Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

A new approach is presented for the application of single‐molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single‐molecule measurements. Cell‐derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution‐based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single‐molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single‐molecule studies to previously inaccessible classes of receptors.     

Multimerization of an Apoptogenic TRAIL‐Mimicking Peptide by Using Adamantane‐Based Dendrons

We have developed a straightforward strategy to multimerize an apoptogenic peptide that mimics the natural tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) by using adamantane‐based dendrons as multivalent scaffolds. The selective binding affinity of the ligands to TRAIL receptor 2 (TR2) was studied by surface plasmon resonance, thus demonstrating that the trimeric and hexameric forms of the peptide exert an increased affinity of about 1500‐ and 20 000‐fold, respectively, relative to the monomer. Moreover, only the trimeric and hexameric ligands were able to induce cell death in TR2 expressing cells (BJAB), thus confirming that a multivalent form of the peptide is necessary to trigger a substantial TR2‐dependent apoptotic response in vitro. These results provide interesting insight into the multivalency effect on biological ligand/receptor interactions for future therapeutic applications.     

Site‐Specific Encoding of Photoactivity in Antibodies Enables Light‐Mediated Antibody–Antigen Binding on Live Cells

Antibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody–antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site‐specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light‐mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12‐EGFR binding affinity by over 20‐fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12‐EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR‐positive live cancer cells in a light‐dependent manner.     

A Constant Potential Molecular Dynamics Simulation Study of the Atomic‐Scale Structure of Water Surfaces Near Electrodes

Novel and technologically important processes and phenomena arise at water surfaces in the presence of electric fields. However, experimental measurements on water surfaces are challenging, and the results are scarce and inconclusive. In this work, the constant potential molecular dynamics method, in which the electrode charges are allowed to fluctuate to keep the electric potential fixed, was implemented in the study of a near‐electrode water surface systems. This simulation system was set up with a vapor/liquid‐water/vapor slab and two electrodes under different sets of applied electrostatic potential, yielding i) a detailed characterization of the external E‐field dependent electrostatic potential/density/dipole moment density profiles, and ii) the relationship between the water surface width and the applied electrode voltage differences which has been rarely reported. The adjustments in the number density profiles in the vicinity of water surfaces due to external E‐fields were observed, while the capillary interfacial widths for the surfaces near both cathode and anode were found with different increment rates under increasing E‐fields. By examining dipole density profiles across the water surfaces, we found that external E‐field induced polarization occurs in both bulk and surface regimes, yet the surface polarization densities vary asymmetrically with respect to the increasing E‐fields. Detailed discussions were carried out to explain the correlation between water surface tension and surface widths, as well as the interplay between the surface polarization densities and the hydrogen bond network structure. We conclude that the mechanical and structural properties of the water surfaces could be tuned by both magnitude and direction of the strong external E‐fields. We also recognize that more surface properties with application value, such as dielectric permittivity tensor or surface potential, could also be regulated by the external E‐fields.     

Directed Supramolecular Surface Assembly of SNAP‐tag Fusion Proteins

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site‐selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site‐selectively labeled with bisadamantane by SNAP‐tag technology. The assembly of the bisadamantane functionalized SNAP‐fusion proteins on cyclodextrin‐coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10⁶ M ^?1 as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest‐functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP‐tag labeling of proteins thus allows for assembly of modified proteins through a host–guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP‐tag with designed supramolecular elements.     

The Polarization Effect in Surface‐Plasmon‐Induced Photocatalysis on Au/TiO2 Nanoparticles

Controlling the interaction of polarization light with an asymmetric nanostructure such as a metal/semiconductor heterostructure provides opportunities for tuning surface plasmon excitation and near‐field spatial distribution. However, light polarization effects on interfacial charge transport and the photocatalysis of plasmonic metal/semiconductor photocatalysts are unclear. Herein, we reveal the polarization dependence of plasmonic charge separation and spatial distribution in Au/TiO₂ nanoparticles under 45° incident light illumination at the single‐particle level using a combination of photon‐irradiated Kelvin probe force microscopy (KPFM) and electromagnetic field simulation. We quantitatively uncover the relationship between the local charge density and polarization angle by investigating the polarization‐dependent surface photovoltage (SPV). The plasmon‐induced photocatalytic activity is enhanced when the polarization direction is perpendicular to the Au/TiO₂ interface.     

