Photofunctional Nanomodulators for Bioexcitation

A single organism comprises diverse types of cells. To acquire a detailed understanding of the biological functions of each cell, comprehensive control and analysis of homeostatic processes at the single‐cell level are required. In this study, we develop a new type of light‐driven nanomodulator comprising dye‐functionalized carbon nanohorns (CNHs) that generate heat and reactive oxygen species under biologically transparent near‐infrared (NIR) laser irradiation. By exploiting the physicochemical properties of the nanohorns, cellular calcium ion flux and membrane currents were successfully controlled at the single‐cell level. In addition, the nanomodulator allows a remote bioexcitation of tissues during NIR laser exposure making this system a powerful tool for single‐cell analyses and innovative cell therapies.     

High‐Throughput Isolation of Cell Protrusions with Single‐Cell Precision for Profiling Subcellular Gene Expression

Invading cancer cells extend cell protrusions, which guide cancer‐cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single‐cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high‐throughput isolation of cell protrusions at single‐cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell‐protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single‐cell precision for subsequent RNA‐Seq analysis, which was further validated by RT‐qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.     

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea‐grafted polyethylenimine (πPEI) with affinity‐purified His‐tagged proteins pre‐organized onto a nickel‐immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His‐tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His‐tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single‐chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.     

Probing Low‐Copy‐Number Proteins in a Single Living Cell

Single‐cell analysis techniques are essential for understanding the microheterogeneity and functions of cells. Low‐copy‐number proteins play important roles in cell functioning, but their measurement in single cells remains challenging. Herein, we report an approach, called plasmonic immunosandwich assay (PISA), for probing low‐copy‐number proteins in single cells. This approach combined in vivo immunoaffinity extraction and plasmon‐enhanced Raman scattering (PERS). Target proteins were specifically extracted from the cells by microprobes modified with monoclonal antibody or molecularly‐imprinted polymer (MIP), followed by labeling with Raman‐active nanotags. The PERS detection, with Raman intensity enhanced by 9 orders of magnitude, provided ultrasensitive detection at the single‐molecule level. Using this approach, we found that alkaline phosphatase and survivin were expressed in distinct levels in cancer and normal cells, and that extended culture passage resulted in reduced expression of survivin. We further developed acupuncture needle‐based PISA for probing low‐copy‐number proteins in living bodies.     

A valve‐based microfluidic device for on‐chip single cell treatments

Assays toward single‐cell analysis have attracted the attention in biological and biomedical researches to reveal cellular mechanisms as well as heterogeneity. Yet nowadays microfluidic devices for single‐cell analysis have several drawbacks: some would cause cell damage due to the hydraulic forces directly acting on cells, while others could not implement biological assays since they could not immobilize cells while manipulating the reagents at the same time. In this work, we presented a two‐layer pneumatic valve‐based platform to implement cell immobilization and treatment on‐chip simultaneously, and cells after treatment could be collected non‐destructively for further analysis. Target cells could be encapsulated in sodium alginate droplets which solidified into hydrogel when reacted with Ca²⁺. The size of hydrogel beads could be precisely controlled by modulating flow rates of continuous/disperse phases. While regulating fluid resistance between the main channel and passages by the integrated pneumatic valves, on‐chip capture and release of hydrogel beads was implemented. As a proof of concept for on‐chip single‐cell treatments, we showed cellular live/dead staining based on our devices. This method would have potential in single cell manipulation for biochemical cellular assays.     

Highly Multiplexed Single‐Cell In Situ Protein Analysis with Cleavable Fluorescent Antibodies

Limitations on the number of proteins that can be quantified in single cells in situ impede advances in our deep understanding of normal cell physiology and disease pathogenesis. Herein, we present a highly multiplexed single‐cell in situ protein analysis approach that is based on chemically cleavable fluorescent antibodies. In this method, antibodies tethered to fluorophores through a novel azide‐based cleavable linker are utilized to detect their protein targets. After fluorescence imaging and data storage, the fluorophores coupled to the antibodies are efficiently cleaved without loss of protein target antigenicity. Upon continuous cycles of target recognition, fluorescence imaging, and fluorophore cleavage, this approach has the potential to quantify over 100 different proteins in individual cells at optical resolution. This single‐cell in situ protein profiling technology will have wide applications in signaling network analysis, molecular diagnosis, and cellular targeted therapies.     

Reaction‐Diffusion Systems in Intracellular Molecular Transport and Control

Chemical reactions make cells work only if the participating chemicals are delivered to desired locations in a timely and precise fashion. Most research to date has focused on active‐transport mechanisms, although passive diffusion is often equally rapid and energetically less costly. Capitalizing on these advantages, cells have developed sophisticated reaction‐diffusion (RD) systems that control a wide range of cellular functions—from chemotaxis and cell division, through signaling cascades and oscillations, to cell motility. These apparently diverse systems share many common features and are “wired” according to “generic” motifs such as nonlinear kinetics, autocatalysis, and feedback loops. Understanding the operation of these complex (bio)chemical systems requires the analysis of pertinent transport‐kinetic equations or, at least on a qualitative level, of the characteristic times of the constituent subprocesses. Therefore, in reviewing the manifestations of cellular RD, we also describe basic theory of reaction‐diffusion phenomena.     

