. The authors further demonstrate that the use of immeNPs confers susceptibility to anti-PD-1 therapy in immune checkpoint blockade-resistant breast and ovarian tumors, and thereby provide a promising strategy to expand the potential of conventional immunotherapy.

Stimuli-Responsive Hydrogel Microcapsules Harnessing the COVID-19 Immune Response for Cancer Therapeutics.

The combination of gene therapy and immunotherapy concepts, along recent advances in DNA nanotechnology, have the potential to provide important tools for cancer therapies. We present the development of stimuli-responsive microcapsules, loaded with a viral immunogenetic agent, harnessing the immune response against the Coronavirus Disease 2019, COVID-19, to selectively attack liver cancer cells (hepatoma) or recognize breast cancer or hepatoma, by expression of green fluorescence protein, GFP. The pH-responsive microcapsules, modified with DNA-tetrahedra nanostructures, increased hepatoma permeation by 50 %. Incorporation of a GFP-encoding lentivirus vector inside the tumor-targeting pH-stimulated miRNA-triggered and Alpha-fetoprotein-dictated microcapsules enables the demonstration of neoplasm selectivity, with approximately 5,000-, 8,000- and 50,000-fold more expression in the cancerous cells, respectively. The incorporation of the SARS-CoV-2 spike protein in the gene vector promotes specific recognition of the immune-evading hepatoma by the COVID-19-analogous immune response, which leads to cytotoxic and inflammatory activity, mediated by serum components taken from vaccinated or recovered COVID-19 patients, resulting in effective elimination of the hepatoma (>85 % yield).

Inhalable pH-responsive DNA tetrahedron nanoplatform for boosting anti-tumor immune responses against metastatic lung cancer.

Despite advancements in the treatment of pulmonary cancer, the existence of mucosal barriers in lung still hampered the penetration and diffusion of therapeutic agents and greatly limited the therapeutic benefits. In this work, we reported a novel inhalable pH-responsive tetrahedral DNA nanomachines with simultaneous delivery of immunomodulatory CpG oligonucleotide and PD-L1-targeting antagonistic DNA aptamer (CP@TDN) for efficient treatment of pulmonary metastatic cancer. By precisely controlling the ratios of CpG and PD-L1 aptamer, the obtained CP@TDN could specifically release PD-L1 aptamer to block PD-1/PD-L1 immune checkpoint axis in acidic tumor microenvironment, followed by endocytosis by antigen-presenting cells to generate anti-tumor immune activation and secretion of anti-tumor cytokines. Moreover, inhalation delivery of CP@TDN showed highly-efficient lung deposition with greatly enhanced intratumoral accumulation, ascribing to the DNA tetrahedron-mediated penetration of pulmonary mucosa. Resultantly, CP@TDN could significantly inhibit the growth of metastatic orthotopic lung tumors via the induction of robust antitumor responses. Therefore, our work presents an attractive approach by virtue of biocompatible DNA tetrahedron as the inhalation delivery system for effective treatment of metastatic lung cancer.

Edge Length-Programmed Single-Stranded RNA Origami for Predictive Innate Immune Activation and Therapy.

Ligands targeting nucleic acid-sensing receptors activate the innate immune system and play a critical role in antiviral and antitumoral therapy. However, ligand design for in situ stability, targeted delivery, and predictive immunogenicity is largely hampered by the sophisticated mechanism of the nucleic acid-sensing process. Here, we utilize single-stranded RNA (ssRNA) origami with precise structural designability as nucleic acid sensor-based ligands to achieve improved biostability, organelle-level targeting, and predictive immunogenicity. The natural ssRNAs self-fold into compact nanoparticles with defined shapes and morphologies and exhibit resistance against RNase digestion in vitro and prolonged retention in macrophage endolysosomes. We find that programming the edge length of ssRNA origami can precisely regulate the degree of macrophage activation via a toll-like receptor-dependent pathway. Further, we demonstrate that the ssRNA origami-based ligand elicits an anti-tumoral immune response of macrophages and neutrophils in the tumor microenvironment and retards tumor growth in the mouse pancreatic tumor model. Our ssRNA origami strategy utilizes structured RNA ligands to achieve predictive immune activation, providing a new solution for nucleic acid sensor-based ligand design and biomedical applications.

Single-Stranded RNA Origami-Based Epigenetic Immunomodulation.

