Inhalable Polymeric Microparticles for Phage and Photothermal Synergistic Therapy of Methicillin-Resistant Staphylococcus aureus Pneumonia.

Acute methicillin-resistant Staphylococcus aureus (MRSA) pneumonia is a common and serious lung infection with high morbidity and mortality rates. Due to the increasing antibiotic resistance, toxicity, and pathogenicity of MRSA, there is an urgent need to explore effective antibacterial strategies. In this study, we developed a dry powder inhalable formulation which is composed of porous microspheres prepared from poly(lactic-co-glycolic acid) (PLGA), internally loaded with indocyanine green (ICG)-modified, heat-resistant phages that we screened for their high efficacy against MRSA. This formulation can deliver therapeutic doses of ICG-modified active phages to the deep lung tissue infection sites, avoiding rapid clearance by alveolar macrophages. Combined with the synergistic treatment of phage therapy and photothermal therapy, the formulation demonstrates potent bactericidal effects in acute MRSA pneumonia. With its long-term stability at room temperature and inhalable characteristics, this formulation has the potential to be a promising drug for the clinical treatment of MRSA pneumonia.

Accurate Cancer Screening and Prediction of PD-L1-Guided Immunotherapy Efficacy Using Quantum Dot Nanosphere Self-Assembly and Machine Learning.

Accurate quantification of exosomal PD-L1 protein in tumors is closely linked to the response to immunotherapy, but robust methods to achieve high-precision quantitative detection of PD-L1 expression on the surface of circulating exosomes are still lacking. In this work, we developed a signal amplification approach based on aptamer recognition and DNA scaffold hybridization-triggered assembly of quantum dot nanospheres, which enables bicolor phenotyping of exosomes to accurately screen for cancers and predict PD-L1-guided immunotherapeutic effects through machine learning. Through DNA-mediated assembly, we utilized two aptamers for simultaneous ultrasensitive detection of exosomal antigens, which have synergistic roles in tumor diagnosis and treatment prediction, and thus, we achieved better sample classification and prediction through machine-learning algorithms. With a drop of blood, we can distinguish between different cancer patients and healthy individuals and predict the outcome of immunotherapy. This approach provides valuable insights into the development of personalized diagnostics and precision medicine.

One-Step Dual-Color Labeling of Viral Envelope and Intraviral Genome with Quantum Dots Harnessing Virus Infection.

Labeling the genome and envelope of a virus with multicolor quantum dots (QDs) simultaneously enables real-time monitoring of viral uncoating and genome release, contributing to our understanding of virus infection mechanisms. However, current labeling techniques require genetic modification, which alters the virus's composition and infectivity. To address this, we utilized the CRISPR/Cas13 system and a bioorthogonal metabolic method to label the Japanese encephalitis virus (JEV) genome and envelopes with different-colored QDs in situ. This technique allows one-step two-color labeling of the viral envelope and intraviral genome with QDs harnessing virus infection. In combination with single-virus tracking, we visualized JEV uncoating and genome release in real time near the endoplasmic reticulum of live cells. This labeling strategy allows for real-time visualization of uncoating and genome release at the single-virus level, and it is expected to advance the study of other viral infection mechanisms.

Near Infrared-II Excited Triplet Fusion Upconversion with Anti-Stokes Shift Approaching the Theoretical Limit.

The anti-Stokes shift represents the capacity of photon upconversion to convert low-energy photons to high-energy photons. Although triplet exciton-mediated photon upconversion presents outstanding performance in solar energy harvesting, photoredox catalysis, stereoscopic 3D printing, and disease therapeutics, the interfacial multistep triplet exciton transfer leads to exciton energy loss to suppress the anti-Stokes shift. Here, we report near infrared-II (NIR-II) excitable triplet exciton-mediated photon upconversion using a hybrid photosensitizer consisting of lead sulfide quantum dots (PbS QDs) and new surface ligands of thiophene-substituted diketopyrrolopyrrole (Th-DPP). Under 1064 nm excitation, this photon upconversion revealed a record-corrected upconversion efficiency of 0.37% (normalized to 100%), with the anti-Stokes shift (1.07 eV) approaching the theoretical limit (1.17 eV). The observation of this unexpected result is due to our discovery of the presence of a weak interaction between the sulfur atom on Th-DPP and Pb2+ on the PbS QDs surface, facilitating electronic coupling between PbS QDs and Th-DPP, such that the realization of triplet exciton transfer efficiency is close to 100% even when the energy gap is as small as 0.04 eV. With this premise, this photon upconversion as a photocatalyst enables the production of standing organic gel via photopolymerization under 1064 nm illumination, displaying NIR-II photon-driven photoredox catalysis. This research not only establishes the foundation for enhancing the performance of NIR-II excitable photonic upconversion but also promotes its development in photonics and photoredox catalysis.