Morphometric Cell Classification for Single‐Cell MALDI‐Mass Spectrometry Imaging

The large‐scale and label‐free molecular characterization of single cells in their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single‐cell‐specific molecular profiles. This approach provides a new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features.     

Mannose‐6‐Phosphate Receptor: A Target for Theranostics of Prostate Cancer

The development of personalized and non‐invasive cancer therapies based on new targets combined with nanodevices is a major challenge in nanomedicine. In this work, the over‐expression of a membrane lectin, the cation‐independent mannose 6‐phosphate receptor (M6PR), was specifically demonstrated in prostate cancer cell lines and tissues. To efficiently target this lectin a mannose‐6‐phosphate analogue was synthesized in six steps and grafted onto the surface of functionalized mesoporous silica nanoparticles (MSNs). These MSNs were used for in vitro and ex vivo photodynamic therapy to treat prostate cancer cell lines and primary cell cultures prepared from patient biopsies. The results demonstrated the efficiency of M6PR targeting for prostate cancer theranostic.     

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere‐Mediated Microfluidic Platform

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single‐cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere‐mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single‐cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral‐readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.     

Reversible Re‐programing of Cell–Cell Interactions

The ability to engineer and re‐program the surfaces of cells would provide an enabling synthetic biological method for the design of cell‐ and tissue‐based therapies. A new cell surface‐engineering strategy is described that uses lipid‐chemically self‐assembled nanorings (lipid‐CSANs) that can be used for the stable and reversible modification of any cell surface with a molecular reporter or targeting ligand. In the presence of a non‐toxic FDA‐approved drug, the nanorings were quickly disassembled and the cell–cell interactions reversed. Similar to T‐cells genetically engineered to express chimeric antigen receptors (CARS), when activated peripheral blood mononuclear cells (PBMCs) were functionalized with the anti‐EpCAM‐lipid‐CSANs, they were shown to selectively kill antigen‐positive cancer cells. Taken together, these results demonstrate that lipid‐CSANs have the potential to be a rapid, stable, and general method for the reversible engineering of cell surfaces and cell–cell interactions.     

Fluorescent PEGulated Oligourethane Nanoparticles for Long‐Term Cellular Tracing

We have introduced a new ABA‐type amphiphilic block copolymer consisting of functional oligourethane hydrophobic blocks and two polyethylene glycol (PEG) hydrophilic blocks. The polymer was synthesized in a single step by step‐growth polymerization between two monomers, namely tetraphenylethylene (TPE)‐diol and hexamehylene di‐isocyanate in the presence of a monofunctional impurity PEG‐2000. The polymer exhibits facile self‐assembly in water by synergistic effects of H‐bonding and π–π interaction among the oligourethane core, leading to the formation of robust nanoparticles with remarkable aggregation‐induced emission (AIE). These nanoparticles show very low critical aggregation concentration, stability over a large pH window, and excellent biocompatibility as revealed by an MTT assay. Cellular imaging with cancer cells showed facile cellular uptake and, more importantly, retention of AIE in cellular milieu for long times, which was successfully utilized for long‐term cancer cell tracking.     

Lysosome‐targeted potent half‐sandwich iridium(III) α‐diimine antitumor complexes

Fifteen organometallic Ir(III) half‐sandwich complexes ( 1A – 5C ) having the general formula [(η⁵‐Cp^x)Ir(N^N)Cl]PF₆ (Cp^x = Cp*, tetramethyl(phenyl)cyclopentadienyl (Cp^xph) or tetramethyl(biphenyl)cyclopentadienyl (Cp^xbiph); N^N = diamine) have been synthesized and characterized. The molecular structure of 1A was determined using single‐crystal X‐ray diffraction analysis. The hydrolysis of 1A – 5C was monitored using UV–visible spectra. Complexes 3A – 3C showed catalytic activity for the oxidation of NADH to NAD⁺, where 3C showed the highest turnover number of 29.9 within 450 min. Cytotoxicity examination by MTT assay was carried out against two human cancer cell lines (HeLa and A549) after 24 or 48 h drug treatment. The complexes showed high potency, where the most potent complex ( 3C ; IC₅₀ = 3.4 μM) was six times more active than cisplatin against A549 cells after 24 h drug exposure. Cytotoxic potency towards A549 cells increased with phenyl substitution on Cp ring: Cp^xbiph > Cp^xph > Cp*. In addition, the biological studies showed that 3C caused cell apoptosis and cell cycle arrest at G₁ phase in A549 cancer cells. Moreover, 3C increased the level of reactive oxygen species markedly after 24 h, which may provide an important basis for killing cancer cells. Confocal laser scanning microscopy was used to track 3C in A549 cells. The cellular localization experiment showed that 3C targeted lysosomes and caused lysosomal damage.     

