Biomolecular Ligands Research Articles

The effects of an RGD-PAMAM dendrimer conjugate in 3D spheroid culture on cell proliferation, expression and aggregation

By presenting biomolecular ligands on the surface in high density, ligand-decorated dendrimers are capable of binding to membrane receptors and cells with specificity and avidity. Despite the various uses, fundamental investigations on ligand-dendrimer conjugates have mainly focused on their binding behavior with cells, whereas their potential bioactivity and applications in multicellular systems, especially in three-dimensional (3D) culture systems, remains untapped. In this study, a typical adhesive peptide ligand – RGD – was modified to generation 4 polyamidoamine (PAMAM), and the bioactivity of suspended RGD-PAMAM conjugates was investigated on cells cultured as multicellular spheroids. Our results demonstrate that the RGD-PAMAM conjugates, after being incorporated into the 3D spheroids, were able to promote cellular proliferation and aggregation, and affect the mRNA expression of extracellular factors by NIH 3T3 cells. These bioactive functions were multivalency-dependent, as none of similar effects was observed for monovalent RGD ligand. Our study suggests that multivalent ligand-dendrimer conjugates may act as a unique type of artificial factors to mediate the cellular microenvironment in 3D culture, a property attributable to the spatial organization of the ligands and possible “cell-gluing” function of multivalent conjugates. This new finding opens the door for further exploring multivalent ligand-dendrimer conjugates for applications in 3D cell culture and tissue engineering.

Correction to “Interaction of Metal Ions with Biomolecular Ligands: How Accurate Are Calculated Free Energies Associated with Metal Ion Complexation?”

Correction to “Interaction of Metal Ions with Biomolecular Ligands: How Accurate Are Calculated Free Energies Associated with Metal Ion Complexation?”

Surface modification of gold–carbon nanotube nanohybrids under the influence of near-infrared laser exposure

The development of multifunctional hybrid nanostructures that can be remotely activated is an attractive strategy for a diverse range of applications ranging from electronics, cancer therapeutics, and drug delivery platforms to sophisticated biosensors. In this study, the authors examined the systematic capture of biomolecular targets onto single-walled carbon nanotubes (SWNTs), site-specific labeling with gold nanoparticles (GNPs) of three different sizes (10, 30, 60 nm), and the subsequent effects upon exposure to 1064 nm near-infrared (NIR) laser irradiation. The authors demonstrate that the SWNT-GNP hybrids containing the smallest GNPs experience greater heating and subsequent GNP release upon NIR laser irradiation compared to SWNT surfaces modified with larger 60 nm GNPs. The authors hypothesize that the greater attachment efficiency of the smaller GNPs to the biomolecules allows increased heat transduction. Therefore, it is possible to physically modify the surface of hybrid nanostructures remotely via NIR laser irradiation. It is anticipated that targeted NIR strategies will benefit from the robustness of novel material combinations, such as SWNT-GNP hybrid nanostructures, as well as interchangeable biomolecular ligands.

Protection and functionalisation of silver as an optical sensing platform for highly sensitive SPR based analysis

Silver thin films are well known as the most sensitive material for surface plasmon resonance (SPR) based analysis. However, the use of silver for this purpose is limited by three main issues, namely poor adhesion to plastic substrates, chemical instability in both air and aqueous environments and hence the difficulty in functionalizing the silver coated substrate for immobilizing biomolecular ligands by conventional liquid phase methods. In this work, we have successfully addressed these problems using gas-phase coating processes. We demonstrate highly adherent sputter-deposited silver coatings on low cost polymer substrates using a sputter-deposited thin gold adhesion layer. The problems of chemical instability and functionalisation have been addressed by using the gas phase process of plasma enhanced chemical vapour deposition (PECVD) to deposit thin films with a base SiO(x)C(y)H(z) layer (using tetraethyl orthosilicate precursor) functionalised with carboxylic acid (from sequential deposition with acrylic acid precursor). The resultant coating serves as a protective layer against degradation of the optical properties of silver under long term storage and use in ambient conditions. The reactive carboxyl functionality is used for the covalent immobilization of biomolecules. The successful stabilisation and functionalization of silver films on plastic sensor chips is demonstrated by mouse IgG immunoassays. The expected superior performance of the silver thin films over gold thin films for SPR analysis is demonstrated.

