A Guanidinobenzol-Rich Polymer Overcoming Cascade Delivery Barriers for CRISPR-Cas9 Genome Editing.

The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.

Engineered Bio-Based Hydrogels for Cancer Immunotherapy.

Immunotherapy represents a revolutionary paradigm in cancer management, showcasing its potential to impede tumor metastasis and recurrence. Nonetheless, challenges including limited therapeutic efficacy and severe immune-related side effects are frequently encountered, especially in solid tumors. Hydrogels, a class of versatile materials featuring well-hydrated structures widely used in biomedicine, offer a promising platform for encapsulating and releasing small molecule drugs, biomacromolecules, and cells in a controlled manner. Immunomodulatory hydrogels present a unique capability for augmenting immune activation and mitigating systemic toxicity through encapsulation of multiple components and localized administration. Notably, hydrogels based on biopolymers have gained significant interest owing to their biocompatibility, environmental friendliness, and ease of production. This review delves into the recent advances in bio-based hydrogels in cancer immunotherapy and synergistic combinatorial approaches, highlighting their diverse applications. It is anticipated that this review will guide the rational design of hydrogels in the field of cancer immunotherapy, fostering clinical translation and ultimately benefiting patients.

Tumor Microenvironment-Responsive Nanoparticles Amplifying STING Signaling Pathway for Cancer Immunotherapy.

Insufficient activation of the stimulator of interferon genes (STING) signaling pathway and profoundly immunosuppressive microenvironment largely limits the effect of cancer immunotherapy. Herein, tumor microenvironment (TME)-responsive nanoparticles (PMM NPs) are exploited that simultaneously harness STING and Toll-like receptor 4 (TLR4) to augment STING activation via TLR4-mediated nuclear factor-kappa B signaling pathway stimulation, leading to the increased secretion of type I interferons (i.e., 4.0-fold enhancement of IFN-ß) and pro-inflammatory cytokines to promote a specific T cell immune response. Moreover, PMM NPs relieve the immunosuppression of the TME by decreasing the percentage of regulatory T cells, and polarizing M2 macrophages to the M1 type, thus creating an immune-supportive TME to unleash a cascade adaptive immune response. Combined with an anti-PD-1 antibody, synergistic efficacy is achieved in both inflamed colorectal cancer and noninflamed metastatic breast tumor models. Moreover, rechallenging tumor-free animals with homotypic cells induced complete tumor rejection, indicating the generation of systemic antitumor memory. These TME-responsive nanoparticles may open a new avenue to achieve the spatiotemporal orchestration of STING activation, providing a promising clinical candidate for next-generation cancer immunotherapy.

Manganese-Enriched Zinc Peroxide Functional Nanoparticles for Potentiating Cancer Immunotherapy.

Immunotherapies have shown high clinical success, however, the therapeutical efficacy is largely restrained by insufficient immune activation and an immunosuppressive microenvironment. Herein, we report tumor microenvironment (TME)-responsive manganese-enriched zinc peroxide nanoparticles (MONPs) for synergistic cancer immunotherapy by inducing the immunogenic death (ICD) of cancer cells and activating the stimulator of the interferon gene (STING) pathway. MONPs especially disassociate upon exposure to acidic tumor tissue and in situ generate •OH for the ICD effect. Moreover, Mn2+ activated the STING and synergistically induced the secretion of type I interferon and inflammatory cytokines for specific T cell responses. Meanwhile, MONPs relieved the immunosuppression of TME through decreasing Tregs and polarizing M2 macrophages to the M1 type to unleash a cascade adaptive immune response. In combination with the anti-PD-1 antibody, MONPs showed superior efficacy in inhibiting tumor growth and preventing lung metastasis. Our study demonstrates the feasibility of functional nanoparticles to amplify STING innate stimulation, showing a prominent strategy for cancer immunotherapy.

Imaging Guided Endogenic H2 -Augmented Electrochemo-Sonodynamic Domino Co-therapy of Tumor in Vivo.

Precise and on-demand release of sufficient hydrogen (H2 ) to tumor sites remains a key challenge for emerging H2 -oncotherapy, and little is known about the physiological effects of "abundant" H2 on complex tumor microenvironments (TME). Here, a highly efficient and cost-effective imaging-guided/-assessed H2 -therapy of tumors based on a joint electrochemo-sonodynamic treatment (H2 -EC/SD co-therapy) with strong "domino effect" triggered by endogenous H2 generation at tumor sites is reported to speedily eliminate tumor tissue (≤80 mm3 ) within 1 day. Adequate H2 is controllably generated in tumor sites through mild electrochemistry in vivo due to acidic TME by using clinical acupuncture Fe needles as electrodes. Besides starvation damage due to gas blockage/destruction of vessels, nano-/micro-bubbles of H2 formed in situ can elevate the tumor's internal temperature and burst vessels to further destroy the tumor under ultrasound irradiation. Remarkably, vulnerable homeostasis of TME is disturbed as H2 also participates in the physiological activity of tumor cells, leading to tumor dysfunction. Last but not least, the body's inflammatory response to cancer is reduced after the treatment, which is beneficial for the body's immune system during post-treatment recovery. Based on all of these merits, the H2 -EC/SD co-therapy provides a potentially safe and viable therapeutic strategy for future clinical applications.

