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.

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.

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.

Live-Cell Pyrophosphate Imaging by in Situ Hot-Spot Generation.

Controlling the electromagnetic hot-spot generation is essential for surface-enhanced Raman scattering (SERS) assays. Current hot-spot-based SERS assays have been extensively studied in solutions or on substrates. However, probing biospecies by controlling the hot-spot assembly in living systems has not been demonstrated thus far. Herein, we report a background-free SERS probe for imaging pyrophosphate (PPi), a biochemically significant anion, in living cells. Intracellular PPi is able to induce the nanoparticle dimerization, thus creating an intense electromagnetic hot spot and dramatically enhancing the signal of the Raman reporters residing in the hot spot. More impressively, the reporter we used in this study provides a strong and sharp single peak in the cellular Raman-silent region (1800-2800 cm-1), thus eliminating the possible background interference. This strategy could be readily extended to detect other biomarkers by only replacing the recognition ligands.

High-Precision Profiling of Sialic Acid Expression in Cancer Cells and Tissues Using Background-Free Surface-Enhanced Raman Scattering Tags.

Precise profiling of the sialic acid (SA) expression on the membrane of cancer cells is critical for early identification of cancers and assessment of cancer metastasis. However, the complex physiological environments often result in false positives with currently available imaging technologies. Herein, we have established a background-free surface-enhanced Raman scattering (SERS) imaging platform that allows high-precision profiling of SA expression in cancer cells and differentiation of clinically relevant cancer tissues with various metastasis degrees. Three-dimensional Raman imaging technique provided a deeper insight into visualizing the probe distribution and thus the SA expression at the single-cell level, without destructing the cells. This noninvasive, high-precision imaging technique could favor early diagnosis, staging, and monitoring therapeutic responses of cancers that are highly essential in clinical settings.

Gadolinium-doped Au@prussian blue nanoparticles as MR/SERS bimodal agents for dendritic cell activating and tracking.

In vivo tracking of dendritic cell (DC) migration to the lymphatic system is essential for evaluating the outcome of DC-based immunotherapies. Novel multimodal imaging strategies with high analytical performance are urgently needed to supply complementary information about the migration and colonization of DCs. In this study, we designed a bimodal imaging agent, namely Au@Prussian blue-Gd@ovalbumin nanoparticles (APG@OVA NPs), for activating DCs and real-time tracking of DC migration process by magnetic resonance imaging (MRI). Moreover, the distribution of the colonized DCs in the lymphatic system was profiled at the single-cell levels based on surface-enhanced Raman scattering (SERS) technique. Methods: In this strategy, PBs as cyanide (CN)-bridged coordination blocks were assembled onto the gold nanoparticles core to provide SERS signal in the Raman-silent region (1800 and 2800 cm-1), which could avoid background signal interference. The doping Gd3+ located in the lattice of PB enables the MRI ability with high relaxivity of the probe. Ovalbumin, an egg allergen, was used as an antigen to activate DCs due to its immunological properties. The prepared APG@OVA NP agents were used to activate DCs with high efficacy and to track their migration and distribution in vivo through SERS/MR bimodal imaging. Results: The APG@OVA NP agents could not only enable DC activating and labeling, but also achieve real-time monitoring of DC migration in vivo and accurate profiling of DC distribution in the lymphatic system. MR imaging indicated the time-dependent migration of the APG@OVA NP-labeled DCs from the footpad to the sentinel lymph node. The background-free Raman mapping of the lymph node tissue slice demonstrated that the activated DCs have successfully colonized to the sentinel lymph node. Conclusion: Concerning the high activating efficacy, dual complementary imaging readouts, and low biological toxicity, the APG@OVA NPs act as high-performance tracking agents for DC-based immunotherapies.

Peptide interdigitation-induced twisted nanoribbons as chiral scaffolds for supramolecular nanozymes.