Plasmon Coupling Enhanced Micro-Spectroscopy and Imaging for Sensitive Discrimination of Membrane Domains of a Single Cell

Different cell membrane domains play different roles in many cell processes, and the discrimination of these domains is of considerable importance for the elucidation of cellular functions. However, the strategies available for distinguishing these cell membrane domains are limited. A novel technique called plasmon coupling enhanced micro-spectroscopy and imaging to discriminate basal and lateral membrane domains of a single cell combines the application of an additional plasmonic silver film for surface plasmon (SP) excitation to selectively excite and enhance the basal membranes in the near-field with directional enhanced microscopic imaging and spectroscopy. The SP and critical evanescent fields are induced upon excitation through a silver-coated semitransparent coverslip at the surface plasmon resonance and critical angles, respectively. The basal and lateral membrane domains located within the SP and critical evanescent fields can be selectively excited and distinguished by adjusting the incident angle of laser irradiation. Moreover, the brighter images and more intense spectra of membrane-targeting fluorescence-Raman probes under directional excitation than in conventional EPI mode allow clear identification of the membrane domains.     

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.     

Probing the Functionalization of Gold Surfaces and Protein Adsorption by PM‐IRRAS

The control of morphology and coating of metal surfaces is essential for a number of organic electronic devices including photovoltaic cells and sensors. In this study, we monitor the functionalization of gold surfaces with 11‐mercaptoundecanoic acid (MUA, HS(CH₂)₁₀CO₂H) and cysteamine, aiming at passivating the surfaces for application in surface plasmon resonance (SPR) biosensors. Using polarization‐modulated infrared reflection–absorption spectroscopy (PM‐IRRAS), cyclic voltammetry, atomic force microscopy and quartz crystal microbalance, we observed a time‐dependent organization process of the adsorbed MUA monolayer with alkyl chains perpendicular to the gold surface. Such optimized condition for surface passivation was obtained with a systematic search for experimental parameters leading to the lowest electrochemical signal of the functionalized gold electrode. The ability to build supramolecular architectures was also confirmed by detecting with PM‐IRRAS the adsorption of streptavidin on the MUA‐functionalized gold. As the approaches used for surface functionalization and its verification with PM‐IRRAS are generic, one may now envisage monitoring the fabrication of tailored electrodes for a variety of applications.     

Block copolymer micelles conjugated with anti‐EGFR antibody for targeted delivery of anticancer drug

Antiepidermal growth factor receptor antibody (anti‐EGFR antibody) was conjugated with the block copolymer micelle based on poly(ethylene glycol) (PEG) and poly(ε‐caprolactone) (PCL) for active targeting to EGFR overexpressing cancer cells. Doxorubicin (DOX) was encapsulated in the core of the block copolymer (MePEG‐b‐PCL) micelle (DOX‐micelle). The mean diameters of the DOX‐micelle and the anti‐EGFR‐PEG‐b‐PCL copolymer micelles loaded with DOX (DOX‐anti‐EGFR‐micelle) were about 25 and 31 nm, respectively. The RKO human colorectal cancer cells expressing moderate degree of EGFR were incubated with free DOX, DOX‐micelle, or DOX‐anti‐EGFR‐micelle to study the distribution of DOX in the cells. When cells were incubated with free DOX, moderate degree of DOX fluorescence was observed in the nuclei. In the cells treated with DOX‐micelle, the DOX fluorescence intensity in the cytoplasm was much greater than that in the nuclei. On the other hand, the nuclei of the cells treated with DOX‐anti‐EGFR‐micelle exhibited DOX fluorescence intensity similar to that in the cytoplasm. The cytotoxicity of DOX‐anti‐EGFR‐micelle to induce apoptosis in RKO cells was significantly greater than that of free DOX or DOX‐micelle. These results demonstrated that the presence of anti‐EGFR antibody on the DOX‐micelle surface (DOX‐anti‐EGFR‐micelle) increased the internalization of the DOX‐micelle and nuclear accumulation of DOX, and enhanced the DOX‐induced cell death. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7321–7331, 2008     

Novel optical techniques for the analysis of polymer surfaces and thin films

Summary We report on novel optical techniques, based on evanescent waves, for the characterization of polymer surfaces and thin films. We first describe photo-ablation studies with polysilane films investigated by surface plasmon microscopy, a technique which is particularly well-suited for ultrathin samples. Thicker films that are homogeneous enough to carry optical waveguide modes can be characterized with high lateral resolution by the recently developed waveguide microscopy. We demonstrate this for a thin film of a solid polyelectrolyte. Finally, we report on surface plasmon field-enhanced Raman-spectroscopic and -imaging investigations of ultrathin Langmuir-Blodgett-Kuhn-layers of cadmium arachidate.W. Hickel is now with HOECHST AG, Angewandte Physik, W-6230 Frankfurt 80     

