A double-switch pHLIP system enables selective enrichment of circulating tumor microenvironment-derived extracellular vesicles.

Circulating tumor microenvironment-derived extracellular vesicles (cTME-EVs) are gaining considerable traction in cancer research and liquid biopsy. However, the study of cTME-EVs is largely limited by the dearth of a general isolation technique to selectively enrich cTME-EVs from biological fluids for downstream analysis. In this work, we broke through this dilemma by presenting a double-switch pH-low insertion peptide (D-S pHLIP) system to exclusively harvest cTME-EVs from the blood serum of tumor mouse models. This D-S pHLIP system consists of a highly sensitive pH-driven conformational switch (pKa ≈ 6.8) that allows specific installation of D-S pHLIP on the EV membranes in TME (pH 6.5 to 6.8) and a unique hook-like switch to "lock" the peptide securely on the cTME-EVs during the systemic circulation. The D-S pHLIP-anchored cTME-EVs were magnetically enriched and then analyzed with high-resolution messenger RNA sequencing, by which more than 18 times the number of TME-related differentially expressed genes and 10 times the number of hub genes were identified, compared with those achieved by the gold-standard ultracentrifugation. This work could revolutionize basic TME research as well as clinical liquid biopsy for cancer.

In Vivo Fluorescent Labeling of Foam Cell-Derived Extracellular Vesicles as Circulating Biomarkers for In Vitro Detection of Atherosclerosis.

Real-time monitoring of the development of atherosclerosis (AS) is key to the management of cardiovascular disease (CVD). However, existing laboratory approaches lack sensitivity and specificity, mostly due to the dearth of reliable AS biomarkers. Herein, we developed an in vivo fluorescent labeling strategy that allows specific staining of the foam cell-derived extracellular vesicles (EVs) in atherosclerotic plaques, which are released into the blood as circulating biomarkers for in vitro detection of AS. This strategy relies on a self-assembled nanoprobe that could recognize foam cells specifically, where the probe is degraded by the intracellular HClO to produce a trifluoromethyl-bearing boron-dipyrromethene fluorophore (termed B-CF3), a lipophilic dye that can be transferred to the exosomal membranes. These circulating B-CF3-stained EVs can be detected directly on a fluorescence spectrometer or microplate reader without resorting to any sophisticated analytical method. This liquid-biopsy format enables early detection and real-time differentiation of lesion vulnerability during AS progression, facilitating effective CVD management.

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.

Self-Referenced Synthetic Urinary Biomarker for Quantitative Monitoring of Cancer Development.

Urinary monitoring of diseases has gained considerable attention due to its simple and non-invasive sampling. However, urinalysis remains limited by the dearth of reliable urinary biomarkers and the intrinsically enormous heterogeneity of urine samples. Herein, we report, to our knowledge, the first renal-clearable Raman probe encoded by an internal standard (IS)-conjugated reporter that acts as a quantifiable urinary biomarker for reliable monitoring of cancer development, simultaneously eliminating the impact of sample heterogeneity. Upon delivery of the probes into tumor microenvironments, the endogenously overexpressed ß-glucuronidase (GUSB) can cleave the target-responsive residues of the probes to produce IS-retained gold nanoclusters, which were excreted into host urine and analyzed by Au growth-based surface-enhanced Raman spectroscopy. As a result, the in vivo GUSB activity was transformed into in vitro quantitative urinary signals. Based on this IS-encoded synthetic biomarker, both the cancer progression and therapy efficacy were quantitatively monitored, potentiating clinical implications.

A Novel Diagnostic Model for Early Hepatitis B Virus-Related Hepatocellular Carcinoma Diagnosis.

