Securing LYTAC with Logic-Identification System for Cancer Cell-Selective Membrane Protein Degradation.

Lysosome-targeting chimera (LYTAC) links proteins of interest (POIs) with lysosome-targeting receptors (LTRs) to achieve membrane protein degradation, which is becoming a promising therapeutic modality. However, cancer cell-selective membrane protein degradation remains a big challenge considering expressions of POIs in both cancer cells and normal cells, as well as broad tissue distribution of LTRs. Here a logic-identification system is designed, termed Logic-TAC, based on cell membrane-guided DNA calculations to secure LYTAC selectively for cancer cells. Logic-TAC is designed as a duplex DNA structure, with both POI and LTR recognition regions sealed to avoid systematic toxicity during administration. MCF-7 and MCF-10A are chosen as sample cancer cell and normal cell respectively. As input 1 for logic-identification, membrane proteins EpCAM, which is highly expressed by MCF-7 but barely by MCF-10A, reacts with Logic-TAC to expose POI recognition region. As input 2 for logic-identification, Logic-TAC binds to POI, membrane protein MUC1, to expose LTR recognition region. As output, MUC1 is connected to LTR and degraded via lysosome pathway selectively for cancer cell MCF-7 with little side effect on normal cell MCF-10A. The logic-identification system also demonstrated satisfactory in vivo therapeutic results, indicating its promising potential in precise targeted therapy.

Cancer Cell-Selective PD-L1 Inhibition via a DNA Safety Catch to Enhance Immunotherapy Specificity.

Immune checkpoint protein blockade (ICB) has emerged as a powerful immunotherapy approach, but suppressing immune-related adverse events (irAEs) for noncancerous cells and normal tissues remains challenging. Activatable ICB has been developed with tumor microenvironment highly-expressed molecules as stimuli, but they still lack precision and efficiency considering the diffusion of stimuli molecules in whole tumor tissue. Here we assemble PD-L1 with a duplex DNA strand, termed as "safety catch", to regulate its accessibility for ICB. The safety catch remains at "on" status for noncancerous cells to prevent ICB binding to PD-L1. Cancer cell membrane protein c-Met acts as a trigger protein to react with safety catch, which selectively exposes its hybridization region for ICB reagent. The ICB reagent is a retractable DNA nanostring with repeating hairpin-structural units, whose contraction drives PD-L1 clustering with endocytosis-guided degradation. The safety catch, even remained at "safety on" status, is removed from the cell membrane via a DNA strand displacement reaction to minimize its influence on noncancerous cells. This strategy demonstrates selective and potent immunotherapeutic capabilities only against cancer cells both in vitro and in vivo, and shows effective suppression of irAEs in normal tissues, therefore would become a promising approach for precise immunotherapy in mice.

Cancer Cell-Selective Membrane Receptor Clustering Driven by VEGF Secretion for In Vivo Therapy.

Clustering of cell membrane receptors regulates cell behaviors. Although receptor clustering plans have achieved wide applications in cancer therapy, it still remains challenging to manipulate receptor clustering selectively for cancer cells with little influence on normal cells. Here, we design a Raji cell Selective MAnipulation of Receptor Clustering (SMARC) strategy for CD20, which is driven by endogenous secretion of Raji cells. Retractable DNA nanostrings with repeating hairpin-structured units are anchored to the cell membrane CD20, which contract in response to Raji cell-secreted vascular endothelial growth factor (VEGF) with corresponding CD20 clustering. The contraction of DNA nanostrings is intensified via a VEGF amplifier including DNA cyclic reactions to continuously trigger the foldings of hairpin-structured units in DNA nanostrings. The SMARC strategy shows selective and efficient apoptosis of Raji cells with little interference to normal B cells and demonstrates good in vivo therapeutic efficacy, which provides a promising tool for precise cancer therapy.

A NIR Programmable In Vivo miRNA Magnifier for NIR-II Imaging of Early Stage Cancer.

Aberrant expressions of biomolecules occur much earlier than tumor visualized size and morphology change, but their common measurement strategies such as biopsy suffer from invasive sampling process. In vivo imaging of slight biomolecule expression difference is urgently needed for early cancer detection. Fluorescence of rare earth nanoparticles (RENPs) in second near-infrared (NIR-II) region makes them appropriate tool for in vivo imaging. However, the incapacity to couple with signal amplification strategies, especially programmable signal amplification strategies, limited their application in lowly expressed biomarkers imaging. Here we develop a 980/808 nm NIR programmed in vivo microRNAs (miRNAs) magnifier by conjugating activatable DNAzyme walker set to RENPs, which achieves more effective NIR-II imaging of early stage tumor than size monitoring imaging technique. Dye FD1080 (FD1080) modified substrate DNA quenches NIR-II downconversion emission of RENPs under 808 nm excitation. The miRNA recognition region in DNAzyme walker is sealed by a photo-cleavable strand to avoid "false positive" signal in systemic circulation. Upconversion emission of RENPs under 980 nm irradiation activates DNAzyme walker for miRNA recognition and amplifies NIR-II fluorescence recovery of RENPs via DNAzyme catalytic reaction to achieve in vivo miRNA imaging. This strategy demonstrates good application potential in the field of early cancer detection.

