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.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Molecularly Imprinted Nanobeacons Redirect Innate Immune Killing towards Triple Negative Breast Cancer.

Harnessing innate immunity is an appealing strategy for cancer treatment. Herein, we report a new strategy called molecularly imprinted nanobeacons (MINBs) for redirecting innate immune killing towards triple-negative breast cancer (TNBC). The MINBs were molecularly imprinted nanoparticles with the N-epitope of glycoprotein nonmetastatic B (GPNMB) as the template and grafted with plentiful fluorescein moieties as the hapten. The MINBs could tag the TNBC cells via binding with GPNMB and thereby provide navigation for recruiting hapten-specific antibodies. The gathered antibodies could further trigger effective Fc-domain-mediated immune killing towards the tagged cancer cells. In vivo experiments showed that the TNBC growth was significantly inhibited after MINBs treatment by intravenous injection as compared with control groups. This study not only opens a new access for redirecting innate immunity towards TNBC but also paves the way for innate immunity-based therapy of other diseases.

Molecularly imprinted and cladded polymers for constructing a portable plasmonic immunoassay for peptides in biofluids.

Using two molecularly imprinted and cladded polymers (cMIPs), an inexpensive, fast and portable plasmonic immuno-sandwich assay (PISA) was rationally developed for high-specificity and ultra-sensitive detection of C-peptide in urine. The dual cMIPs-based PISA allowed healthy individuals to be distinguished from diabetes patients and exhibited several significant merits over existing immunoassays, holding great promise in clinical diagnosis.

Targeted degradation of ABCG2 for reversing multidrug resistance by hypervalent bispecific gold nanoparticle-anchored aptamer chimeras.

Hypervalent bispecific gold nanoparticle-anchored aptamer chimeras (AuNP-APTACs) were designed as a new tool of lysosome-targeting chimeras (LYTACs) for efficient degradation of the ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) to reverse multidrug resistance (MDR) of cancer cells. The AuNP-APTACs could effectively increase the accumulation of drugs in drug-resistant cancer cells and provide comparable efficacy to small-molecule inhibitors. Thus, this new strategy provides a new way to reverse MDR, holding great promise in cancer therapy.

Molecular imprinting and cladding produces antibody mimics with significantly improved affinity and specificity.

Molecularly imprinted polymers (MIPs), as important mimics of antibodies, are chemically synthesized by polymerization in the presence of a target compound. MIPs have found wide applications in important fileds. However, the current molecular imprinting technology suffers from a dilemma; there is often a compromise between the best affinity and the best specificity for MIPs prepared under optimized conditions. Herein, we proposed a new strategy called molecular imprinting and cladding (MIC) to solve this issue. The principle is straightforward; after molecular imprinting, a chemically inert cladding thinlayer is generated to precisely cover non-imprinted area. We further proposed a special MIC approach for controllably engineering protein binders. The prepared cladded MIPs (cMIPs) exhibited significantly improved affinity and specificity. The general applicability of the proposed strategy and method was verified by engineering of cMIPs for the recognition of a variety of different proteins. The feasibility of cMIPs for real applications was demonstrated by fluorescence imaging of cancer cells against normal cells and immunoassay of C-peptide in human urine. This study opened up a new avenue for controllably engineering protein-specific antibody mimics with excellent recognition properties, holding great prospective in important applications such as disease diagnosis and nanomedicine.

Rational development of molecularly imprinted nanoparticles for blocking PD-1/PD-L1 axis.

Blocking the PD-1/PD-L1 immune checkpoint has emerged as a promising strategy in cancer immunotherapy, in which monoclonal antibodies are predominately used as inhibitors. Despite their remarkable success, monoclonal antibody-based therapeutics suffer from drawbacks due to the use of antibodies, such as high cost, low stability and high frequency of immune-related adverse effects. Therefore, novel anti-PD-1/PD-L1 therapeutics that can address these issues are of significant importance. Herein, we report a molecularly imprinted polymer (MIP) based PD-1 nano inhibitor for blocking the PD-1/PD-L1 axis. The anti-PD-1 nanoMIP was rationally designed and engineered by epitope imprinting using the N-terminal epitope of PD-1 as the binding site. The anti-PD-1 nanoMIP showed good specificity and high affinity towards PD-1, yielding a disassociation constant at the 10-8 M level, much better than that between PD-1 and PD-L1. Via steric hindrance, this inhibitor could effectively block PD-1/PD-L1 interaction. Besides, it could effectively reactivate T cells and reverse the chemoresistance of tumor cells. Therefore, this present study not only provides a novel and promising immune checkpoint blockade inhibitor but also boosts further development of MIPs for cancer immunotherapy.

Molecularly Imprinted and Cladded Nanotags Enable Specific SERS Bioimaging of Tyrosine Phosphorylation.

Tyrosine phosphorylation is an important post-translational modification of proteins, and its accurate analysis is of vital importance. However, due to limited abundance of tyrosine phosphorylation as well as severe interference of serine/threonine phosphorylation and other phosphate-containing species, approaches that can directly analyse tyrosine phosphorylation on the cell membrane still remain limited. Herein, we report the rational development of molecularly imprinted and cladded Raman nanotags and their successful application in surface enhanced Raman spectroscopy (SERS) imaging of tyrosine phosphorylation on cancerous cells and tissues. The prepared molecularly imprinted and cladded SERS nanotags could specifically recognize phosphotyrosine and thereby allowed for distinguishing phosphotyrosine from other phosphate-containing species on cancerous cells and tissues by SERS imaging. Therefore, the molecularly imprinted and cladded nanotags-based SERS imaging can be a promising tool for tyrosine phosphorylation analysis and tyrosine phosphorylation-related studies, showing great potential for biomedical applications.