Targeted Degradation of Transcription Coactivator SRC‐1 through the N‐Degron Pathway

Aberrantly elevated steroid receptor coactivator‐1 (SRC‐1) expression and activity are strongly correlated with cancer progression and metastasis. Here we report, for the first time, the development of a proteolysis targeting chimera (PROTAC) that is composed of a selective SRC‐1 binder linked to a specific ligand for UBR box, a unique class of E3 ligases recognizing N‐degrons. We showed that the bifunctional molecule efficiently and selectively induced the degradation of SRC‐1 in cells through the N‐degron pathway. Importantly, given the ubiquitous expression of the UBR protein in most cells, PROTACs targeting the UBR box could degrade a protein of interest regardless of cell types. We also showed that the SRC‐1 degrader significantly suppressed cancer cell invasion and migration in vitro and in vivo. Together, these results demonstrate that the SRC‐1 degrader can be an invaluable chemical tool in the studies of SRC‐1 functions. Moreover, our findings suggest PROTACs based on the N‐degron pathway as a widely useful strategy to degrade disease‐relevant proteins.     

Antibody modified Bovine Serum Albumin microspheres for targeted delivery of anticancer agent Gemcitabine

Inhibition of the EGFR signaling pathway is one of the attractive therapeutic targets for pancreatic cancer as recent studies demonstrated that EGFR is over‐expressed in pancreatic cancer. In this article we have demonstrated the design of targeted drug delivery system containing Bovine Serum Albumin (BSA) microspheres as delivery vehicle, gemcitabine as anticancer drug and anti‐EGFR (epidermal growth factor receptor) monoclonal antibody as targeting agent. The conjugated BSA microspheres were characterized by several physico‐chemical techniques such as scanning electron microscope, optical microscopy, fluorescent microscopy etc. Administration of these BSA microspheres containing gemcitabine and anti‐EGFR (BSA‐Gem‐EGFR) shows significant inhibition of pancreatic cancer cells (AsPC1) compared to the cells treated with only BSA microspheres, BSA with gemcitabine (BSA‐Gem), and free gemcitabine. This strategy could be used as a generalized approach for the treatment of pancreatic cancer along with other cancers which overexpress EGFR on cell surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Surface‐Specific Functionalization of Nanoscale Metal–Organic Frameworks

A method for modifying the external surfaces of a series of nanoscale metal–organic frameworks (MOFs) with 1,2‐dioleoyl‐sn‐glycero‐3‐phosphate (DOPA) is presented. A series of zirconium‐based nanoMOFs of the same topology (UiO‐66, UiO‐67, and BUT‐30) were synthesized, isolated as aggregates, and then conjugated with DOPA to create stably dispersed colloids. BET surface area analysis revealed that these structures maintain their porosity after surface functionalization, providing evidence that DOPA functionalization only occurs on the external surface. Additionally, dye‐labeled ligand loading studies revealed that the density of DOPA on the surface of the nanoscale MOF correlates to the density of metal nodes on the surface of each MOF. Importantly, the surface modification strategy described will allow for the general and divergent synthesis and study of a wide variety of nanoscale MOFs as stable colloidal materials.     

Integrated Hollow Mesoporous Silica Nanoparticles for Target Drug/siRNA Co‐Delivery

A hollow mesoporous silica nanoparticle (HMSNP) based drug/siRNA co‐delivery system was designed and fabricated, aiming at overcoming multidrug resistance (MDR) in cancer cells for targeted cancer therapy. The as‐prepared HMSNPs have perpendicular nanochannels connecting to the internal hollow cores, thereby facilitating drug loading and release. The extra volume of the hollow core enhances the drug loading capacity by two folds as compared with conventional mesoporous silica nanoparticles (MSNPs). Folic acid conjugated polyethyleneimine (PEI‐FA) was coated on the HMSNP surfaces under neutral conditions through electrostatic interactions between the partially charged amino groups of PEI‐FA and the phosphate groups on the HMSNP surfaces, blocking the mesopores and preventing the loaded drugs from leakage. Folic acid acts as the targeting ligand that enables the co‐delivery system to selectively bind with and enter into the target cancer cells. PEI‐FA‐coated HMSNPs show enhanced siRNA binding capability on account of electrostatic interactions between the amino groups of PEI‐FA and siRNA, as compared with that of MSNPs. The electrostatic interactions provide the feasibility of pH‐controlled release. In vitro pH‐responsive drug/siRNA co‐delivery experiments were conducted on HeLa cell lines with high folic acid receptor expression and MCF‐7 cell lines with low folic acid receptor expression for comparison, showing effective target delivery to the HeLa cells through folic acid receptor meditated cellular endocytosis. The pH‐responsive intracellular drug/siRNA release greatly minimizes the prerelease and possible side effects of the delivery system. By simultaneously delivering both doxorubicin (Dox) and siRNA against the Bcl‐2 protein into the HeLa cells, the expression of the anti‐apoptotic protein Bcl‐2 was successfully suppressed, leading to an enhanced therapeutic efficacy. Thus, the present multifunctional nanoparticles show promising potentials for controlled and targeted drug and gene co‐delivery in cancer treatment.     