Multihierarchical Regulation To Achieve Quantum Dot Nanospheres with a Photoluminescence Quantum Yield Close to 100.

Quantum dots (QDs) exhibit superior brightness and photochemical stability, making them the preferred option for highly sensitive single-molecule detection compared with fluorescent dyes or proteins. Nevertheless, their high surface energy leads to nonspecific adsorption and poor colloidal stability. In the past decades, we have found that QD-based fluorescent nanoparticles (FNs) can not only address these limitations but also enhance detection sensitivity. However, the photoluminescence quantum yield (PLQY) of FNs is significantly lower compared with that of original QDs. It is urgent to develop a strategy to solve the issue, aiming to further enhance detection sensitivity. In this study, we found that the decrease of PLQY of FNs prepared by free radical polymerization was attributed to two factors: (1) generation of defects that can cause nonradiative transitions resulting from QD-ligands desorption and QD-shell oxidation induced by free radicals; (2) self-absorption resulting from aggregation caused by incompatibility of QDs with polymers. Based on these, we proposed a multihierarchical regulation strategy that includes: (1) regulating QD-ligands; (2) precisely controlling free radical concentration; and (3) constructing cross-linked structures of polymer to improve compatibility and to reduce the formation of surface defects. It is crucial to emphasize that the simultaneous coordination of multiple factors is essential. Consequently, a world-record PLQY of 97.6% for FNs was achieved, breaking through the current bottleneck at 65%. The flexible application of this regulatory concept paves the way for the large-scale production of high-brightness QD-polymer complexes, enhancing their potential applications in sensitive biomedical detection.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

Biosynthesis of NIR-II Ag2 Se Quantum Dots with Bacterial Catalase for Photoacoustic Imaging and Alleviating-Hypoxia Photothermal Therapy.

Developing the second near-infrared (NIR-II) photoacoustic (PA) agent is of great interest in bioimaging. Ag2 Se quantum dots (QDs) are one kind of potential probe for applications in NIR-II photoacoustic imaging (PAI). However, the surfaces with excess anions of Ag2 Se QDs, which increase the probability of nonradiative transitions of excitons benefiting PA imaging, are not conducive to binding electron donor ligands for potential biolabeling and imaging. In this study, Staphylococcus aureus (S. aureus) cells are driven for the biosynthesis of Ag2 Se QDs with catalase (CAT). Biosynthesized Ag2 Se (bio-Ag2 Se-CAT) QDs are produced in Se-enriched environment of S. aureus and have a high Se-rich surface. The photothermal conversion efficiency of bio-Ag2 Se-CAT QDs at 808 and 1064 nm is calculated as 75.3% and 51.7%, respectively. Additionally, the PA signal responsiveness of bio-Ag2 Se-CAT QDs is ≈10 times that of the commercial PA contrast agent indocyanine green. In particular, the bacterial CAT is naturally attached to bio-Ag2 Se-CAT QDs surface, which can effectively relieve tumor hypoxia. The bio-Ag2 Se-CAT QDs can relieve heat-initiated oxidative stress while undergoing effective photothermal therapy (PTT). Such biosynthesis method of NIR-II bio-Ag2 Se-CAT QDs opens a new avenue for developing multifunctional nanomaterials, showing great promise for PAI, hypoxia alleviation, and PTT.

Enhancing Circularly Polarized Luminescence in Quantum Dots through Chiral Coordination-Mediated Growth at the Liquid/Liquid Interface.

Here, we develop a novel methodology for synthesizing chiral CdSe@ZnS quantum dots (QDs) with enhanced circularly polarized luminescence (CPL) by incorporating l-/d-histidine (l-/d-His) ligands during ZnS shell growth at the water/oil interface. The resulting chiral QDs exhibit exceptional absolute photoluminescence quantum yield of up to 67.2%, surpassing the reported limits of 40.0% for chiral inorganic QDs, along with absorption dissymmetry factor (|gabs|) and luminescence dissymmetry factor (|glum|) values of 10-2, exceeding the range of 10-5-10-3 and 10-4-10-2, respectively. Detailed investigations of the synthetic pathway reveal that the interface, as a binary synthetic environment, facilitates the coordinated ligand exchange and shell growth mediated by chiral His-Zn2+ coordination complexes, leading to a maximum fluorescent brightness and chiroptical activities. The growth process, regulated by the His-Zn2+ coordination complex, not only reduces trap states on the CdSe surface, thereby enhancing the fluorescence intensity, but also significantly promotes the orbital hybridization between QDs and chiral ligands, effectively overcoming the shielding effect of the wide bandgap shell and imparting pronounced chirality. The proposed growth pathway elucidates the origin of chirality and provides insights into the regulation of the CPL intensity in chiral QDs. Furthermore, the application of CPL QDs in multilevel anticounterfeiting systems overcomes the limitations of replication in achiral fluorescence materials and enhances the system's resistance to counterfeiting, thus opening new opportunities for chiral QDs in optical anticounterfeiting and intelligent information encryption.

