Insights into <i>In Vivo</i> Environmental Effects on Quantitative Biochemistry in Single Cells

Biomacromolecules exist and function in a crowded and spatially confined intracellular milieu. Single-cell analysis has been an essential tool for deciphering the molecular mechanisms of cell biology and cellular heterogeneity. However, a sound understanding of in vivo environmental effects on single-cell quantification has not been well established. In this study, via cell mimicking with giant unilamellar vesicles and single-cell analysis by an approach called plasmonic immunosandwich assay (PISA) that we developed previously, we investigated the effects of two in vivo environmental factors, i.e., molecular crowding and spatial confinement, on quantitative biochemistry in the cytoplasm of single cells. We find that molecular crowding greatly affects the biomolecular interactions and immunorecognition-based detection while the effect of spatial confinement in cell-sized space is negligible. Without considering the effect of molecular crowding, the results by PISA were found to be apparently under-quantitated, being only 29.5–50.0% of those by the calibration curve considering the effect of molecular crowding. We further demonstrated that the use of a calibration curve established with standard solutions containing 20% (wt) polyethylene glycol 6000 can well offset the effect of intracellular crowding and thereby provide a simple but accurate calibration for the PISA measurement. Thus, this study not only sheds light on how intracellular environmental factors influence biomolecular interactions and immunorecognition-based single-cell quantification but also provides a simple but effective strategy to make the single-cell analysis more accurate

Molecularly Imprinted and Cladded Nanoparticles Provide Better Phosphorylation Recognition

Phosphorylation is one of the most frequently occurring post-translation modifications in mammals. Because abnormal protein phosphorylation is related to many diseases, phosphorylation analysis is essential for a sound understanding of protein phosphorylation and its relationship with diseases. Among several types of reagents for phosphorylation recognition, molecularly imprinted polymers (MIPs), as synthetic mimics of antibodies, have exhibited unique strengths that can overcome the drawbacks of biological reagents. However, the performance of current MIPs has remained unideal. Meanwhile, while the currently existing imprinting methods have permitted the production of several material formats, such as crushed particles and mesoporous nanoparticles, a general method allowing for the preparation of monodispersed molecularly imprinted nanoparticles has not been developed yet. Herein, we report a new approach called reverse microemulsion template docking surface imprinting and cladding (RMTD-SIC) for facile preparation of monodispersed imprinted nanoparticles for better phosphorylation recognition. Through rational design and controllable engineering, monodisperse imprinted and cladded nanoparticles specific to general phosphorylation and tyrosine phosphorylation were synthesized, which yield the highest imprinting factors as compared with published studies. The prepared nanomaterials exhibited excellent specificity and affinity, allowing for specific enrichment and improved mass spectrometric identification of target phosphorylated peptides from complex samples containing 100-fold more abundant interfering peptides. Therefore, the RMTD-SIC approach holds great potential for phosphorylation analysis and phosphorylation recognition-based applications

Single-Cell Plasmonic Immunosandwich Assay Reveals the Modulation of Nucleocytoplasmic Localization Fluctuation of ABL1 on Cell Migration

Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1’s shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1’s to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein–protein interactions between 14-3-3 and ABL1 modulate ABL1’s nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1’s nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer

Hierarchically Structured Molecularly Imprinted Nanotransducers for Truncated HER2-Targeted Photodynamic Therapy of Therapeutic Antibody-Resistant Breast Cancer

Antibodies have been a mainstream class of therapeutics for clinical treatment of various diseases, especially cancers. However, mutation in cancer cells leads to resistance to therapeutic antibodies, hyperactivity of proliferation of cancer cells, and difficulty in the development of therapeutic antibodies. Herein, we present a strategy termed molecularly imprinted nanotransducer (MINT) for targeted photodynamic therapy (PDT) of mutated cancers. The MINT is a rationally engineered nanocomposite featuring a core of an upconversion nanoparticle, a shell of a thin layer of molecularly imprinted polymer, and a photosensitizer modified on the surface. As a proof-of-principle, truncated HER2 (P95HER2) overexpressed breast cancer, a challenging cancer lacking effective targeted therapeutics, was used as the cancer model. The designed structure, properties, functions, and anticancer efficacy of MINT were systematically investigated and experimentally confirmed. The MINT could not only specifically target P95HER2+ cancer cells in vitro and in vivo but also efficiently transfer the irradiated light and generate excited-state oxygen, resulting in efficient targeted cancer killing. Therefore, the MINT strategy provides a promising therapeutic for targeted PDT of drug-resistant cancers caused by target mutation

Redox-Responsive Molecularly Imprinted Nanoparticles for Targeted Intracellular Delivery of Protein toward Cancer Therapy