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is closely related to the diagnostic stage. Due to the difficulty diagnosing early-stage HCC, most patients with HCC are diagnosed at the advanced stage. In this study, we used protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein (AFP) combined with aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-Bil) to establish a novel diagnostic model for early-stage hepatitis B virus (HBV)-related HCC. METHODS: The serum levels of PIVKA-II, AFP, AST, ALT, and T-Bil were measured in 148 patients with early-stage HBV-related HCC and 940 patients with chronic hepatitis B. The receiver operating characteristic (ROC) curves were used to verify the diagnostic efficacy of the novel diagnostic model for early-stage HBV-related HCC. RESULTS: The mathematical model of [1.5 x PIVKA-II/(AST x T-Bil) + AFP/(ALT x T-Bil)] was selected as the novel diagnostic model. The areas under ROC curves (AUROCs) of the novel diagnostic model for detecting early-stage HBV-related HCC were significantly higher than those of PIVKA-II, AFP, and PIVKA-II combined with AFP (HCC ≤ 5 cm: 0.925 vs. 0.826, 0.666, and 0.821; HCC < 3 cm: 0.896 vs. 0.741, 0.651, and 0.765, respectively) (all p < 0.001). Using serum levels of AFP ≥ 20 ng/mL, the diagnostic model had the highest AUROC values of 0.960 and 0.933 for HCC ≤ 5 cm (89 cases) and HCC < 3 cm (40 cases), respectively, with a sensitivity of 83.15%, and 77.50% and specificity of 95.34% and 90.69%, respectively. CONCLUSIONS: The novel diagnostic model is superior to PIVKA-II and AFP for diagnosing early-stage HBV-related HCC, especially in patients with abnormal serum AFP levels.

Trojan Horse Delivery of Spherical Nucleic Acid Probes into the Cytoplasm for High-Fidelity Imaging of MicroRNAs.

We report a Trojan horse strategy to efficiently deliver the spherical nucleic acid probes (namely, nanoflares) into the cytoplasm for microRNA (miRNA) imaging with high fidelity, breaking through the cytoplasmic transport dilemma of RNA probes in living cells. The nanoflare is encapsulated into a "Trojan horse" consisting of zwitterionic choline phosphates (CPs) and acid-degradable crosslinkers; the former effectively promotes cell uptake and the latter triggers instantaneous liberation of the nanoflare probes from the lysosome to the cytoplasm. The exposed nanoflares in the cytoplasm can be lightened up by the target miRNAs specifically. Compared with the conventional nanoflares as well as the improved ones in previous reports, the "Trojan horse" nanoflares avoid nuclease degradation and thiol displacement during the delivery process, providing unprecedentedly high accuracy for intracellular miRNA imaging.

Cancer-derived small extracellular vesicles: emerging biomarkers and therapies for pancreatic ductal adenocarcinoma diagnosis/prognosis and treatment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers worldwide with high mortality, which is mainly due to the lack of reliable biomarkers for PDAC diagnosis/prognosis in the early stages and effective therapeutic strategies for the treatment. Cancer-derived small extracellular vesicles (sEVs), which carry various messages and signal biomolecules (e.g. RNAs, DNAs, proteins, lipids, and glycans) to constitute the key features (e.g. genetic and phenotypic status) of cancer cells, are regarded as highly competitive non-invasive biomarkers for PDAC diagnosis/prognosis. Additionally, new insights on the biogenesis and molecular functions of cancer-derived sEVs pave the way for novel therapeutic strategies based on cancer-derived sEVs for PDAC treatment such as inhibition of the formation or secretion of cancer-derived sEVs, using cancer-derived sEVs as drug carriers and for immunotherapy. This review provides a comprehensive overview of the most recent scientific and clinical research on the discovery and involvement of key molecules in cancer-derived sEVs for PDAC diagnosis/prognosis and strategies using cancer-derived sEVs for PDAC treatment. The current limitations and emerging trends toward clinical application of cancer-derived sEVs in PDAC diagnosis/prognosis and treatment have also been discussed.

pH-Mediated Clustering of Exosomes: Breaking Through the Size Limit of Exosome Analysis in Conventional Flow Cytometry.

Exosomes have recently emerged as some of the most promising biomarkers for disease diagnosis. Due to their small sizes and composition heterogeneity, exosomes are difficult to detect by currently available platforms. Here, we report a pH-mediated assembly system that converts single nanosized exosomes into microsized clusters, which can be directly analyzed by conventional flow cytometry (FCM), breaking through the size limit of exosome analysis. We demonstrated the clinical utility of the pH-mediated clustering system by profiling the exosomal proteins from blood plasma samples of 33 cancer patients and 11 benign controls. The results indicated that the combination of MUC-1 and PD-L1 could serve as a new biomarker panel for the early diagnosis of liver cancer with high clinical accuracy. This pH-mediated assembly strategy allows rapid, sensitive, and high-throughput analysis of exosome biomarkers by conventional FCM, which can be easily refined for use in both basic and clinical settings.