Screening T-Cell Activity via a Photodetachable DNA-Copolymer Nanocage and Its Therapeutic Application.

Screening T-cell activity and selecting active ones from large ex vivo-expanded populations before reinfusion is important for the success of T-cell therapy. Cytokine secretion is the evaluation criterion of cell immune activity. Cell membrane-anchored probes and microchamber-based techniques have been used to screen cytokine secretion at the single-cell level. However, they are either easily affected by nearby cells' secretion or lack of single-cell encapsulation efficiency. Here, we design a photodetachable DNA-copolymer nanocage on the cell membrane for screening the activities of ex vivo-expanded T cells by in-situ monitoring cytokine interferon-gamma (IFN-γ) secretion. The ones with good immune activity are selected for therapeutic application. DNA-copolymer nanocage is self-assembled on a cell membrane to encapsulate a single T cell. A self-quenched IFN-γ recognition aptamer is contained in the DNA-copolymer nanocage, which recovers fluorescence in response to IFN-γ secretion to indicate individual T-cell activity. The active T cells are collected after fluorescence-activated cell sorting, irradiated with 5 min UV light to detach nanocage from the cell membrane, and continuously cocultured with downstream cells. The selected Jurkat cells and CD19 CAR-T cells showed improved capabilities for downstream cell activation and cancer cell killing. The cell membrane-detachable DNA-copolymer nanocage-based T-cell activity screening and selection would have promising applications in T-cell therapy.

Activating a DNA Nanomachine via Computation across Cancer Cell Membranes for Precise Therapy of Solid Tumors.

Taking advantage of cancer cells' endogenous characters, the responsive activation of DNA nanomachines has achieved great success in tumor therapy. Combining with extra stimuli such as external light irradiation provided spatiotemporal control of DNA nanomachine activation. However, specific activation at the cellular level is still challenging considering the macroscopic-scale exposure area of usual light sources. DNA logic gates located at the cell membrane contributed to cellular specificity, but the free diffusion of input DNA strands during the operation process would impair efficiency and result in side effects to circumjacent normal cells in solid tumors. Here we design a transmembrane DNA logical computation strategy to activate a DNA nanomachine only in cancer cells from a complex solid tumor microenvironment. The DNA nanomachine multishell UCNPs-DNA is prepared by modifying DNA strands on upconversion nanoparticles. LA-apt, a DNA strand anchoring to a cancer cell membrane overexpressed receptor, and intracellular miRNA-21 served as inputs 1 and 2, respectively. Hybridization with input 1 at the cell membrane not only exposes the miRNA-21 recognition region at the DNA nanomachine, but also delivers it into cancer cells. The cascade hybridization with intracellular input 2 completes the "AND" gate operation and releases a DNA strand L2 as output. L2 acts as the trigger to operate the DNA nanomachine and correspondingly activates the photosensitizer Rose Bengal for reactive oxygen species generation. Through the "AND" gate operation of the DNA nanomachine across the cancer cell membrane, highly precise therapy only to cancer cells is achieved in a complex solid tumor microenvironment, which could become a promising modality for precise therapy of solid tumors.

In Situ Protease Secretion Visualization and Metastatic Lymph Nodes Imaging via a Cell Membrane-Anchored Upconversion Nanoprobe.

Matrix metalloproteinase (MMP) secretion is highly associated with tumor invasion and metastasis; therefore, monitoring MMP secretion is important for disease progression study and therapy choosing. Though working well for intracellular MMP imaging, the performance of current MMP detection probes is impaired in secretion monitoring due to the diffusion of MMP in an extracellular environment after secretion and low secreted amount. Here, we design a cell membrane-anchored ratiometric upconversion nanoprobe (UCNPs-Cy3/Pep-QSY7/Ab) for in situ MMP secretion visualization. Anti-EGFR is functionalized on the nanoprobe to provide specific recognition to tumor cells and guarantee fast response to MMP2 in the local place of secretion. MMP-responsive cleavage of Pep-QSY7 results in Cy3 luminescence recovery at 580 nm, which is ratioed over an internal standard of UCNP emission at 654 nm for MMP2 detection. The presented cell membrane-anchored ratiometric upconversion nanoprobe demonstrated that satisfactory results for in situ monitoring of MMP2 secretion from MDA-MB-231 cells and MCF-7 cells, as well as in vivo imaging of metastatic lymph nodes, would provide a universal platform for protease secretion study and contribute to tumor invasiveness assessment.