Although protein therapeutics is of significance in therapeutic intervention of cancers, controlled delivery of therapeutic proteins still faces substantial challenges including susceptibility to degradation and denaturation and poor membrane permeability. Herein, we report a sialic acid (SA)-imprinted biodegradable silica nanoparticles (BS-NPs)-based protein delivery strategy for targeted cancer therapy. Cytotoxic ribonuclease A (RNase A) was effectively caged in the matrix of disulfide-hybridized silica NPs (encapsulation efficiency of ∼64%), which were further functionalized with cancer targeting capability via surface imprinting with SA as imprinting template. Such nanovectors could not only maintain high stability in physiological conditions but also permit redox-triggered biodegradation for both concomitant release of the loaded therapeutic cargo and in vivo clearance. In vitro experiments confirmed that the SA-imprinted RNase A@BS-NPs could selectively target SA-overexpressed tumor cells, promote cells uptake, and subsequently be cleaved by intracellular glutathione (GSH), resulting in rapid release kinetics and enhanced cell cytotoxicity. In vivo experiments further confirmed that the SA-imprinted RNase A@BS-NPs had specific tumor-targeting ability and high therapeutic efficacy of RNase A in xenograft tumor model. Due to the specific targeting and traceless GSH-stimulated intracellular protein release, the SA-imprinted BS-NPs provided a promising platform for the delivery of biomacromolecules in cancer therapy

Single-Cell Plasmonic Immunosandwich Assay Reveals the Modulation of Nucleocytoplasmic Localization Fluctuation of ABL1 on Cell Migration

Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1’s shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1’s to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein–protein interactions between 14-3-3 and ABL1 modulate ABL1’s nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1’s nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer

Dual Biomimetic Recognition-Driven Plasmonic Nanogap-Enhanced Raman Scattering for Ultrasensitive Protein Fingerprinting and Quantitation

Protein assays with fingerprints and high sensitivity are essential for biomedical research and applications. However, the prevailing methods mainly rely on indirect or labeled immunoassays, failing to provide fingerprint information. Herein, we report a dual biomimetic recognition-driven plasmonic nanogap-enhanced Raman scattering (DBR-PNERS) strategy for ultrasensitive protein fingerprinting and quantitation. A pair of molecularly imprinted nanoantennas were rationally engineered for specifically trapping a target protein into well-defined plasmonic nanogaps through dual-terminal recognition for ultrahigh Raman signal amplification. Meanwhile, a Raman-active small molecule was embedded into the nanoantenna as an internal standard to provide a ratiometric assay for robust quantitation. DBR-PNERS exhibited several significant merits over existing approaches, including fingerprinting, ultrahigh sensitivity, quantitation robustness, speed, sample consumption, and so on. Therefore, it can be a promising tool for a protein assay and holds a great perspective in important applications

Single-Cell Plasmonic Immunosandwich Assay Reveals the Modulation of Nucleocytoplasmic Localization Fluctuation of ABL1 on Cell Migration

Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1’s shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1’s to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein–protein interactions between 14-3-3 and ABL1 modulate ABL1’s nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1’s nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer

Boosting Chemodynamic Therapy by Tumor-Targeting and Cellular Redox Homeostasis-Disrupting Nanoparticles

Chemodynamic therapy (CDT) that kills tumor cells by converting low-reactivity H2O2 into highly toxic hydroxyl radicals (•OH) is an emerging tumor therapeutic modality, but its therapeutic efficacy is largely limited by both the lack of tumor targeting and redox homeostasis in tumor cells. Herein, we report Cu2+-encapsulated and GalNAc-imprinted biodegradable silica nanoparticles (nanoMIP) for boosting CDT. In this strategy, the Cu2+ was first encapsulated into disulfide-bridged silica nanoparticles with a high loading capacity of ∼18.3%, followed by in situ functionalization via molecular imprinting using GalNAc as a template. Such a nanovector could specifically target tumor cells overexpressing the Tn antigen to promote the cellular uptake. After internalization into tumor cells, the degradation of nanoMIP occurred in response to the tumor microenvironment, spontaneously releasing Cu2+/Cu+ via redox cycles, which in turn promoted highly potent GSH depletion and triggered •OH generation by a Fenton-like reaction. Notably, we found that the catalase activity could be effectively inhibited by the produced Cu+, which indirectly upregulated the endogenous H2O2 level. As a result, the “maladjusted” tumor cells lost the resistance against •OH damage, finally resulting in the apoptosis of tumor cells. In vitro and in vivo experiments demonstrated that our nanoMIP exhibited excellent cytotoxicity against tumor cells and high efficacy of tumor inhibition in the xenograft tumor model with negligible side effects. Taken together, our study provides not only a promising strategy for maximizing the CDT efficacy but also a new insight for developing MIP-based nanomedicine