Preparation of multifunctional glyconanoparticles as a platform for potential carbohydrate-based anticancer vaccines

A novel platform for anticancer vaccines has been prepared using glyconanotechnology recently developed in our laboratory. Ten different multifunctional gold glyconanoparticles incorporating sialylTn and Lewis(y) antigens, T-cell helper peptides (TT) and glucose in well defined average proportions and with differing density have been synthesised in one step and characterised using NMR and TEM. Size and nature of the linker were crucial to control kinetics of S-Au bond formation and to achieve the desired ligand ratio on the gold clusters. The technology presented here opens the way for tailoring polyvalent anticancer vaccines candidates and drug delivery carriers with defined average chemical composition.     

Albumin as a versatile platform for drug half-life extension

Background

Albumin is the most abundant plasma protein, is highly soluble, very stable and has an extraordinarily long circulatory half-life as a direct result of its size and interaction with the FcRn mediated recycling pathway. In contrast, many therapeutic molecules are smaller than the renal filtration threshold and are rapidly lost from the circulation thereby limiting their therapeutic potential. Albumin can be used in a variety of ways to increase the circulatory half-life of such molecules.

Scope of review

This article will review the mechanisms which underpin albumin's extraordinarily long circulatory half-life and how the understanding of these processes are currently being employed to extend the circulatory half-life of drugs which can be engineered to bind to albumin, or are conjugated to, or genetically fused to, albumin.

Major conclusions

The recent and growing understanding of the pivotal role of FcRn in maintaining the extended circulatory half-life of albumin will necessitate a greater and more thorough investigation of suitable pre-clinical model systems for assessing the pharmacokinetic profiles of drugs associated, conjugated or fused to albumin.

General significance

Association, conjugation or fusion of therapeutic drugs to albumin is a well-accepted and established half-life extension technology. The manipulation of the albumin–FcRn interaction will facilitate the modulation of the circulatory half-life of albumin-enabled drugs, leading to superior pharmacokinetics tailored to the disease state and increased patient compliance. This article is part of a Special Issue entitled Serum Albumin.     

Exploiting cholera vaccines as a versatile antigen delivery platform

The development of safe, immunogenic and protective cholera vaccine candidates makes possible their use as a versatile antigen delivery platform. Foreign antigens can be delivered to the immune system with cholera vaccines by expressing heterologous antigens in live attenuated vectors, as fusion proteins with cholera toxin subunits combined with inactivated Vibrio cholerae whole cells or by exposing them on the surface of V. cholerae ghosts. Progress in our understanding of the genes expressed by V. cholerae during infection creates unprecedented opportunities to develop an improved generation of vaccine vectors to induce immune protection against a broad range of pathogenic organisms.     

Caenorhabditis elegans: a versatile platform for drug discovery

Drug discovery and drug target identification are two intimately linked facets of intervention strategies aimed at effectively combating pathological conditions in humans. Simple model organisms provide attractive platforms for devising and streamlining efficient drug discovery and drug target identification methodologies. The nematode worm Caenorhabditis elegans has emerged as a particularly convenient and versatile tool that can be exploited to achieve these goals. Although C. elegans is a relatively modern addition to the arsenal of model organisms, its biology has already been investigated to an exceptional level. This, coupled with effortless handling and a notable low cost of cultivation and maintenance, allows seamless implementation of high-throughput drug screening approaches as well as in-depth genetic and biochemical studies of the molecular pathways targeted by specific drugs. In this review, we introduce C. elegans as a model organism with significant advantages toward the identification of molecular drug targets. In addition, we discuss the value of the worm in the development of drug screening and drug evaluation protocols. The unique features of C. elegans, which greatly facilitate drug studies, hold promise for both deciphering disease pathogenesis and formulating educated and effective therapeutic interventions.     

Sulfur metabolism: a versatile platform for launching defence operations

Sulfur-containing defence compounds (SDCs) are crucial for the survival of plants under biotic and abiotic stress. SDCs include elemental sulfur (S(0)), H(2)S, glutathione, phytochelatins, various secondary metabolites and sulfur-rich proteins. Their constitutive and/or stress-induced formation is intimately dependent on demand-driven sulfate uptake and assimilation. Here, we highlight the complex network of plant SDCs and report on recent breakthroughs in our understanding of sulfur assimilation and how its regulation impinges on SDC function. These new insights have led us to revisit the hypothesis of 'sulfur-induced resistance', which claimed a prominent role for 'extra' sulfur nutrition in the defence potential of plants.     

Oligonucleotide-coated metallic nanoparticles as a flexible platform for molecular imaging agents

Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotide-based approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.     

In vivo imaging platform for tracking immunotherapeutic cells

Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a 'conventional' (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. We injected labeled DCs into tissue or intravenously in mice and then tracked the cells in vivo using (19)F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo.     

Suspension-cultured plant cells as a platform for obtaining recombinant proteins

Production of recombinant proteins in suspension cultures of genetically modified plant cells is a promising and rapidly developing area of plant biotechnology. In the present review article, advantages related to using plant systems for expression of recombinant proteins are considered. Here, the main focus is covering the literature on optimization of cultivation conditions of suspension-cultured plant cells to obtain a maximal yield of target proteins. In particular, certain examples of successful use of such cells to produce pharmaceuticals were described.     

Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.     