In-Sequence High-Specificity Dual-Reporter Unlocking of Fluorescent Probe Enables the Precise Identification of Atherosclerotic Plaques.

The formation of atherosclerotic plaques is the root cause of various cardiovascular diseases (CVDs). Effective CVD interventions thus call for precise identification of the plaques to aid clinical assessment, diagnosis, and treatment of such diseases. In this study, we introduce a dual-target sequentially activated fluorescence reporting system, termed in-sequence high-specificity dual-reporter unlocking (iSHERLOCK), to precisely identify the atherosclerotic plaques in vivo and ex vivo. ISHERLOCK was achieved by creating a three-in-one fluorescent probe that permits highly specific and sensitive detection of lipid droplets and hypochlorous acid via "off-on" and ratiometric readouts, respectively. Based on this format, the upregulated lipid accumulation and oxidative stress-the two hallmarks of atherosclerosis (AS)-were specifically measured in the atherosclerotic plaques, breaking through the barrier of precise tissue biopsy of AS and thus aiding effective CVD stewardship.

Multichannel Stimulus-Responsive Nanoprobes for H2O2 Sensing in Diverse Biological Milieus.

Hydrogen peroxide (H2O2) is widely involved in various physiological or pathological processes such as cell differentiation, proliferation, tumorigenesis, and immune responses. The accurate detection of H2O2 is highly required in many situations ranging from chemical sensing to biomedical diagnosis. However, it is exceedingly challenging to develop a single sensor that can respond to H2O2 in different conditions. Herein, a three-in-one stimulus-responsive nanoplatform (Au@MnO2@Raman reporter) was designed for colorimetry/SERS/MR tri-channel H2O2 detection which satisfied different applications. The MnO2 shell acted as a distance mediator between the gold nanoparticle (Au NP) core and the Raman reporter layer. In the presence of H2O2, the MnO2 shell is degraded, thus releasing the Mn2+ and Au NP core, which act as magnetic resonance (MR) and colorimetry signals, respectively. Simultaneously, the Raman reporters adsorb on the exposed Au NPs, resulting in the surface-enhanced Raman scattering (SERS) effect. The Au NP-based colorimetric assay was employed as H2O2 sensors for glucose detection while the turn-on signals of SERS and MR were used for H2O2 sensing and imaging in live cells and tumors, showing great versatility and flexibility of the multichannel probes in diverse situations.

Reliable Quantification of pH Variation in Live Cells Using Prussian Blue-Caged Surface-Enhanced Raman Scattering Probes.

Intracellular pH is an important parameter that is highly associated with diverse physiological processes. The reliable measurement of pH values inside cells remains a formidable challenge because of the complexity of cytoplasm. Herein, we report a robust Prussian blue (PB)-caged pH-responsive surface-enhanced Raman scattering (SERS) probe for precisely mapping the dynamic pH values in live cells. The PB shell has a subnanoscale porous structure that allows only very small biospecies such as H+ or OH- to pass freely through the shell and react with the encased pH-responsive SERS probe, while physically resisting the entry of large biomolecules. This probe achieved unmatched detection linearity (R2 > 0.999) for pH measurements in diverse complex biological samples. Moreover, the nitrile (C≡N) in PB shows a sharp band in the cellular Raman-silent region, which serves as a background-free internal standard for accurate profiling of the probe distribution inside the cells. We applied the proposed probe to monitor the dynamic pH changes during cellular autophagy induced by different stimuli and thereby demonstrated that the PB-caged probe can reliably quantify subtle intracellular pH variations, providing an effective tool for revealing the relationship between abnormal intracellular pH and cellular functions.

Chemoenzymatic Labeling of Extracellular Vesicles for Visualizing Their Cellular Internalization in Real Time.