Interaction of Metal Ions with Biomolecular Ligands: How Accurate Are Calculated Free Energies Associated with Metal Ion Complexation?

To address fundamental questions in bioinorganic chemistry, such as metal ion selectivity, accurate computational protocols for both the gas-phase association of metal-ligand complexes and solvation/desolvation energies of the species involved are needed. In this work, we attempt to critically evaluate the performance of the ab initio and DFT electronic structure methods available and recent solvation models in calculations of the energetics associated with metal ion complexation. On the example of five model complexes ([M(II)(CH(3)S)(H(2)O)](+), [M(II)(H(2)O)(2)(H(2)S)(NH(3))](2+), [M(II)(CH(3)S)(NH(3))(H(2)O)(CH(3)COO)], [M(II)(H(2)O)(3)(SH)(CH(3)COO)(Im)], [M(II)(H(2)S)(H(2)O)(CH(3)COO)(PhOH)(Im)](+) in typical coordination geometries) and four metal ions (Fe(2+), Cu(2+), Zn(2+), and Cd(2+); representing open- and closed-shell and the first- and second-row transition metal elements), we provide reference values for the gas-phase complexation energies, as presumably obtained using the CCSD(T)/aug-cc-pVTZ method, and compare them with cheaper methods, such as DFT and RI-MP2, that can be used for large-scale calculations. We also discuss two possible definitions of interaction energies underlying the theoretically predicted metal-ion selectivity and the effect of geometry optimization on these values. Finally, popular solvation models, such as COSMO-RS and SMD, are used to demonstrate whether quantum chemical calculations can provide the overall free enthalpy (ΔG) changes in the range of the expected experimental values for the model complexes or match the experimental stability constants in the case of three complexes for which the experimental data exist. The data presented highlight several intricacies in the theoretical predictions of the experimental stability constants: the covalent character of some metal-ligand bonds (e.g., Cu(II)-thiolate) causing larger errors in the gas-phase complexation energies, inaccuracies in the treatment of solvation of the charged species, and difficulties in the definition of the reference state for Jahn-Teller unstable systems (e.g., [Cu(H(2)O)(6)](2+)). Although the agreement between the experimental (as derived from the stability constants) and calculated values is often within 5 kcal·mol(-1), in more complicated cases, it may exceed 15 kcal·mol(-1). Therefore, extreme caution must be exercised in assessing the subtle issues of metal ion selectivity quantitatively.

Aptamers for safety and quality assurance in the food industry: detection of pathogens

SummaryAptamers are biomolecular ligands composed of nucleic acids. They can be developed to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. Methods for the production of aptamers and their potential applications to the food industry in the form of rapid assays and biosensors are discussed. In contrast to antibody‐based diagnostics, aptamers can be produced in animal‐free systems which have clear ethical and financial benefits. This review identifies a need for the development of new aptamers with specificity against micro‐organisms and highlights their potential use for the detection of food‐borne pathogens.

Synthesis and Biomedical Application of SiO<sub>2</sub>/Au Nanofluid Based on Laser-Induced Surface Plasmon Resonance Thermal Effect

We described the synthesis of Au coated SiO2 nanoshells linked with NH2 biomolecular ligands using a simple wet chemical method with a particular application for laser tissue soldering. Tunable nanoshells were prepared by using different gold colloidal concentrations. The nanoshells are characterized by UV-vis spectroscopy, FTIR, XRD and AFM. The FTIR results confirmed the functionalized surfaces of silica nanoparticles with NH2 terminal groups. A broad absorption was observed between 470 - 600 nm with a maximum range between 530 - 560 nm. Based on the XRD results three main peaks of Au (111), (200) and (220) were identified. In addition, AFM results showed that the diameter of silica core was between 90 - 110 nm with gold shell thickness between 10 - 30 nm. A possible tissue soldering using gold nanoshells and laser-induced thermal effect based on surface plasmon resonance is demonstrated. In our case this corresponds to 90?C (i.e. below vaporization) using the higher gold concentration (2 ml) at 60 W·cm–2.

