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Fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) region holds great promise for in vivo bioimaging. However, it is challenging to develop a brilliant donor-acceptor-donor (D-A-D) type NIR-II fluorophore with maximal absorption beyond 1000 nm in aqueous solution. Herein, we report a bright D-A-D type BOIMPY-based NIR-II dye (NK1143) with peak absorption/emission at 1005/1143 nm for in vivo bioimaging. Co-assembly of NK1143, SC12 (intermolecular steric hindrance modulator), and DSPE-PEG2000 effectively inhibits H-aggregation of NK1143 in aqueous solution and enhances the brightness simultaneously up to 53-fold by leveraging synergistic steric regulation strategy. Notably, this strategy allows for deep optical penetration of 8 mm and high-resolution blood vessels imaging in vivo, displaying high signal-to-background ratio of 7.8/1 under 980 nm excitation. More importantly, the BOIMPY-based nanoprobe can passively target and clearly visualize broad types of tumor xenografts, further improving intraoperative NIR-II fluorescence-guided resection of tiny metastases of less than 1 mm. This work provides an effective strategy for the development of BOIMPY-based NIR-II organic fluorophores with broad applications.

Bacterial infectious diseases are common clinical diseases that seriously threaten human health, especially in countries and regions with poor environmental hygiene. Due to the lack of characteristic clinical symptoms and signs, it is a challenge to distinguish a bacterial infection from other infections, leading to misdiagnosis and antibiotic overuse. Therefore, there is an urgent need to develop a specific method for detection of bacterial infections. Herein, utilizing ultrabright fluorescent nanospheres (FNs) as reporters, immunochromatographic dyad test strips are developed for the early detection of bacterial infections and distinction of different stages of bacterial infectious diseases in clinical samples. C-reactive protein (CRP) and heparin-binding protein (HBP) are quantified and assayed because their levels in plasma are varied dynamically and asynchronously during the progression of the disease. The detection limits of CRP and HBP can reach as low as 0.51 and 0.65 ng/mL, respectively, due to the superior fluorescence intensity of each FN, which is 570 times stronger than that of a single quantum dot. The assay procedure can be achieved in 22 min, fully meeting the needs of rapid and ultrasensitive detection in the field. This constructed strip has been successfully used to profile the stage and severity of bacterial infections by monitoring the levels of CRP and HBP in human plasma samples, showing great potential as a point-of-care biosensor for clinical diagnosis. In addition to bacterial infections, the developed ultrabright FN-based point-of-care testing can be readily expanded for rapid, quantitative, and ultrasensitive detection of other trace substances in complex systems.

Generally speaking, it is difficult to keep nanomaterials encapsulated in amphiphilic polymers like octylamine-grafted poly(acrylic acid) (OPA) compact in coating-layer, with a small hydrodynamic size. Here, we prepared stable hydrophilic quantum dots (QDs) via encapsulation in ∼3 nm-long amphiphilic and zwitterionic (AZ) molecules. After encapsulation with AZ molecules, the coated QDs are only 2.1 nm thicker in coating, instead of 5.4 nm with OPA. Meanwhile, the hydrodynamic sizes of CdSe/CdS, ZnCdSeS, ZnCdSe/ZnS, and CdSe/ZnS QDs encapsulated in AZ molecules (AZ-QDs) are less than 15 nm, and 6-7 nm smaller than those of QDs in OPA (OPA-QDs). Notably, both extracellular and intracellular nonspecific binding of AZ-QDs is approximately 100-folds lower than that of OPA-QDs.

Mammalian cell display technology uses eukaryotic protein expression system to display proteins on cell surfaces and has become an important method in biological research. Although mammalian cell display technology has many advantages and development potential, certain attributes of the displayed protein remain uncharacterized, such as whether the displayed proteins re-enter the cell and how displayed proteins move into the cell. Here, we present the endocytosis mechanism, motility behavior, and transport kinetics of displayed proteins determined using HaloTag as the displayed protein and quantum dot-based single-particle tracking. The displayed protein enters the cell through clathrin-mediated endocytosis and is transported through the cell in three stages, which is dependent on microfilaments and microtubules. The dynamic information obtained in this study provides answers to questions about endocytosis and postendocytosis transport of displayed proteins and, therefore, is expected to facilitate the development of surface display technology.

Maximizing the therapeutic capacity of drugs by allowing them to escape lysosomal degradation is a long-term challenge for nanodrug delivery. Japanese encephalitis virus (JEV) has evolved the ability to escape the endosomal region to avoid degradation of internal genetic material by lysosomes and further induce upregulation of cellular autophagy for the purpose of their mass reproduction. In this work, to exploit the lysosome escape and autophagy-inducing properties of JEV for cancer therapy, we constructed a virus-mimicking nanodrug consisting of anti-PDL1 antibody-decorated JEV-mimicking virosome encapsulated with a clinically available autophagy inhibitor, hydroxychloroquine (HCQ). Our study indicated that the nanodrug can upregulate the autophagy level and inhibit the autophagic flux, thereby inducing the apoptosis of tumor cells, and further activating the immune response, which can greatly improve the antitumor and tumor metastasis suppression effects and provide a potential therapeutic strategy for tumor treatment.