Extracellular vesicles (EVs) are intercellular communicators that are heavily implicated in diverse pathological processes. However, it is poorly understood how EVs interact with recipient cells due to the lack of appropriate tracking techniques. Here, we report a robust chemoenzymatic labeling technique for visualizing the internalization process of EVs into target cells in real time. This method uses phospholipase D (PLD) to catalyze the in situ exchange of choline by alkyne in the native EV phosphatidylcholine. Subsequent alkyne-azide click chemistry allows conjugation of Cy5 dyes for visualizing EVs internalization by confocal fluorescence microscopy. The fluorescent labeling of EVs was accomplished in an efficient and biocompatible way, without affecting both the morphology and biological activity of EVs. We applied this chemoenzymatic labeling strategy to monitor the cellular uptake of cancer cell-derived EVs in real time and to further reveal multiple internalization mechanisms. This robust, biocompatible labeling strategy provides an essential tool for EV-related studies ranging from chemical biology to drug delivery.

Click RNA for Rapid Capture and Identification of Intracellular MicroRNA Targets.

Rapid capture and identification of the intracellular target genes of microRNAs (miRNAs) are the key to understanding miRNA functions and development of RNA-based therapeutics. However, developing biochemical tools that can fish out the target genes of miRNAs in live cells is a significant technical challenge. Here, we report a remarkably simple yet powerful technology capable of loading virtually any miRNA into Ago2 of the RNA-induced silencing complexes (RISCs). This surprising discovery enables rapid capture and identification of target mRNAs and long noncoding RNAs. It is achieved by linking dibenzocyclooctyne (DBCO), a classical chemical moiety in copper-free click chemistry, to the 3' end of miRNAs. DBCO serves as a high-affinity tag to the Ago2 protein, thus boosting the formation of RISCs with miRNA target genes in living cells. Upon cell lysing, DBCO's routine function in click chemistry allows rapid enrichment of target genes for analysis without the need of additional molecular handles. A series of miR-21 and miR-27a target genes that were previously unknown were pulled down from various cell lines and identified with qRT-PCR, demonstrating the utility of this innovative technology in both transcriptomic research and RNA-based studies.

General Approach to Engineering Extracellular Vesicles for Biomedical Analysis.

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that play critical roles in numerous pathophysiological processes. Enrichment and detection of EVs are technically challenging due to the lack of appropriate modification strategies. Herein, we propose a general, facile, and robust approach to engineering EVs by installation of maleimide (Mal) moieties onto EV surfaces based on a hydrophobic insertion strategy. Mal serves as a high-efficiency clickable handle for functionalizing EVs without influencing their structural integrity and biological activity. The Mal-installed EVs were applied into three biomedical applications: (i) labeling with a fluorescent dye for monitoring the EV-mediated cellular communication, (ii) rapid enrichment by magnetic particles (MPs) for high-efficiency EVs isolation, and (iii) conjugation with gold nanoparticles (AuNPs) for Raman detection of the surface components of EVs in situ. This technique would greatly facilitate the applications of EVs in both basic studies and clinical uses.

Encapsulating a Single Nanoprobe in a Multifunctional Nanogel for High-Fidelity Imaging of Caspase Activity in Vivo.

The formation of a protein corona on nanoprobes in the blood can not only reduce the delivery efficiency to their destination but also inhibit the functions of the nanoprobes. Herein, we report a multifunctional nanogel that can shield a single gold nanoparticle (AuNP) probe from interaction with the serum proteins, virtually eliminating protein corona formation on the nanoprobes. As a result, the delivery efficiency of the nanogel-encapsulated nanoprobes to tumors was dramatically enhanced. When the probes are delivered into target cells, the nanogel shells are degraded in acidic endosomes, where a proton sponge effect occurs instantaneously to release the AuNP probes into the cytoplasm to realize their bioimaging functions. We demonstrated the applicability of these probes for high-fidelity, noninvasive imaging of caspase activity in both cancer cells and in tumors. This strategy offers an exciting opportunity to design high-efficacy nanoprobes for in vivo imaging.