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.

Rationally Screened and Designed ABCG2-Binding Aptamers for Targeting Cancer Stem Cells and Reversing Multidrug Resistance.

The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2), as an important member of ABC transporters, plays a key role in multidrug resistance (MDR) in cancer and has been widely considered as a marker of cancer stem cells (CSC). Reagents capable of simultaneously targeting ABCG2 and reversing MDR have great clinical application values, but their development is highly challenging. Herein, ABCG2 glycosylated extracellular region-binding aptamers were efficiently screened by a cladded molecularly imprinted polymer (cMIP)-based in vitro screening method and further rationally engineered into cyclic bivalent aptamers. Experiments showed that both the monovalent and cyclic bivalent aptamers could specifically bind ABCG2 and thereby specially target CSC of human colorectal carcinomas (CoCSC), while the latter could effectively reverse MDR in drug-resistant liver cancer cells (HepG2/ADR). Different from currently predominant small molecule inhibitors, the reversal of MDR relied on a different mechanism; the cyclic bivalent aptamers bound the two monomers of ABCG2 dimers simultaneously and thereby blocked the ABCG2-mediated drug-pumping channel, resulting in increased intracellular accumulation of substrate drugs. This study opened a new access to the development of affinity reagents for targeting CSC and reversing MDR, holding great prospects in cancer diagnosis and treatment.

Controllable Engineering and Functionalizing of Nanoparticles for Targeting Specific Proteins towards Biomedical Applications.

Nanoparticles have been widely used in important biomedical applications such as imaging, drug delivery, and disease therapy, in which targeting toward specific proteins is often essential. However, current targeting strategies mainly rely on surface modification with bioligands, which not only often fail to provide desired properties but also remain challenging. Here an unprecedented approach is reported, called reverse microemulsion-confined epitope-oriented surface imprinting and cladding (ROSIC), for facile, versatile, and controllable engineering coreless and core/shell nanoparticles with tunable monodispersed size as well as specific targeting capability toward proteins and peptides. Via engineering coreless imprinted and cladded silica nanoparticles, the effectiveness and superiority over conventional imprinting of the proposed approach are first verified. The prepared nanoparticles exhibit both high specificity and high affinity. Using quantum dots, superparamagnetic nanoparticles, silver nanoparticles, and upconverting nanoparticles as a representative set of core substrates, a variety of imprinted and cladded single-core/shell nanoparticles are then successfully prepared. Finally, using imprinted and cladded fluorescent nanoparticles as probes, in vitro targeted imaging of triple-negative breast cancer (TNBC) cells and in vivo targeted imaging of TNBC-bearing mice are achieved. This approach opens a new avenue to engineering of nanoparticles for targeting specific proteins, holding great prospects in biomedical applications.

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.

Dendritic Mesoporous Silica Nanospheres: Toward the Ultimate Minimum Particle Size for Ultraefficient Liquid Chromatographic Separation.

Use of smaller particle size of packing materials in liquid chromatography leads to faster separation and higher efficiency. This basic law has driven the evolution of packing materials for several generations. However, the use of nanoscale packing materials has been severely hampered by extremely high back pressure. Here, we report a new possibility of solving this issue via introducing novel nanomaterials with highly favorable structures. n-Octyl-modified monodispersed dendritic mesoporous silica nanospheres (DMSNs) with an unprecedentedly small diameter (ca. 170 nm) and appropriate pore size (5.6 nm) were controllably synthesized and demonstrated to be a practically applicable packing material offering ultrahigh efficiency. The center-radial centrosymmetric mesopore channels significantly improved the permeability of packed capillaries, enabling column packing and capillary electrochromatographic separation on regular instruments. Due to the unique morphology, very tiny particle size, and highly uniform packing, the packed column exhibited ultrahigh efficiency up to 3 500 000 plates/m. Powerful separation capability was demonstrated with glycan profiling of cancerous and normal cells, which revealed that cancerous cells exhibited characteristic N-glycans. Because DMSNs with tunable particle size and mesopores can be controllably prepared, DMSNs hold great potential to be a new record toward the ultimate generation of packing materials for ultraefficient liquid chromatographic separation.

Efficient Screening of Glycan-Specific Aptamers Using a Glycosylated Peptide as a Scaffold.

Abnormal glycan structures are valuable biomarkers for disease states; the development of glycan-specific binders is thereby significantly important. However, the structural homology and weak immunogenicity of glycans pose major hurdles in the evolution of antibodies, while the poor availability of complex glycans also has extremely hindered the selection of anti-glycan aptamers. Herein, we present a new approach to efficiently screen aptamers toward specific glycans with a complex structure, using a glycosylated peptide as a scaffold. In this method, using peptide-imprinted magnetic nanoparticles (MNPs) as a versatile platform, a glycopeptide tryptically digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection. Alternating positive and negative selection steps were carried out to guide the directed evolution of glycan-binding aptamers. As proof of the principle, the biantennary digalactosylated disialylated N-glycan A2G2S2, against which there have been no antibodies and lectins so far, was employed as the target. With the glycoprotein transferrin as a source of target glycan, two satisfied anti-A2G2S2 aptamers were selected within seven rounds. Since A2G2S2 is upregulated in cancerous liver cells, carboxyfluorescein (FAM)-labeled aptamers were prepared as fluorescent imaging reagents, and successful differentiation of cancerous liver cells over normal liver cells was achieved, which demonstrated the application feasibility of the selected aptamers. This approach obviated a tedious glycan preparation process and allowed favorable expose of the intrinsic flexible conformation of natural glycans. Therefore, it holds great promise for developing glycan-specific aptamers for challenging applications such as cancer targeting.