Engineered Nanoprobes for Immune Activation Monitoring.

The activation of the immune system is critical for cancer immunotherapy and treatments of inflammatory diseases. Non-invasive visualization of immunoactivation is designed to monitor the dynamic nature of the immune response and facilitate the assessment of therapeutic outcomes, which, however, remains challenging. Conventional imaging modalities, such as positron emission tomography, computed tomography, etc., were utilized for imaging immune-related biomarkers. To explore the dynamic immune monitoring, probes with signals correlated to biomarkers of immune activation or prognosis are urgently needed. These emerging molecular probes, which turn on the signal only in the presence of the intended biomarker, can improve the detection specificity. These probes with "turn on" signals enable non-invasive, dynamic, and real-time imaging with high sensitivity and efficiency, showing significance for multifunctionality/multimodality imaging. As a result, more and more innovative engineered nanoprobes combined with diverse imaging modalities were developed to assess the activation of the immune system. In this work, we comprehensively review the recent and emerging advances in engineered nanoprobes for monitoring immune activation in cancer or other immune-mediated inflammatory diseases and discuss the potential in predicting the efficacy following treatments. Research on real-time in vivo immunoimaging is still under exploration, and this review can provide guidance and facilitate the development and application of next-generation imaging technologies.

Optoplasmonic Modulation of Cell Metabolic State Promotes Rapid Cell Differentiation.

Cell differentiation plays a vital role in mediating organ formation and tissue repair and regeneration. Although rapid and effective methods to stimulate cell differentiation for clinical purposes are highly desired, it remains a great challenge in the medical fields. Herein, a highly effective and conceptual optical method was developed based on a plasmonic chip platform (made of 2D AuNPs nanomembranes). through effective light-augmented plasmonic regulation of cellular bioenergetics (CBE) and an entropy effect at bionano interfaces, to promote rapid cell differentiation. Compared with traditional methods, the developed optoplasmonic method greatly shortens cell differentiation time from usually more than 10 days to only about 3 days. Upon the optoplasmonic treatment of cells, the conformational and vibration entropy changes of cell membranes were clearly revealed through theoretical simulation and fingerprint spectra of cell membranes. Meanwhile, during the treatment process, bioenergetics levels of cells were elevated with increasing mitochondrial membrane potential (Δψm), which accelerates cell differentiation and proliferation. The developed optoplasmonic method is highly efficient and easy to implement, provides a new perspective and avenue for cell differentiation and proliferation, and has potential application prospects in accelerating tissue repair and regeneration.

Photoactivated Bacteriorhodopsin/SiNx Nanopore-Based Biological Nanofluidic Generator with Single-Protein Sensitivity.

Nanofluidics is an emerging hot field that explores the unusual behaviors of ions/molecules transporting through nanoscale channels, which possesses a broad application prospect. However, in situ probing bioactivity of functional proteins on a single-molecule level by a nanofluidic device has not been reported, and it is still a big challenge in the field. Herein, we reported a biological nanofluidic device with a single-protein sensitivity, based on natural proton-pumping protein, bacteriorhodopsin (bR), and a single SiNx nanopore. Nanofluidic single-molecule probing of bR proton-pumping activity and its light response were achieved under applied voltage of 0 V, by biologically self-powered steady-state ionic current nanopore sensing. Green-light irradiation of the device led to the monitoring of a steady-state proton current of ∼3.51 pA/per bR trimer, corresponding to charge density of 815 µC/cm2 generated by each bR monomer, which far exceeded the previously reported value of 1.4 µC/cm2. This finding and method would promote the development of artificial biological and hybrid nanofluidic devices in biosensing and energy conversion applications.

Smart Tumor Microenvironment-Responsive Nano-Prodrug for Disulfiram Toxification In Situ and the Exploration of Lethal Mechanisms in Cells.