Self-Assembly of Biocompatible FeSe Hollow Nanostructures and 2D CuFeSe Nanosheets with One- and Two-Photon Luminescence Properties.

Transition metal chalcogenides are investigated for catalyst, intermediary agency, and particular optical properties because of their distinguished electron-vacancy-transfer (EVT) process toward different applications. In this work, one convenient approach for making pure-phased FeSe nanocrystals (NCs) and doped CuFeSe nanosheets (NSs) through a wet chemistry method in mixed solvents is illustrated. The surface modification of each product is realized by using a peptide molecule glutathione (GSH), in which the thiol group (-SH) is ascribed to be the in situ reducer and bonding agency between the crystalline surface and surfactant in whole constructing processes. Due to the functional groups in biological GSH, highly aggregated NCs are rebuilt in the form of an FeSe hollow structure through amino and carboxyl cross-linking functions through a spontaneous assembly procedure. Owing to the coupling procedure of Cu and Fe in the growth process, it generates enhanced EVT. Additionally, it shows the emission spectra of λEM-PL = 436 nm (FeSe) and 452 nm (CuFeSe) while λEX-PL = 356 nm, it also conveys two-photon phenomenon while λEX-PL = 720 nm. Moreover, it also shows strong off-resonant luminescence due to two-photon absorption, which should be valuable for biological applications.

Two-step signal amplification for high-sensitivity detection of biomarkers using gold nanoparticle-based conjugates.

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based, two-step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96-well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)-conjugated detection antibodies were labeled on large-size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high-sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate-specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.

Cross-Linked Poly(ethylene glycol) Shells for Nanoparticles: Enhanced Stealth Effect and Colloidal Stability.

Preventing protein corona formation and macrophage uptake is the key to improving the delivery efficiency of nanocarriers. Herein, we present a kind of cross-linking poly(ethylene glycol) (CL-PEG) shell-wrapped gold nanoparticles (namely, Au@CL-PEG NPs), which show much enhanced stealth effect and colloidal stability in physiological environments. Compared to the AuNPs coated with conventional linear PEGs (namely, Au@PEG NPs), Au@CL-PEG NPs have a greater ability to resist protein adsorption and thus show reduced cellular uptake by macrophages. In addition, the Au@CL-PEG NPs show higher chemical and colloidal stability under different extreme conditions than the conventional Au@PEG NPs. The CL-PEGylation strategy provides a new window for the surface functionalization of nanomaterials, indicating great promise for the development of high-performance nanomedicines.

In Vivo Tracking of Multiple Tumor Exosomes Labeled by Phospholipid-Based Bioorthogonal Conjugation.

Exosomes are cell-secreted nanoscale membrane vesicles that play critical roles in many pathophysiological processes. The clinical value of exosomes is under intense investigation, yet current knowledge regarding their in vivo properties is very limited because of the lack of efficient labeling techniques. Here, we report a phospholipid-based bioorthogonal labeling strategy to endow exosomes with optical probes without influencing their native biological functions. We investigated the dynamic in vivo biodistribution and organotropic uptake of multiple tumor exosomes in a single mouse. The results indicate that the exosomes derived from different cell lines show specific organotropic uptake. This phospholipid-based labeling strategy opens a new window to directly visualize and monitor exosome trafficking in living systems and holds great promise for exploring exosome-involved biological events such as cancer metastasis.

Dual-Responsive Self-Assembled Monolayer for Specific Capture and On-Demand Release of Live Cells.

We report a dual-responsive self-assembled monolayer (SAM) on a well-defined rough gold substrate for dynamic capture and release of live cells. By incorporating 5'-triphosphate (ATP) aptamer into a SAM, we can accurately isolate specific cell types and subsequently release captured cells at either population or desired-group (or even single-cell) levels. On one hand, the whole SAMs can be disassembled through addition of ATP solution, leading to the entire release of the captured cells from the supported substrate. On the other hand, desired cells can be selectively released using near-infrared light irradiation, with relatively high spatial and temporal precision. The proposed dual-responsive cell capture-and-release system is biologically friendly and is reusable with another round of modification, showing great usefulness in cancer diagnosis and molecular analysis.