Photostable Fluorescent Tracker for Imaging Mitochondria with Super Resolution.

The power factories in cells, mitochondria, play important roles in all physiological processes. It is reported that progressive mitochondrial swelling and outer mitochondrial membrane rupture could be induced by a wide variety of apoptotic and necrotic stimuli. Regrettably, although a variety of mitochondrial probes have been developed, most of them are based on the detection of active species in mitochondria. Probes that can monitor the status and distribution of mitochondria for a long time are still urgently needed. In this study, a fluorescent sensor with excellent properties, EtNBEn, is described. Outstanding performance allows it to be observed not only in cells but also in living Daphnia and zebrafish under confocal microscopy for a long time. Moreover, the swelling process of mitochondria under light stimulation is also visualized under super-resolution (SR) microscopy. All these results suggest that EtNBEn could be employed for tagging mitochondria in various physiological processes, which makes a great contribution to the cure of diseases.

Triply Enhanced Immunotherapy via Dual Glycan Reforming Integrated with Perforation.

The enhancement of immunotherapy is an emerging direction to develop highly effective and practical cancer therapeutic methods. Here a triply enhanced immunotherapy drug (TEID) is designed for ingeniously integrating in situ dual glycan reforming with perforation on cell membrane. The TEID is composed of galactose and neuraminidase conjugated streptolysin O (SLO-Gal and SLO-NEU), which are encapsulated in a hyaluronic acid (HA) shell for targeted recognition to tumor tissue via cell surface CD44. After targeted delivery and HAase-mediated degradation in the tumor region, the TEID releases SLO-Gal and SLO-NEU, which can easily anchor Gal and NEU on the tumor cell membrane via the perforation of SLO to perform dual glycan reforming for the introduction of Gal and the cleavage of sialic acid. The former can activate immune cells to secret cytokines for immune-killing, and the latter can weaken the immune inhibition to improve the immunotherapeutic efficacy. Meanwhile, the perforation of SLO can promote the delivery of cytokines into the tumor cells to further enhance the efficacy. The designed triply enhanced immunotherapy strategy opens a significant and promising route to promote clinical immunotherapy of cancer.

Single-cell HER2 quantification via instant signal amplification in microdroplets.

Accurate and ultrasensitive evaluation of human epidermal growth factor receptor 2 (HER2) protein is key to early diagnosis and subtype differentiation of breast cancer. Single-cell analyses to reduce ineffective targeted therapies due to breast cancer heterogeneity and improve patient survival remain challenging. Herein, we reported a novel droplet microfluidic combined with an instant cation exchange signal amplification strategy for quantitative analysis of HER2 protein expression on single cells. In the 160 µm droplets produced by a tapered capillary bundle, abundant Immuno-CdS labeled on HER2-positive cells were replaced by Ag + to obtain Cd2+ that stimulated Rhod-5N fluorescence. This uniformly distributed and instantaneous fluorescence amplification strategy in droplets improves sensitivity and reduces signal fluctuation. Using HER2 modified PS microsphere to simulate single cells, we obtained a linear fitting of HER2-modified concentration and fluorescence intensity in microdroplets with the limit detection of 11.372 pg mL-1. Moreover, the relative standard deviation (RSD) was 4.2-fold lower than the traditional immunofluorescence technique (2.89% vs 12.21%). The HER2 protein on SK-BR-3 cells encapsulated in droplets was subsequently quantified, ranging from 9862.954 pg mL-1 and 205.26 pg mL-1, equivalent to 9.795 × 106 and 2.038 × 105 protein molecules. This detection system provides a universal platform for single-cell sensitive quantitative analysis and contributes to the evaluation of HER2-positive tumors.

Single cell multi-miRNAs quantification with hydrogel microbeads for liver cancer cell subtypes discrimination.

The simultaneous quantification of multi-miRNAs in single cells reveals cellular heterogeneity, and benefits the subtypes discrimination of cancer cells . Though micro-droplet techniques enable successful single cell encapsulation, the isolated and restricted reaction space of microdroplets causes cross-reactions and inaccuracy for simultaneous multi-miRNAs quantification. Herein, we develop a hydrogel microbead based strategy for the simultaneous sensitive quantification of miRNA-21, 122 and 222 in single cells. Single cells are encapsulated and undergo cytolysis in hydrogel microbeads. The three target miRNAs are retained in the microbead by pre-immobilized capture probes, and activate rolling circle amplification (RCA) reactions. The RCA products are hybridized with corresponding dye labelled DNA reporters, and the respective fluorescence intensities are recorded for multi-miRNA quantification. The porous structure of the hydrogel microbeads allows the free diffusion of reactants and easy removal of unreacted DNA strands, which effectively avoids nonspecific cross-reactions. Clear differentiation of cellular heterogeneity and subpopulation discrimination are achieved for three kinds of liver cancer cells and one normal liver cell.