A Nanobody‐Conjugated DNA Nanoplatform for Targeted Platinum‐Drug Delivery

The targeted delivery of chemotherapeutic drugs is a major challenge in the clinical treatment of cancer. Herein, we constructed a multifunctional DNA nanoplatform as a versatile carrier of the highly potent platinum‐based DNA intercalator, 56MESS. In our rational design, 56MESS was efficiently loaded into the double‐bundle DNA tetrahedron through intercalation with the DNA duplex. With the integration of a nanobody that both targets and blocks epidermal growth factor receptor (EGFR), the DNA nanocarriers exhibit excellent selectivity for cells with elevated EGFR expression (a common biomarker related to tumor formation) and combined tumor therapy without obvious systemic toxicity. This DNA‐based platinum‐drug delivery system provides a promising strategy for the treatment of tumors.     

The Mechanism of Ligand‐Induced Activation or Inhibition of μ‐ and κ‐Opioid Receptors

G‐protein‐coupled receptors (GPCRs) are important targets for treating severe diseases. However why certain molecules act as activators whereas others, with similar structures, block GPCR activation, is poorly understood since the same molecule can activate one receptor subtype while blocking another closely related receptor. To shed light on these central questions, we used all‐atom, long‐time‐scale molecular dynamics simulations on the κ‐opioid and μ‐opioid receptors (κOR and μOR). We found that water molecules penetrating into the receptor interior mediate the activating versus blocking effects of a particular ligand–receptor interaction. Both the size and the flexibility of the bound ligand regulated water influx into the receptor. The solvent‐accessible inner surface area was found to be a parameter that can help predict the function of the bound ligand.     

Re‐engineering the Immune Response to Metastatic Cancer: Antibody‐Recruiting Small Molecules Targeting the Urokinase Receptor

Developing selective strategies to treat metastatic cancers remains a significant challenge. Herein, we report the first antibody‐recruiting small molecule (ARM) that is capable of recognizing the urokinase‐type plasminogen activator receptor (uPAR), a uniquely overexpressed cancer cell‐surface marker, and facilitating the immune‐mediated destruction of cancer cells. A co‐crystal structure of the ARM‐U2/uPAR complex was obtained, representing the first crystal structure of uPAR complexed with a non‐peptide ligand. Finally, we demonstrated that ARM‐U2 substantially suppresses tumor growth in vivo with no evidence of weight loss, unlike the standard‐of‐care agent doxorubicin. This work underscores the promise of antibody‐recruiting molecules as immunotherapeutics for treating cancer.     

Tumor‐Targeting of EGFR Inhibitors by Hypoxia‐Mediated Activation

The development of receptor tyrosine‐kinase inhibitors (TKIs) was a major step forward in cancer treatment. However, the therapy with TKIs is limited by strong side effects and drug resistance. The aim of this study was the design of novel epidermal growth factor receptor (EGFR) inhibitors that are specifically activated in malignant tissue. Thus, a Co^III‐based prodrug strategy for the targeted release of an EGFR inhibitor triggered by hypoxia in the solid tumor was used. New inhibitors with chelating moieties were prepared and tested for their EGFR‐inhibitory potential. The most promising candidate was coupled to Co^III and the biological activity tested in cell culture. Indeed, hypoxic activation and subsequent EGFR inhibition was proven. Finally, the compound was tested in vivo, also revealing potent anticancer activity.     

Investigating cellular signaling reactions in single attoliter vesicles

Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or cell populations. Here, we show how to monitor cellular signaling reactions at a yet unreached miniaturization level. On the basis of our observations, cytochalasin induces mammalian cells to extrude from their plasma membrane submicrometer-sized native vesicles. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic proteins in the vesicle interior. As a prototypical example, ligand binding to the ionotropic 5-HT(3) receptor and subsequent transmembrane Ca(2+) signaling were monitored in single attoliter vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signaling reactions under physiological conditions. Because a single cell delivers about 50 native vesicles, which can be isolated and addressed as individuals, our concept allows multiple functional analyses of individual cells having a limited availability and opens new vistas for miniaturized bioanalytics.