Quantum dots bearing lectin-functionalized nanoparticles as a platform for in vivo brain imaging

Delivery of imaging agents to the brain is highly important for the diagnosis and treatment of central nervous system (CNS) diseases, as well as the elucidation of their pathophysiology. Quantum dots (QDs) provide a novel probe with unique physical, chemical, and optical properties, and become a promising tool for in vivo molecular and cellular imaging. However, their poor stability and low blood-brain barrier permeability severely limit their ability to enter into and act on their target sites in the CNS following parenteral administration. Here, we developed a QDs-based imaging platform for brain imaging by incorporating QDs into the core of poly(ethylene glycol)-poly(lactic acid) nanoparticles, which was then functionalized with wheat germ agglutinin and delivered into the brain via nasal application. The resulting nanoparticles, with high payload capacity, are water-soluble, stable, and showed excellent and safe brain targeting and imaging properties. With PEG functional terminal groups available on the nanoparticles surface, this nanoprobe allows for conjugation of various biological ligands, holding considerable potential for the development of specific imaging agents for various CNS diseases.     

Maleic anhydride copolymers--a versatile platform for molecular biosurface engineering

A platform of thin polymer coatings was introduced for the functional modulation of immobilized bioactive molecules at solid/liquid interfaces. The approach is based on covalently attached alternating maleic acid anhydride copolymers with a variety of comonomers and extended through conversion of the anhydride moieties by hydrolysis, reaction with functional amines, and other conversions of the anhydride moieties. We demonstrate that these options permit control of the physicochemical constraints for bioactive molecules immobilized at interfaces to influence important performance characteristics of biofunctionalized materials for medical devices and molecular diagnostics. Examples concern the impact of the substrate-anchorage of fibronectin on the formation of cell-matrix adhesions, the orientation of endothelial cells according to lateral anti-adhesive micropatterns using grafted poly(ethylene oxide), and the spacer-dependent activity of immobilized synthetic thrombin inhibitors.     

Chitosan fibers: versatile platform for nickel-mediated protein assembly

Fibers are a versatile platform because standard methods are available for the hierarchical assembly of individual fibers into controllable patterns (e.g., fabrics). Here, we report a method to biofunctionalize individual fibers by the reversible binding of proteins, and we suggest the potential of fiber assemblies by generating simple multifiber structures. Specifically, we use chitosan fibers and show that nickel can mediate assembly of histidine-tagged proteins to these fibers. Initial studies with the model His-GFP demonstrate the concept of nickel-mediated protein assembly. Subsequent studies with a His-tagged streptococcal antibody-binding protein (protein G) demonstrate the assembly of antibodies to generate antibody-presenting fibers. Antibody assembly onto the fiber was shown to be controllable, and antigen-binding to these antibody-presenting fibers was measured. Importantly, antibody and antigen were observed to penetrate substantially into the individual fibers (tens of microns) to allow the assembly of pmole levels of protein per cm of fiber length. Finally, antibody-presenting fibers with different specificities were assembled into simple one- and two-dimensional structures, and individual fibers in these fiber assemblies were observed to capture their respective antigens from antigen mixtures. The potential of fiber assemblies for multiplexed analysis is discussed.     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.     

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.     

The double-stranded RNA-binding motif, a versatile macromolecular docking platform

The double-stranded RNA-binding motif (dsRBM) is an alphabetabetabetaalpha fold with a well-characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM-containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A-form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non-RNA targets (proteins and DNA), and act in combination with other dsRBMs and non-dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM-protein recognition.     

Enzyme integrated silicate-Pt nanoparticle architecture: a versatile biosensing platform

A novel 3-D nanoarchitectured platform based on Pt nanoparticles (nPts) is developed for the sensing of sub-nanomolar levels of hydrogen peroxide and for the fabrication of amperometric biosensor for uric acid, cholesterol and glucose. The nPts have been immobilized on the thiol functional group containing sol-gel silicate 3-D network derived from 3-mercaptopropyltrimethoxysilane (MPTS). The nanoparticles on the 3-D architecture have size distribution between 7 and 10nm. The nPts on the platform efficiently catalyze the oxidation of H(2)O(2) at the potential of +0.45 V in the absence of enzymes and redox mediators. This nanoarchitectured platform is highly sensitive and can detect H(2)O(2) at sub-nanomolar levels (0.1 nM) in neutral solution. The nanoarchitectured platform does not suffer from interference due to other common easily oxidizable interfering agents. Excellent reproducibility, long-term storage and operational stability are observed. This platform is used to determine H(2)O(2) concentration in rainwater and for the fabrication of biosensors. Amperometric oxidase-based biosensing platforms are developed by integrating the enzymes and nPts with the silicate network for the sensing of uric acid cholesterol and glucose. The enzyme encapsulated 3-D architecture retains the enzymatic activity and efficiently detects enzymatically generated H(2)O(2) without any interference. These biosensors are stable and show excellent sensitivity and fast response time. A linear response was obtained for a wide concentration range of all analytes. The practical utilization of the biosensor for the measurement of uric acid, cholesterol and glucose in serum sample is demonstrated. The biological sample analysis was validated with clinical laboratory measurements.     

Protein B: a versatile bacterial Fc-binding protein selective for human IgA

Protein B, a selective bacterial IgA Fc-binding protein isolated from group B streptococci, has been used to quantify fluid phase and immobilized human IgA. Protein B detects both human IgA1 and IgA2 subclasses and is also reactive with secretory IgA. Protein B can be used immobilized to microtiter plates to capture IgA or following biotinylation as a tracer for fluid phase or immobilized human IgA. The studies presented here suggest protein B will prove to be a valuable reagent for quantitative immunochemical procedures involving human IgA antibodies and facilitate a variety of studies of IgA responses in man.