Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

The expression of recombinant antibody fragments on the surface of mammalian cells has recently emerged as a therapeutic strategy, particularly in the treatment of a number of cancers. Screening technologies that allow for the facile characterization of fragments expressed on the cell surface would hasten the identification and isolation of reagents to be used as therapeutics. In this report, we describe a cellular microarray-based platform for the comparative functional analysis of single-chain antibodies (scFvs) expressed on the plasma membrane of mammalian cells. Using the anti-fluorescein monoclonal antibody 4-4-20 as a model system, the native binding site and three mutants were expressed as scFvs on the membrane of HEK 293T/17 cells in a microarray format. Collectively, the equilibrium dissociation constants of the soluble forms of the wild-type scFv and the three mutants spanned nearly 3 orders of magnitude. Expression of the scFvs on the surface of mammalian cells was achieved by the deposition of plasmid DNAs in micrometer-sized spots onto the surface of a glass microscope slide. The addition of cells to the printed array resulted in the expression of the scFvs in clusters of cells in spatially discrete locations. Ligand binding assays performed with a fluorescein−bovine serum albumin conjugate demonstrated the ability of the transfected cell microarray to differentiate the relative binding affinities of the expressed scFvs. Further, the apparent affinities of the membrane-displayed scFvs were within 10-fold of those reported for the soluble forms of the scFvs. The assays described herein demonstrate the potential for cellular microarrays to be used for the high-throughput screening of potential therapeutic reagents. More generally, our work details the utility of transfected cell microarrays in mediating the functional characterization of expressed membrane receptor proteins

Identification of Important Residues in Metal−Chelate Recognition by Monoclonal Antibodies^†

The molecular characterization of antibodies directed against metal−chelate complexes will provide important insights into the design and development of radiotherapeutic and radioimaging reagents. In this study, two monoclonal antibodies directed against different metal−chelate complexes were expressed as recombinant Fab fragments. Covalent modification and site-directed mutagenesis were employed to ascertain those residues important in antigen recognition. Antibody 5B2 was raised to a Pb(II)-loaded isothiocyanatobenzyl-diethylenetriamine pentaacetic acid (DTPA)−protein conjugate. The native antibody bound to complexes of Pb(II)−p-aminobenzyl-DTPA with an affinity of 4.6 × 10-9 M. A monovalent Fab fragment prepared from the native protein and a bivalent recombinant fragment exhibited comparable affinities for the same Pb(II)−chelate complex, approximately 6-fold lower than that of the intact antibody. Covalent modification and molecular modeling predicted that Lys58 in the heavy chain contacted the Pb(II)-chelate ligand. Mutational analysis supported a role for Lys58 in ion pair or hydrogen bond formation with the carboxylate groups on the chelate. Antibody E5 was directed toward an isothiocyanatobenzyl-ethylenediamine tetraacetic acid (EDTA)−protein conjugate loaded with ionic Cd(II). The native immunoglobulin recognized Cd(II)−p-aminobenzyl-EDTA with an affinity of 8.2 × 10-12 M. A proteolytically derived fragment and a bivalent recombinant fragment bound to the same Cd(II)−chelate complex with affinities that were comparable to that of the native antibody. Homology modeling and mutagenesis identified three residues (Trp52 and His96 in the heavy chain and Arg96 in the light chain) that were important for Cd(II)−chelate recognition. His96 likely mediates a direct ligation to the Cd(II) ion and Trp52 appears to be involved in hydrophobic stacking with the benzyl moiety of the chelator. Arg96 appeared to mediate an electrostatic or hydrogen bond to the chelate portion of the complex. These studies demonstrate that antibody recognition of metal−chelate haptens occurs through a limited number of molecular contacts and that these molecular interactions involve both direct ligation between the antibody and the metal ion and interactions between the antibody and the chelator

Peptides for Specifically Targeting Nanoparticles to Cellular Organelles: <i>Quo Vadis</i>?