Establishing reliable strategies for rationally manipulating the organization of peptide building blocks and thereby precisely creating chiral nanostructures is challenging, while meaningful toward development of advanced functional materials. Here we report on a peptide-interdigitating mechanism for the reliable self-assembly of lipid-inspired amphiphiles (LIPIAs) into robust twisted nanoribbons by grafting domains to one alkyl tail of lipids as an extended element. Peptide interdigitation promoted the self-assembly of LIPIAs into twisted or flat nanoribbons driven by antiparallel or parallel ß-sheet hydrogen bonds, respectively, strongly associated with the connecting direction of the incorporated domains. We found that the LIPIAs containing N-terminus-connected domains with either bulky or small side chain groups formed twisted nanoribbons in a broad pH range, thus implying a sequence- and pH-independent strategy for creation of robust chiral nanostructures. Integrating the resulting twisted nanoribbons with gold nanoparticles led to supramolecular nanozymes exhibiting the excellent catalytic activity and enantioselectivity of asymmetric oxidation of 3,4-dihyroxy-phenylalanine molecules. Our finding demonstrates that the peptide-interdigitating mechanism is a reliable strategy for precise creation of chiral nanostructures serving as chiral matrices for supramolecular nanozymes with improved catalytic performance, thus potentially paving the way towards advanced biomimetic systems resembling natural systems.

Nanozyme-assisted sensitive profiling of exosomal proteins for rapid cancer diagnosis.

The proteins expressed on exosomes have emerged as promising liquid-biopsy biomarkers for cancer diagnosis. However, molecular profiling of exosomal proteins remains technically challenging. Herein, we report a nanozyme-assisted immunosorbent assay (NAISA) that enables sensitive and rapid multiplex profiling of exosomal proteins. This NAISA system is based on the installation of peroxidase-like nanozymes onto the phospholipid membranes of exosomes, thus avoiding the need for post-labelling detection antibodies. The exosomal proteins are determined by a sensitive nanozyme-catalyzed colorimetric assay less than 3 h, without the need for multi-step incubation and washing operations. Using NAISA to profile exosomal proteins from different cell lines and clinical samples, we reveal that tumor-associated exosomal proteins can serve as promising biomarkers for accurate cancer diagnosis in a cooperative detection pattern. Methods: Exosomes were engineered with DSPE-PEG-SH through hydrophobic interaction, and then were assembled with gold nanoparticles (2 nm) to produce Exo@Au nanozyme. The proteins on Exo@Au could be selectively captured by their specific antibodies seeded into a 96-well plate. The immobilized Exo@Au shows peroxidase-like activity to perform colorimetric assays by reaction with 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2. The protein levels of exosomes were recorded on a microplate reader. Results: The NAISA platform is capable of profiling multiple exosomal proteins from both cancer cell lines and clinical samples. The expression levels of exosomal proteins, such as CD63, CEA, GPC-3, PD-L1 and HER2, were used to classify different cancer cell lines. Moreover, the protein profiles have been applied to differentiate healthy donors, hepatitis B patients, and hepatic cell carcinoma (HCC) patients with high accuracy. Conclusion: The NAISA nanozyme was allowed to rapidly profile multiple exosomal proteins and could have great promise for early HCC diagnosis and identification of other cancer types.

When Prussian Blue Meets Porous Gold Nanoparticles: A High Signal-to-Background Surface-Enhanced Raman Scattering Probe for Cellular Biomarker Imaging.

Profiling cellular biomarkers without the interference of endogenous signals could facilitate the investigation of complex intracellular biological events and provide new possibilities for precision disease diagnosis. Herein, a surface-enhanced Raman scattering (SERS) probe with a high signal-to-background ratio (SBR) for cellular biomarker imaging is constructed. The probes are prepared by incorporating Prussian blue (PB) with porous gold nanoparticles (p-Au NPs). Due to their rich built-in Raman hotspots, the p-Au NPs are excellent SERS substrates that can significantly amplify the signals of the incorporated PB. In parallel, PB shows a single peak in the cellular silent region, where the signals from the probes and endogenous molecules can be completely resolved without the need of complex spectral unmixing. As a consequence, the combination of probe signal enhancement and background elimination endows the SERS probes with an extremely high SBR. To evaluate their performance in biomarker imaging, the high-SBR SERS probes are utilized to profile folic acids at a single-cell level. This background-free, high-precision imaging technique is conducive to early diagnosis and therapeutic response of cancer that is of great importance in clinical settings.