Real-Time Imaging of Single Viral mRNA Translation in Live Cells Using CRISPR/dCas13.

Translation is one of the many critical cellular activities regulated by viruses following host-cell invasion, and studies of viral mRNA translation kinetics and subcellular localization require techniques for the dynamic, real-time visualization of translation. However, conventional tools for imaging mRNA translation often require coding region modifications that may affect native translation. Here, we achieve dynamic imaging of translation with a tool that labels target mRNAs with unmodified coding regions using a CRISPR/dCas13 system with specific complementary paired guide RNAs. This system enables a real-time dynamic visualization of the translation process and is a promising tool for further investigations of the mechanisms of translation.

Combining Upconversion Luminescence, Photothermy, and Electrochemistry for Highly Accurate Triple-Signal Detection of Hydrogen Sulfide by Optically Trapping Single Microbeads.

The detection of hydrogen sulfide (H2S), the third gas signaling molecule, is a promising strategy for identifying the occurrence of certain diseases. However, the conventional single- or dual-signal detection can introduce false-positive or false-negative results, which ultimately decreases the diagnostic accuracy. To address this limitation, we developed a luminescent, photothermal, and electrochemical triple-signal detection platform by optically trapping the synthetic highly doped upconversion coupled SiO2 microbeads coated with metal-organic frameworks H-UCNP-SiO2@HKUST-1 (H-USH) to detect the concentration of H2S. The H-USH was first synthesized and proved to have stable structure and excellent luminescent, photothermal, and electrochemical properties. Under 980 nm optical trapping and 808 nm irradiation, H-USH showed great detection linearity, a low limit of detection, and high specificity for H2S quantification via triple-signal detection. Moreover, H-USH was captured by optical tweezers to realize quantitative detection of H2S content in serum of acute pancreatitis and spontaneously hypertensive rats. Finally, by analyzing the receiver operating characteristic (ROC) curve, we concluded that triple-signal detection of H2S was more accurate than single- or dual-signal detection, which overcame the problem of false-negative/positive results in the detection of H2S in actual serum samples.

Acid-Resistant Near-Infrared II Ag2Se Quantum Dots for Gastrointestinal Imaging.

With the development of near-infrared II (NIR-II) fluorescence imaging, Ag2Se quantum dots (QDs) have become promising label candidates due to their negligible toxicity and narrow band gap. Despite their potential for gastrointestinal (GI) imaging, the application of Ag2Se QDs still presents significant challenges due to issues such as fluorescence extinction or poor stability in the complex digestive microenvironment. Herein, we have proposed a novel approach to the continuous production of Se precursors using glutathione (GSH) as the reductant under acidic conditions, realizing the continuous growth of water-dispersible Ag2Se QDs. The Ag2Se QDs emitting at 600-1100 nm have been successfully synthesized. Meanwhile, the silver-rich surface of the synthesized NIR-II Ag2Se QDs has been passivated well with the dense GSH, resulting in exceptional colloidal stability and photostability and endowing them with acid resistance. As a result, the obtained NIR-II Ag2Se QDs have exhibited remarkable stability in gastric acid, thus enabling their utilization for long-term real-time monitoring of GI peristalsis via NIR-II fluorescence imaging. Moreover, in contrast to conventional barium meal-based X-ray imaging, NIR-II fluorescence imaging with as-prepared NIR-II Ag2Se QDs can offer clearer visualization of fine intestinal structures, with a width as small as 1.07 mm. The developed strategy has offered a new opportunity for the synthesis of acid-resistant nanocrystals, and the acid-resistant, low-toxicity, and biocompatible NIR-II Ag2Se QDs synthesized in this work show a great promise for GI imaging and diagnosis of GI diseases in vivo.

Redox Imbalance Triggered Intratumoral Cascade Reaction for Tumor "turn on" Imaging and Synergistic Therapy.