Disulfiram (DSF) is a clinical antialcoholism drug that has been confirmed to show anticancer bioactivity after chelating with Cu2+. Therefore, how to co-deliver DSF and Cu2+ to tumor tissues and generate a smart response to the tumor microenvironment (TME) are the focus of repurposing DSF for the effective treatment of cancer. Herein, we fabricated facilely a smart nanosystem by coating tannic acid (TA) and Cu2+ network on DSF, denoted as DSF@TA-Cu, which responses well to TME and forms CuET complex in situ. In such a way, besides the chemotherapy effect of CuET, the anticancer efficacy of the resulting nano-prodrug can further be augmented by a continuous Fenton-like reaction. We then tested the cytotoxicity DSF@TA-Cu with normal and cancerous cell lines. Finally, by constructing mitochondria-targeted nanoprobes, we monitored the changes in mitochondrial metabolism and explored the lethal mechanisms in A549 cells. We found that DSF@TA-Cu showed higher toxicity to cancerous cells. By analyzing the fluorescence images and surface-enhanced Raman scattering (SERS) spectra of mitochondria, we found that the DNA damage and the decrease in mitochondrial membrane potential (MMP) were closely related to the generation and accumulation of reactive oxygen species (ROS). Although activated related pathways try to counteract the effects of elevation of ROS, excessive ROS inevitably leads to apoptosis of cancer cells.

Label-Free Analysis of Cell Membrane Proteins via Evanescent Field Excited Surface-Enhanced Raman Scattering.

Challenges in studying the structures and functions of cell membrane proteins lie in their lipophilicity, which makes them hard to be stabilized, crystallized, and expressed by E. coli. Herein, we propose an evanescent field excited surface-enhanced Raman scattering (EF-SERS) strategy for label-free analysis of membrane proteins in situ. Extracted cell membranes tightly wrapped the metal nanoparticles by an extruder, which ensures the SERS signals of the membrane proteins precisely benefit from the localized surface plasmons (LSPs). The leaky mode of a waveguide was employed to improve the plasmon excitation coupling. Thus, the LSPs and waveguide modes together enable the achievement of high-quality SERS profiles of membrane proteins. By spectral analysis, the structural changes of membrane proteins can be deeply understood at the molecular level. This method has broader applicability in establishing the Ramanomics of membrane proteins and unraveling the exact changes of membrane proteins during physiological processes.

Wet-Chemical Electro-Plasmonic Modulation of Metasurfaced Cell-Electrode Interfaces for Effective and Selective Entropic Killing of Cancer Cells.

Surface plasmons (SPs) of metallic nanostructures excited by optical ways have been extensively utilized for versatile sensing, biomedical, catalysis, and energy conversion applications. Nevertheless, utilizing the electrically excited plasmonic field (effect) of metallic nanostructures (and electrodes) in wet-chemical conditions, for catalytic and energy conversion, especially for potential biological and biomedical applications, is still poorly studied. Herein, we report a conceptual and biocompatible wet-chemical platform and approach to utilize the electrically excited plasmonic field (effect) of metasurfaced plasmonic electrodes (without light irradiation) for cell fate regulation on electrode surfaces. By using self-assembled two-dimensional (2D) ordered-plasmonic AuNP- or Au@SiO2 NP-nanomembrane as a metasurfaced electrode, the cancer cells cultured on it can be selectively and effectively killed (due to the enhanced stimulus current and related entropic effects) via wet-chemical electro-plasmonic modulation (WC-EPM) of the cell-electrode interfaces. Biological conformational and configurational entropic change information from the cell membrane during the WC-EPM of the cell-electrode interface has also been revealed by label-free in situ surface-enhanced Raman spectroscopy. The developed approach and results can be guides for the WC-EPM regulation of biological interfaces to achieve cell fate regulation and disease treatment and is also constructive for the design of 2D plasmonic nanomaterials and devices for efficient electrochemical energy conservation and biomedical applications.

Plasmon-enhanced unidirectional charge transfer for efficient solar water oxidation.

Precise modulation and nano-engineering of photoelectrochemical (PEC) materials, with high-speed charge separation efficiency and broad spectral response, are of significant importance in improving the PEC catalytic activities. Herein, by rational design of material structures, 3D-coaxial plasmonic hetero-nanostructures (carbon cloth@TiO2@SrTiO3-Au, CC@TiO2@SrTiO3-Au) are tactfully fabricated, which exhibit superior solar energy conversion efficiency in PEC water splitting with a current density reaching up to 23.56 mA cm-2 (1.23 V vs. RHE). More specific research and in-depth simulations reveal that the enhanced PEC performance should be attributed to the high-speed charge transfer channels of CC@TiO2@SrTiO3 and excellent light utilization ability stemming from the surface plasmon resonance and strong light-scattering of the 3D-coaxial frameworks. This study provides new strategies for the design of plasmon-enhanced PEC nanocatalysts and will benefit the development of photoelectric energy conversion.

Label-Free Single-Particle Surface-Enhanced Raman Spectroscopy Detection of Phosphatidylserine Externalization on Cell Membranes with Multifunctional Micron-Nano Composite Probes.