Viral encephalitis is an inflammatory disease of the brain parenchyma and caused by various viral infections. In vivo monitoring of the progression of viral infections can aid accurate diagnosis of viral encephalitis and effective intervention. We developed an activatable and reversible virus-mimicking near-infrared II nanoprobe consisting of an Fe

Nuclear trafficking of viral genome is an essential cellular process in the life cycles of viruses. Despite substantial progress in uncovering a wide variety of complicated mechanisms of virus entry, intracellular transport of viral components, virus assembly, and egress, the temporal and spatial dynamics of viral genes trafficking within the nucleus remains poorly understood. Herein, using single-particle tracking, we explored the real-time dynamic nuclear trafficking of influenza A virus (IAV) genes packaged as the viral ribonucleoprotein complexes (vRNPs) by combining a four-plasmid DNA transfection system for the reconstruction of green fluorescent protein (GFP)-labeled vRNPs and a spinning disk super-resolution fluorescence microscope. We found that IAV infection significantly induced the formation of actin microfilaments (F-actin) in the nucleus. In combination with the fluorescent protein-tagged nuclear F-actin probe, we visualized the directed movement of GFP-labeled vRNPs foci along the nuclear F-actin with a speed of 0.18 μm/s, which is similar to the microfilaments-dependent slow directed motion of IAVs in the cytoplasm. The disruption of nuclear F-actin after treatment with microfilament inhibitors caused a considerable decrease in vRNPs motility and suppressed the nuclear export of newly produced vRNPs, indicating that the slow, directed movement plays a crucial role in facilitating the nuclear egress of vRNPs. Our findings identified a nuclear F-actin-dependent pathway for IAV vRNPs transporting from the nucleus into the cytoplasm, which may in turn uncover a novel target for antiviral treatment.

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol independent, whereas the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein that activates actin nucleation, is recruited to the IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of the CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. IMPORTANCE IAV infects cells by harnessing cellular endocytic machineries. A better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in the plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results provide new insights into IAV infection and the pathway/cargo-specific involvement of the cholesterol pool(s).

Single-virus tracking (SVT) offers the opportunity to monitor the journey of individual viruses in real time and to explore the interactions between viral and cellular structures in live cells, which can assist in characterizing the complex infection process and revealing the associated dynamic mechanisms. However, the low brightness and poor photostability of conventional fluorescent tags (e.g., organic dyes and fluorescent proteins) greatly limit the development of the SVT technique, and challenges remain in performing multicolor SVT over long periods of time. Owing to the outstanding photostability, high brightness and narrow emission with tunable color range of quantum dots (QDs), QD-based SVT (QSVT) enables us to follow the fate of individual viruses interacting with different cellular structures at the single-virus level for milliseconds to hours, providing more accurate and detailed information regarding viral infection in live cells. So far, the QSVT technique has yielded spectacular achievements in uncovering the mechanisms associated with virus entry, trafficking and egress. Here, we provide a detailed protocol for QSVT implementation using the viruses that we have previously studied systematically as an example. The specific procedures for performing QSVT experiments in live cells are described, including virus preparation, the QD labeling strategies, imaging approaches, image processing and data analysis. The protocol takes 1-2 weeks from the preparation of viruses and cellular specimens to image acquisition, and 1 d for image processing and data analysis.

Natural killer cells (NKs) hold great promise in cancer treatment, but their application in solid tumors remains a great challenge because current solutions hardly can overcome various difficulties that faced. Herein, we endow NKs with the phytochemical feature for effective immunotherapy of solid tumors. NKs are decorated with natural thylakoid (Tk) membranes through an efficient and convenient membrane fusion strategy. Tk engineering effectively activates NKs, because the antioxidase on Tk induce glycogen synthase kinase-3β inhibition, and subsequently increase the expression of activating receptor and cytotoxic effector molecules in NKs. After systemic administration, the phytochemical NKs (PC-NKs) can target tumor tissues, and then profoundly reprogram tumor microenvironment (TME) with the help of catalase on Tk, resulting in significantly enhanced direct killing of PC-NKs and immune activated TME. Therefore, potent therapeutic effects with few abnormalities are achieved, providing a novel idea for the development of highly efficient NKs for solid tumors.

Influenza A virus (IAV) is internalized into its host cells by endocytosis, which involves many cellular proteins and molecules. In this study, we focus on the function of calcium ion (Ca

In vivo fluorescence imaging can perform real-time, noninvasive, and high spatiotemporal resolution imaging to accurately obtain the dynamic biological information in vivo, which plays significant roles in the early diagnosis and treatment of cancer. However, traditional in vivo fluorescence imaging usually operates in the visible and near-infrared (NIR)-I windows, which are severely interfered by the strong tissue absorption, tissue scattering, and autofluorescence. The emergence of NIR-II imaging at 1000-1700 nm significantly breaks through the imaging limitations in deep tissues, due to less tissue scattering and absorption. Benefiting from the outstanding optical properties of NIR-II quantum dots (QDs), such as high brightness and good photostability, in vivo fluorescence imaging exhibits excellent temporal-spatial resolution and large penetration depth, and QDs have become a kind of promising fluorescent biomarkers in the field of in vivo fluorescence imaging. Herein, the authors review NIR-II QDs from preparation to modification, and summarize recent applications of NIR-II QDs, including in vivo imaging and imaging-guided therapies. Finally, they discuss the special concerns when NIR-II QDs are shifted from in vivo imaging applications to further in-depth applications.