Fluorescent Mimetics of CMP‐Neu5Ac Are Highly Potent,Cell‐Permeable Polarization Probes of Eukaryotic and Bacterial Sialyltransferases and Inhibit Cellular Sialylation

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate‐nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation.     

Design of the First‐in‐Class,Highly Potent Irreversible Inhibitor Targeting the Menin‐MLL Protein–Protein Interaction

The structure‐based design of M‐525 as the first‐in‐class, highly potent, irreversible small‐molecule inhibitor of the menin‐MLL interaction is presented. M‐525 targets cellular menin protein at sub‐nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL‐regulated gene expression in MLL leukemia cells. M‐525 demonstrates high cellular specificity over non‐MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co‐crystal structure of M‐525 in complex with menin firmly establish its mode of action. A single administration of M‐525 effectively suppresses MLL‐regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M‐525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.     

Design of the First‐in‐Class,Highly Potent Irreversible Inhibitor Targeting the Menin‐MLL Protein–Protein Interaction

The structure‐based design of M‐525 as the first‐in‐class, highly potent, irreversible small‐molecule inhibitor of the menin‐MLL interaction is presented. M‐525 targets cellular menin protein at sub‐nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL‐regulated gene expression in MLL leukemia cells. M‐525 demonstrates high cellular specificity over non‐MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co‐crystal structure of M‐525 in complex with menin firmly establish its mode of action. A single administration of M‐525 effectively suppresses MLL‐regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M‐525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.     

Paclitaxel‐Loaded Stabilizer‐Free Poly(D,L‐lactide‐co‐glycolide) Nanoparticles Conjugated with Quantum Dots for Reversion of Anti‐Cancer Drug Resistance and Cancer Cellular Imaging

We prepared the PLGA‐loaded anti‐cancer drug and coated it with quantum dots to make it a dual‐function nanoparticles, and analyzed its potential use in cellular imaging and curing cancers. Two cancer cell lines, paclitaxel‐sensitive KB and paclitaxel‐resistant KB paclitaxel‐50 cervical carcinoma cells, were the relativistic models for analysis of the cytotoxicity of free paclitaxel and paclitaxel‐loaded PLGA conjugated with quantum‐dot nanoparticles. The paclitaxel‐loaded PLGA conjugated with quantum dots nanoparticles were significantly more cytotoxic than the free paclitaxel drug in paclitaxel‐resistant KB paclitaxel‐50 cells. This might have been because the cancer cells developed multi‐drug resistance (MDR), which hampered the action of free paclitaxel by pumping its molecules to extracellular areas. Addition of verapamil, a P‐glycoprotein inhibitor, reversed the MDR mechanism and significantly reduced KB paclitaxel‐50 cell viability. As a result, KB paclitaxel‐50 was highly associated with MDR on the cell membrane. The cytotoxicity results indicated that PLGA nanoparticles served as drug carriers and protected the drugs from MDR‐accelerated efflux. Combined quantum dots with PLGA nanoparticles allowed additional functionality for cellular imaging.     

CD133 and CD133‐regulated nucleophosmin linked to 5‐fluorouracil susceptibility in human colon cancer cell line SW620

Cancer stem cells (CSCs) are known to be resistant to conventional chemotherapy and radiotherapy. Specific CSC targeting and eradication is therefore a therapeutically important challenge. CD133 is a colorectal CSC marker with unknown function(s). Assessing proteomic changes induced by CD133 may provide clues not only to new CD133 functions but also to the chemotherapy and radiation susceptibility of colon cancer cells. To identify the proteins affected by CD133, CD133‐positive (CD133+), and CD133‐negative (CD133–) human colon cancer cells were obtained by cell sorting. Whole proteomes were profiled from SW620/CD133+ and SW620/CD133– cells and analyzed by 2D‐based proteome analysis. Nucleophosmin (NPM1) was identified as a protein regulated by CD133. CD133 protein level was not affected by NPM1, and an interaction between the two proteins was not observed. CD133 and NPM1 protein levels were positively correlated in 11 human colon cancer cell lines. The CD133+ subpopulation percentage or its value normalized against CD133 protein level was only linked to intrinsic susceptibility of human colon cancer cells to 5‐fluorouracil (5‐FU). However, either suppression of CD133 or NPM1 significantly increased 5‐FU susceptibility of SW620. The present study suggests that CD133‐regulated NPM1 protein level may provide a clue to novel CD133 function(s) linked to human colon cancer cell susceptibility to chemotherapy.