Monitoring externalization of phosphatidylserine (PS) and gaining insights into molecular events of cell membrane damage are significant for programmed cell death studies. Herein, by encapsulating zeolitic imidazole frameworks-8 with plasmonic gold nanoparticles to form micron-nano composites and using them as a single-particle surface-enhanced Raman spectroscopy (SERS) substrate, we succeeded in real-time discriminating and monitoring the externalization of PS on cell membranes during electrostimulus-induced apoptosis. The micron-nano composite probe provides rich "hot spots" and robust anchoring capacity for cell membranes, achieving the capture and label-free single-particle SERS detection of the externalized PS. By this method, the dynamic PS externalization process differences between cancerous cells and normal cells were clearly revealed of which the cell membrane damage of cancerous cells was more serious than that of normal cells. This method is versatile and robust for monitoring the externalization of PS and uncovering related cell membrane damage mechanisms. This work also broadens the application of metal-organic framework materials for advanced biomedical applications.

Plasmonic SERS Au Nanosunflowers for Sensitive and Label-Free Diagnosis of DNA Base Damage in Stimulus-Induced Cell Apoptosis.

Molecular diagnosis and accurate damage analysis of complex genomic DNAs in tumor cells are crucial to the theranostics of cancers but still a huge challenge. Herein, by designed preparation of a uniform plasmonic sunflower-like assembly gold (Au) nanostructure that is capable of efficient DNA capture and providing high-density gap-plasmon "hot spots" for adequate surface-enhanced Raman spectroscopy (SERS) enhancement, we succeeded in sensitive and reliable label-free SERS detection of DNA damage in electrostimulus-induced apoptotic cancer cells at the DNA base level for the first time. The SERS results showed that the external electrostimulus (at 1.2 V, for 5 min) was almost harmless to normal healthy cells, but it caused pronounced double strand break and adenine base damage in cancer cell DNAs, which effectively destroyed the reproduction and transcription of DNAs and ultimately induced cell apoptosis. The developed sensing platform and method are promising for cell study of genetically related diseases.

Tumor Microenvironment-Activated Degradable Multifunctional Nanoreactor for Synergistic Cancer Therapy and Glucose SERS Feedback.

Integration of disease diagnosis and therapy in vivo by nanotechnology is a challenge in the design of multifunctional nanocarriers. Herein, we report an intelligent and degradable nanoreactor, an assembly of the 4-mercaptobenzonitrile-decorated silver nanoparticles (AgNPs@MBN) and the glucose oxidase (GOx)-loaded metal-organic-framework (ZIF-8@GOx), which can be activated by tumor microenvironment to start the catalytic cascade-enhanced chemo-starvation synergistic therapy and simultaneous self-sense of cellular glucose level. Under the mild acidic microenvironment of tumor, the nanoreactor will collapse to release GOx that triggers a catalytic cascade reaction in vivo, depleting glucose, etching AgNPs@MBN, and producing toxic H2O2, Ag+, and Zn2+ ions, all of which work together to inhibit tumor growth. The AgNPs@MBN as SERS nanoprobe reads out glucose concentration noninvasively in tumor to achieve instant feedback of therapeutic progression. This work proposes a promising example of using enzyme-encapsulated biomineralized MOFs as an effective anticarcinogen for clinical applications.

Fast Activation and Tracing of Caspase-3 Involved Cell Apoptosis by Combined Electrostimulation and Smart Signal-Amplified SERS Nanoprobes.

Caspase-3 is considered as one of the key proteases that can spontaneously regulate the life activities of cells, and its activation (usually is a slow process) will execute the apoptosis process of cells. Rapid activation of caspase-3 on demand in living-cells is therefore highly desired toward precise cancer therapy but it is still a key challenge. Herein, we applied electrostimulus (ES) to achieve fast activation of caspase-3 and trigger cell apoptosis, and developed a smart magnetic-plasmonic assembly nanoprobes (A-nanoprobes) to real-time trace cellular caspase-3 activation at the single cell level. The designer core-satellite A-nanoprobe, working specific to the activated caspase-3 via a disassembly tactic, provides strong "hot spots" to improve the sensitivity and therefore enables SERS sensing of cellular caspase-3 upon activated by ES. Single-cell analysis revealed that the ES can rapidly activate the apoptosis pathway of caspase-3 on demand to make the DNA fragmentation and ultimately induce the cell apoptosis. Such method and nanoplatform were further used to monitor ES-triggered caspase-3 activation in cell apoptosis process of different cell types, revealing that more caspase-3 will be activated for cancerous cells than normal cells during the ES to induce cells apoptosis. This strategy and platform are promising for detecting cellular caspase-3 and other enzymes in the process of cancer diagnosis and treatments.