Engineered mesenchymal stem cells with self-assembled vesicles for systemic cell targeting

Cell therapy has the potential to impact the quality of life of suffering patients. Systemic infusion is a convenient method of cell delivery; however, the efficiency of engraftment presents a major challenge. It has been shown that modification of the cell surface with adhesion ligands is a viable approach to improve cell homing, yet current methods including genetic modification suffer potential safety concerns, are practically complex and are unable to accommodate a wide variety of homing ligands or are not amendable to multiple cell types. We report herein a facile and generic approach to transiently engineer the cell surface using lipid vesicles to present biomolecular ligands that promote cell rolling, one of the first steps in the homing process. Specifically, we demonstrated that lipid vesicles rapidly fuse with the cell membrane to introduce biotin moieties on the cell surface that can subsequently conjugate streptavidin and potentially any biotinylated homing ligand. Given that cell rolling is a pre-requisite to firm adhesion for systemic cell homing, we examined the potential of immobilizing sialyl Lewis X (SLeX) on mesenchymal stem cells (MSCs) to induce cell rolling on a P-selectin surface, under dynamic flow conditions. MSCs modified with SLeX exhibit significantly improved rolling interactions with a velocity of 8 μm/s as compared to 61 μm/s for unmodified MSCs at a shear stress of 0.5 dyn/cm 2. The cell surface modification does not impact the phenotype of the MSCs including their viability and multi-lineage differentiation potential. These results show that the transitory modification of cell surfaces with lipid vesicles can be used to efficiently immobilize adhesion ligands and potentially target systemically administered cells to the site of inflammation.

Synthesis of Alkyne-Terminated PCDA Linker for Applying Click Chemistry on PDA Layers

An alkyne-terminated PCDA (10,12-pentacosadiynoic acid) linker, which can undergo a simple click reaction with azide compounds, was synthesized for the purpose of immobilizing various kinds of biomolecular surface ligands on PDA (polydiacetylene) layers. In order to confirm the ability of the linker compound to anchor azide-modified surface ligands, we carried out a model reaction with benzyl azide under typical click-reaction conditions. We were able to confirm that the model reaction proceeded smoothly by observing the color change of PDA, including its thermochromic reversibility.

Tri-functionalization of mesoporous silica nanoparticles for comprehensive cancer theranostics—the trio of imaging, targeting and therapy

In this work we report the development of the tri-functionalized mesoporous silica nanoparticles (MSNs) for use as theranostic compounds that orchestrate the trio of imaging, target and therapy in a single particle. The MSNs are functionalized in sequence with (1) contrast agents that enable traceable imaging of particle targeting, (2) drug payloads for therapeutic intervention and, (3) biomolecular ligands for highly-targeted particle delivery. Traceable imaging of nanoparticles was accomplished by directly incorporating a near-infrared (NIR) fluorescent contrast agent, ATTO647N, into the silica framework of MSNs, to exploit the relative transparency of most tissues at NIR wavelengths and maximize MSN surface area available for the subsequent conjugating drugs and targeting ligands. An oxygen-sensing, palladium-porphyrin based photosensitizer (Pd-porphyrin; PdTPP) was incorporated into the MSN's nanochannels, to enable photodynamic therapy (PDT). cRGDyK peptides, tiling the outermost surfaces of MSNs, were used for targeting the overexpressed αvβ3 integrins of cancer cells, and to ensure the internalization of the photosensitizer PdTPP. In vitro cell evaluation of the theranostic platform demonstrated not only excellent targeting specificity and minimal collateral damage, but highly potent therapeutic effect as well.