The integration of functional modules at the molecular level into RNA nanostructures holds great potential for expanding their applications. However, the quantitative integration of nucleoside analogue molecules into RNA nanostructures and their impact on the structure and function of RNA nanostructures remain largely unexplored. Here, we report a transcription-based approach to controllably integrate multiple nucleoside analogues into a 2000 nucleotide (nt) single-stranded RNA (ssRNA) origami nanostructure. The resulting integrated ssRNA origami preserves the morphology and biostability of the original ssRNA origami. Moreover, the integration of nucleoside analogues introduced new biomedical functions to ssRNA origamis, including innate immune recognition and regulation after the precise integration of epigenetic nucleoside analogues and synergistic effects on tumor cell killing after integration of therapeutic nucleoside analogues. This study provides a promising approach for the quantitative integration of functional nucleoside analogues into RNA nanostructures at the molecular level, thereby offering valuable insights for the development of multifunctional ssRNA origamis.

Targeted Reprogramming of Vitamin B3 Metabolism as a Nanotherapeutic Strategy towards Chemoresistant Cancers.

Cancer-associated fibroblasts (CAFs) promote cancer stem cell (CSC)-mediated chemoresistance and immunosuppressive tumor microenvironment. However, direct depletion of CAFs may increase cancer invasiveness and metastasis. As a generalized strategy against chemoresistant cancers, Gemini-like homotypic targeting nanoparticles (NPs) are designed for two-pronged CAF transformation and cancer cell elimination. The CAF-targeted NPs couple vitamin B3 metabolic reprogramming to epigenetic modulation of secreted pro-stemness and immunosuppressive factors, thereby diminishing CSC and suppressive immune cell populations to enhance cancer cell drug susceptibility and cytotoxic T cell infiltration. In mouse models of breast, liver, pancreatic and colorectal cancers that are resistant to their respective first-line chemotherapeutics, a single dose of hydrogel co-delivering the Gemini-like NPs can rehabilitate chemosensitivity, induce immune activation, and achieve tumor regression. Moreover, it stimulates robust T cell memory for long-term protection against tumor rechallenge. This study thus represents an innovative approach with broad applicability for overcoming cancer chemoresistance.

Slimming and Reinvigorating Tumor-Associated Dendritic Cells with Hierarchical Lipid Rewiring Nanoparticles.

Dendritic cells (DCs) are crucial mediators of innate and adaptive antitumor immunity, whereas exogenously and endogenously driven lipid accumulation causes immune tolerance of tumor-associated DCs (TADCs) and thereby diminishes tumor responsiveness to various therapies. Herein, a type of multilevel lipid rewiring nanoparticles (NPs) for TADC revitalization is designed. These self-assembled NPs specifically bind to the lipid transport receptor Msr1 on the TADC surface and orchestrate the restriction of extracellular lipid uptake, cytoplasmic de novo lipid biosynthesis and nuclear lipogenic gene transcription. It is found that the slimming of TADCs via the three-in-one lipid metabolic reprogramming substantially promotes their maturation and rehabilitate their functions in inflammatory cytokine production, cytotoxic T cell recruitment, and tumor inhibition. Significantly, tumor resistance to immune checkpoint blockade therapy is further overcome. The study presents a non-canonical strategy to remodel tumor-infiltrating immune cells and paves a new path for improving the efficacy of cancer immunotherapy.

Precise Epitope Organization with Self-adjuvant Framework Nucleic Acid for Efficient COVID-19 Peptide Vaccine Construction.

Peptide vaccines have advantages in easy fabrication and high safety, but their effectiveness is hampered by the poor immunogenicity of the epitopes themselves. Herein, we constructed a series of framework nucleic acids (FNAs) with regulated rigidity and size to precisely organize epitopes in order to reveal the influence of epitope spacing and carrier rigidity on the efficiency of peptide vaccines. We found that assembling epitopes on rigid tetrahedral FNAs (tFNAs) with the appropriate size could efficiently enhance their immunogenicity. Further, by integrating epitopes from SARS-CoV-2 on preferred tFNAs, we constructed a COVID-19 peptide vaccine which could induce high titers of IgG against the receptor binding domain (RBD) of SARS-CoV-2 spike protein and increase the ratio of memory B and T cells in mice. Considering the good biocompatibility of tFNAs, our research provides a new idea for developing efficient peptide vaccines against viruses and possibly other diseases.