ConspectusThe interfacing of nanomaterials and especially nanoparticles within all aspects of biological research continues to grow at a nearly unabated pace with projected applications focusing on powerful new tools for cellular labeling, imaging, and sensing, theranostic materials, and drug delivery. At the most fundamental level, many of these nanoparticles are meant to target not only very specific cell-types, regardless of whether they are in a culture, tissue, an animal model, or ultimately a patient, but also in many cases a specific subcellular organelle. During this process, these materials will undergo a complex journey that must first find the target cell of interest, then be taken up by those cells across the extracellular membrane, and ultimately localize to a desired subcellular organelle, which may include the nucleus, plasma membrane, endolysosomal system, mitochondria, cytosol, or endoplasmic reticulum. To accomplish these complex tasks in the correct sequence, researchers are increasingly interested in selecting for and exploiting targeting peptides that can impart the requisite capabilities to a given nanoparticle construct. There are also a number of related criteria that need careful consideration for this undertaking centering on the nature and properties of the peptide vector itself, the peptide–nanoparticle conjugate characteristics, and the target cell.Here, we highlight some important issues and key research areas related to this burgeoning field. We begin by providing a brief overview of some criteria for optimal attachment of peptides to nanoparticles, the predominant methods by which nanoparticles enter cells, and some of the peptide sequences that have been utilized to facilitate nanoparticle delivery to cells focusing on those that engender the initial targeting and uptake. Because almost all materials delivered to cells by peptides utilize the endosomal system of vesicular transport and in many cases remain sequestered within the vesicles, we critically evaluate the issue of endosomal escape in the context of some recently reported successes in this regard. Following from this, peptides that have been reported to deliver nanoparticles to specific subcellular compartments are examined with a focus on what they delivered and the putative mechanisms by which they were able to accomplish this. The last section focuses on two areas that are critical to realizing this overall approach in the long term. The first is how to select for peptidyl sequences capable of improved or more specific cellular or subcellular targeting based upon principles commonly associated with drug discovery. The second looks at what has been done to create modular peptides that incorporate multiple desirable functionalities within a single, contiguous sequence. This provides a viable alternative to either the almost insurmountable challenge of finding one sequence capable of all functions or, alternatively, attaching different peptides with different functionalities to the same nanoparticle in different ratios when trying to orchestrate their net effects. Finally, we conclude with a brief perspective on the future evolution and broader impact of this growing area of bionanoscience

Binding and Neutralization of Lipopolysaccharides by Plant Proanthocyanidins

Proanthocyanidins (PACs), polyphenolic metabolites that are widely distributed in higher plants, have been associated with potential positive health benefits including antibacterial, chemotherapeutic, and antiatherosclerotic activities. In this paper, we analyze the binding of PACs from cranberries, tea, and grapes to lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria and the cause of several human illnesses. We demonstrate that in the case of cranberries, the most potent LPS-binding activity is contained within a PAC fraction composed of polymers with an average degree of polymerization of 21. The PAC fraction modestly inhibits the binding of LPS to the surface of HEK 293 cells expressing the full complement of LPS receptors (TLR4/MD2 and CD14), while it significantly abrogates the endocytosis of LPS. This PAC fraction also inhibits LPS-induced nuclear factor-κB activation in a manner that is not readily overcome by excess LPS. Such an effect is mediated through the inhibition of LPS interaction with TLR4/MD2 and the partial abrogation of LPS interaction with CD14. Importantly, PAC concentrations that mediate effective LPS neutralization elicit minimal in vitro cytotoxicity. Our results identify PACs as a new class of LPS-binding compound and suggest that they have potential utility in applications that necessitate either the purification and removal of LPS or the in vivo neutralization of LPS

Modification of Poly(ethylene glycol)-Capped Quantum Dots with Nickel Nitrilotriacetic Acid and Self-Assembly with Histidine-Tagged Proteins

We describe the design and preparation of luminescent quantum dots (QDs) modified with terminal nickel-nitrilotriacetic acid (Ni-NTA) groups via simple EDC (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride) condensation, to form QD-Ni-NTA complexes. The QDs were prepared starting from CdSe-ZnS core−shell QDs surface-capped with carboxy-functionalized poly(ethylene glycol) (PEG) ligands. This modification allowed coupling of a controllable number of oligohistidine-tagged proteins via metal-affinity interactions. In particular, QD-Ni-NTA complexes were coupled to 5×His-tagged maltose binding proteins labeled with Texas red, where effective formation of the QD-MBP-Texas red conjugates was confirmed by agarose gel electrophoresis and fluorescence resonance energy transfer studies. The stability of the QD-MBP conjugates was further tested inside live cells following microinjection directly into the cytoplasm. This design expands on the good colloidal stability afforded by our earlier PEG-based modular ligands which provided QDs with great colloidal stability over a broad range of biologically relevant conditions

Intracellular Bioconjugation of Targeted Proteins with Semiconductor Quantum Dots

Intracellular Bioconjugation of Targeted Proteins with Semiconductor Quantum Dot

Multifunctional Liquid Crystal Nanoparticles for Intracellular Fluorescent Imaging and Drug Delivery

A continuing goal of nanoparticle (NP)-mediated drug delivery (NMDD) is the simultaneous improvement of drug efficacy coupled with tracking of the intracellular fate of the nanoparticle delivery vehicle and its drug cargo. Here, we present a robust multifunctional liquid crystal NP (LCNP)-based delivery system that affords facile intracellular fate tracking coupled with the efficient delivery and modulation of the anticancer therapeutic doxorubicin (Dox), employed here as a model drug cargo. The LCNPs consist of (1) a liquid crystal cross-linking agent, (2) a homologue of the organic chromophore perylene, and (3) a polymerizable surfactant containing a carboxylate headgroup. The NP core provides an environment to both incorporate fluorescent dye for spectrally tuned particle tracking and encapsulation of amphiphilic and/or hydrophobic agents for intracellular delivery. The carboxylate head groups enable conjugation to biologicals to facilitate the cellular uptake of the particles. Upon functionalization of the NPs with transferrin, we show the ability to differentially label the recycling endocytic pathway in HEK 293T/17 cells in a time-resolved manner with minimal cytotoxicity and with superior dye photostability compared to traditional organic fluorophores. Further, when passively loaded with Dox, the NPs mediate the rapid uptake and subsequent sustained release of Dox from within endocytic vesicles. We demonstrate the ability of the LCNPs to simultaneously serve as both an efficient delivery vehicle for Dox as well as a modulator of the drug’s cytotoxicity. Specifically, the delivery of Dox as a LCNP conjugate results in a ∼40-fold improvement in its IC₅₀ compared to free Dox in solution. Cumulatively, our results demonstrate the utility of the LCNPs as an effective nanomaterial for simultaneous cellular imaging, tracking, and delivery of drug cargos

Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc Phthalocyanine-Mediated Photodynamic Therapy

Current challenges in photodynamic therapy (PDT) include both the targeted delivery of the photosensitizer (PS) to the desired cellular location and the maintenance of PS efficacy. Zinc phthalocyanine (ZnPc), a macrocyclic porphyrin and a potent PS for PDT, undergoes photoexcitation to generate reactive singlet oxygen that kills cells efficiently, particularly when delivered to the plasma membrane. Like other commonly employed PS, ZnPc is highly hydrophobic and prone to self-aggregation in aqueous biological media. Further, it lacks innate subcellular targeting specificity. Cumulatively, these attributes pose significant challenges for delivery via traditional systemic drug delivery modalities. Here, we report the development and characterization of a liquid crystal nanoparticle (LCNP)-based formulation for the encapsulation and targeted tethering of ZnPc to the plasma membrane bilayer. ZnPc was coloaded with the organic fluorophore, perylene (PY), in the hydrophobic polymeric matrix of the LCNP core. PY facilitated the fluorescence-based tracking of the LCNP carrier while also serving as a Förster resonance energy transfer (FRET) donor to the ZnPc acceptor. This configuration availed efficient singlet oxygen generation via enhanced excitation of ZnPc from multiple surrounding PY energy donors. When excited in a FRET configuration, cuvette-based assays revealed that singlet oxygen generation from the ZnPc was ∼1.8-fold greater and kinetically 12 times faster compared to when the ZnPc was excited directly. The specific tethering of the LCNPs to the plasma membrane of HEK 293T/17 and HeLa cells was achieved by surface functionalization of the NPs with PEGylated cholesterol. In HeLa cells, LCNPs coloaded with PY and ZnPc, when photoexcited in a FRET configuration, mediated 70% greater cell killing compared to LCNPs containing ZnPc alone (direct excitation of ZnPc). This was attributed to a significant increase of the oxidative stress in the cells during the PDT. Overall, this work details the ability of the LCNP platform to facilitate (1) the specific tethering of the PY-ZnPc FRET pair to the plasma membrane and (2) the FRET-mediated, augmented singlet oxygen generation for enhanced PDT relative to the direct excitation of ZnPc alone

Enhancing the Stability and Biological Functionalities of Quantum Dots via Compact Multifunctional Ligands

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules:  a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging showed that QDs capped with DHLA−PEG−biotin strongly interacted with either NeutrAvidin immobilized on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA−PEG−COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands described here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities

Lipid Raft-Mediated Membrane Tethering and Delivery of Hydrophobic Cargos from Liquid Crystal-Based Nanocarriers

A main goal of bionanotechnology and nanoparticle (NP)-mediated drug delivery (NMDD) continues to be the development of novel biomaterials that can controllably modulate the activity of the NP-associated therapeutic cargo. One of the desired subcellular locations for targeted delivery in NMDD is the plasma membrane. However, the controlled delivery of hydrophobic cargos to the membrane bilayer poses significant challenges including cargo precipitation and lack of specificity. Here, we employ a liquid crystal NP (LCNP)-based delivery system for the controlled partitioning of a model dye cargo from within the NP core into the plasma membrane bilayer. During synthesis of the NPs, the water-insoluble model dye cargo, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), was efficiently incorporated into the hydrophobic LCNP core as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) facilitated the localization of the dye-loaded NPs to lipid raft microdomains in the plasma membrane in HEK 293T/17 cell. Analysis of DiO cellular internalization kinetics revealed that when delivered as a LCNP-PEG-Chol NP, the half-life of DiO membrane residence time (30 min) was twice that of free DiO (DiO_free) (15 min) delivered from bulk solution. Time-resolved laser scanning confocal microscopy was employed to visualize the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer as confirmed by Förster resonance energy transfer (FRET) imaging. Finally, the delivery of DiO as a LCNP-PEG-Chol complex resulted in the attenuation of its cytotoxicity; the NP form of DiO exhibited ∼30–40% less toxicity compared to DiO_free. Our data demonstrate the utility of the LCNP platform as an efficient vehicle for the combined membrane-targeted delivery and physicochemical modulation of molecular cargos using lipid raft-mediated tethering