Aggregated CdS Quantum Dots: Host of Biomolecular Ligands

In this contribution, we have studied structural and photophysical properties of aggregated CdS quantum dots (QDs) capped with 2-mercaptoethanol in aqueous medium. The hydrodynamic diameter of the nanostructures in aqueous solution was found to be approximately 160 nm with the dynamic light scattering (DLS) technique, which is in close agreement with atomic force microscopy (AFM) studies (diameter approximately 150 nm). However, the UV-vis absorption spectroscopy, powder X-ray diffraction (XRD), and transmission electron microscopy (TEM) studies confirm the average particle size (QD) in the nanoaggregate to be 4.0 +/- 0.5 nm. The steady-state and time-resolved photoluminescence studies on the QDs further confirm preservation of electronic band structure of the QDs in the nanoaggregate. To study the nature of the nanoaggregate we have used small fluorescent probes, which are widely used as biomolecular ligands (2,6-p-toluidinonaphthalene sulfonate (TNS) and Oxazine 1), and found the pores of the aggregate to be hydrophobic in nature. The significantly large spectral overlap of the host quantum dots (donor) with that of the guest fluorescent probe Oxazine 1 (acceptor) allows us to carry out Förster resonance energy transfer (FRET) studies to estimate average donor-acceptor distance in the nanostructure, found to be approximately 25 Angstrom. The quantum dot aggregate and the characterization techniques reported here could have implications in the future application of the QD-nanoaggregate as host of small ligand molecules of biological interest.

Engineering Luminescent Quantum Dots for In Vivo Molecular and Cellular Imaging

Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bio-conjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.

Nucleotide-Directed Growth of Semiconductor Nanocrystals

We engineer colloidal quantum dot nanocrystals through the choice of biomolecular ligands responsible for nanoparticle nucleation, growth, stabilization, and passivation. We systematically vary the presence of, and thereby elucidate the role of, phosphate groups and a multiplicity of functionalities on the mononucleotides used as ligands. The results provide the basis for synthesis of nanoparticles using precisely controlled synthetic oligonucleotide sequences.

Synthesis of polymerized thin films for immobilized ligand display in proteomic analysis.

We describe a new material for the display of biomolecular ligands for use in proteomic analysis. We report here on the construction of the first functionalized polymerized diacetylene thin films (PDTFs) for use in displaying immobilized ligands and their application in mass spectral proteomic analysis. Functionalized polymerized thin film surfaces were constructed with diacetylene-containing biotin lipid monomers designed for the capture of proteins (streptavidin) from a complex cellular lysate and detection with mass spectrometry (MS). These materials serve as a prototype for ligand-based spotted arrays amenable to high throughput screening. Functionalized PDTFs can be easily manufactured for customized microarrays and demonstrate high protein specificity and low nonspecific protein adsorption, and the resulting microarrays constructed from these materials are compatible with several different protein analysis platforms. Our results suggest that these materials have broad potential applications for use in mass spectral-based proteomic analysis.

Characterization of metal complexes with metallothioneins by capillary zone electrophoresis (CZE) with ICP-MS and electrospray (ES)-MS detection

The complementarity of CZE-ICP-MS and CZE-ES-MS couplings was evaluated for the characterization of metal complexes with metallothionein (MT) isoforms. The preparations characterized included the rabbit liver MT and its further purified MT-1 and MT-2 isoforms. The CZE-ICP-MS electrophoregrams showed the presence of several well (often baseline) separated Gaussian-shape peaks. ES-MS was used for the identification of these signals; mass spectra taken at the peak apexes were complex and indicated the co-existence of several metal complexes (possibly with different isoforms) within each peak. Many of these complexes could be identified on the basis of the molecular mass. The study highlights the attractive potential of CZE-ICP-MS and CZE-ES-MS when used in parallel for the characterization of metal complexes with biomolecular ligands. The inherent discrepancy between the sensitivities of ICP-MS and ES-MS is partly alleviated by the fact that the metal (detected by ICP-MS) constitutes only a small fraction of the total molecule (detected by ES-MS), and by the smaller dilution factor with the makeup sheath flow in ES-MS than in ICP-MS.