The redox homeostasis in tumors enhances their antioxidant defense ability, limiting reactive oxygen species mediated tumor therapy efficacy. The development of strategies for specific and continuous disruption of the redox homeostasis in tumor cells facilitates the improvement of the cancer therapeutic effect by promoting the apoptosis of tumor cells. Herein, a responsively biodegradable targeting multifunctional integrated nanosphere (HDMn-QDs/PEG-FA) is designed to enhance the anti-tumor efficacy by triggering intratumoral cascade reactions to effectively disrupt intracellular redox homeostasis. Once HDMn-QDs/PEG-FA enters tumor cells, manganese dioxide (MnO2 ) shell on the surface of nanosphere consumes glutathione (GSH) to produce Mn2+ , enabling enhanced chemodynamic therapy (CDT) via a Fenton-like reaction and T1 -weighted magnetic resonance imaging. Meanwhile, the degradation of MnO2 can also cause the fluorescence recovery of quantum dots conjugated on the surface of the shell, realizing "turn-on" fluorescence imaging. In addition, the doxorubicin is released because of the cleavage of the embedded SS bond in the hybrid core framework by GSH. A superior synergistic therapeutic efficiency combined CDT and chemotherapy is shown by HDMn-QDs/PEG-FA in vivo. The tumor-inhibition rate reaches to 94.8% and does not cause normal tissue damage due to the good targeting and tumor microenvironment-specific response.

Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry.

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).

Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

Capture of small clusters by ligand-solvent interaction.

Clusters are considered to become increasingly significant for elaborating the nanocrystal's formation mechanism. However, capturing the clusters with high chemical potential is challenging because of the lack of effective strategies. In this work, the key role of ligand-solvent interaction has been revealed for the stabilization of clusters in silver telluride synthesis. The Flory interaction coefficient that comprehensively regards the temperature and dispersion, polarity, and hydrogen bonding of the solvent has been used to evaluate the ligand-solvent interaction and thus assist in the design of synthetic systems. Small silver telluride clusters have been successfully captured, and the composition of the smallest cluster is determined as Ag7Te8(SCy)2 (SCy represents the ligand). This work provides new insights into the design of cluster/nanocrystal synthesis systems and paves the way to revealing the mechanism of precursor-cluster-nanocrystal conversion.

Inducing Autophagy and Blocking Autophagic Flux via a Virus-Mimicking Nanodrug for Cancer Therapy.

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.

"Dual-Key-and-Lock" NIR-II NSCyanines Enable High-Contrast Activatable Phototheranostics in Extrahepatic Diseases.

Conventional cyanine dyes with a symmetric structure are "always-on", which can easily accumulate in the liver and display high liver background fluorescence, inevitably interfering the accurate diagnosis and therapy in extrahepatic diseases. We herein report a platform of NIR-II non-symmetric cyanine (NSCyanine) dyes by harnessing a non-symmetric strategy, which are extremely sensitive to pH/viscosity and can be activated via a "dual-key-and-lock" strategy. These NSCyanine dyes with a low pKa (<4.0) only show weak fluorescence at lysosome pH (key1), however, the fluorescence can be completely switched on and significantly enhanced by intracellular viscosity (key2) in disease tissues, exhibiting high target-to-liver ratios up to 19.5/1. Notably, high-contrast phototheranostics in extrahepatic diseases are achieved, including intestinal metastasis-imaging, acute gastritis-imaging, bacteria infected wound healing, and tumor ablation via targeted combined photothermal therapy and chemotherapy.

In Situ Self-Assembly of Fluorogenic RNA Nanozipper Enables Real-Time Imaging of Single Viral mRNA Translation.

Real-time visualization of individual viral mRNA translation activities in live cells is essential to obtain critical details of viral mRNA dynamics and to detect its transient responses to environmental stress. Fluorogenic RNA aptamers are powerful tools for real-time imaging of mRNA in live cells, but monitoring the translation activity of individual mRNAs remains a challenge due to their intrinsic photophysical properties. Here, we develop a genetically encoded turn-on 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding RNA nanozipper with superior brightness and high photostability by in situ self-assembly of multiple nanozippers along single mRNAs. The nanozipper enables real-time imaging of the mobility and dynamic translation of individual viral mRNAs in live cells, providing information on the spatial dynamics and translational elongation rate of viral mRNAs.

Uncovering the F-Actin-Based Nuclear Egress Mechanism of Newly Synthesized Influenza A Virus Ribonucleoprotein Complexes by Single-Particle Tracking.

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.

Quantum Dots Tracking Endocytosis and Transport of Proteins Displayed by Mammalian Cells.

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.