Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.
ABSTRACT: CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single-stranded DNA. Nanomaterials can be designed and synthesized in needed sizes and shapes, and they possess unique chemical and physical properties, which make them useful as DNA carriers or assistants, excellent signal reporters, transducers, and amplifiers. When nanomaterials are combined with functional DNAs to create novel assay platforms, highly sensitive biosensing and high-resolution imaging result. For example, gold nanoparticles and graphene oxides can quench fluorescence efficiently to achieve low background and effectively increase the signal-to-background ratio. Meanwhile, gold nanoparticles themselves can be colorimetric reporters because of their different optical absorptions between monodispersion and aggregation. DNA self-assembled nanomaterials contain several properties of both DNA and nanomaterials. Compared with DNA-nanomaterial complexes, DNA self-assembled nanomaterials more closely resemble living beings, and therefore they have lower cytotoxicity at high concentrations. Functional DNA self-assemblies also have high density of DNA for multivalent reaction and three-dimensional nanostructures for cell uptake. Now and in the future, we envision the use of DNA bases in making designer molecules for many challenging applications confronting chemists. With the further development of artificial DNA bases using smart organic synthesis, DNA macromolecules based on elegant molecular assembly approaches are expected to achieve great diversity, additional versatility, and advanced functions.
Project description:CONSPECTUS: Several properties of nanomaterials, such as morphologies (e.g., shapes and surface structures) and distance dependent properties (e.g., plasmonic and quantum confinement effects), make nanomaterials uniquely qualified as potential choices for future applications from catalysis to biomedicine. To realize the full potential of these nanomaterials, it is important to demonstrate fine control of the morphology of individual nanoparticles, as well as precise spatial control of the position, orientation, and distances between multiple nanoparticles. In addition, dynamic control of nanomaterial assembly in response to multiple stimuli, with minimal or no error, and the reversibility of the assemblies are also required. In this Account, we summarize recent progress of using DNA as a powerful programmable tool to realize the above goals. First, inspired by the discovery of genetic codes in biology, we have discovered DNA sequence combinations to control different morphologies of nanoparticles during their growth process and have shown that these effects are synergistic or competitive, depending on the sequence combination. The DNA, which guides the growth of the nanomaterial, is stable and retains its biorecognition ability. Second, by taking advantage of different reactivities of phosphorothioate and phosphodiester backbone, we have placed phosphorothioate at selective positions on different DNA nanostructures including DNA tetrahedrons. Bifunctional linkers have been used to conjugate phosphorothioate on one end and bind nanoparticles or proteins on the other end. In doing so, precise control of distances between two or more nanoparticles or proteins with nanometer resolution can be achieved. Furthermore, by developing facile methods to functionalize two hemispheres of Janus nanoparticles with two different DNA sequences regioselectively, we have demonstrated directional control of nanomaterial assembly, where DNA strands with specific hybridization serve as orthogonal linkers. Third, by using functional DNA that includes DNAzyme, aptamer, and aptazyme, dynamic control of assemblies of gold nanoparticles, quantum dots, carbon nanotubes, and iron oxide nanoparticles in response to one or more stimuli cooperatively have been achieved, resulting in colorimetric, fluorescent, electrochemical, and magnetic resonance signals for a wide range of targets, such as metal ions, small molecules, proteins, and intact cells. Fourth, by mimicking biology, we have employed DNAzymes as proofreading units to remove errors in nanoparticle assembly and further used DNAzyme cascade reactions to modify or repair DNA sequences involved in the assembly. Finally, by taking advantage of different affinities of biotin and desthiobiotin toward streptavidin, we have demonstrated reversible assembly of proteins on DNA origami.
Project description:Nanomaterials have shown great promise in advancing biomedical and environmental analysis because of the unique properties originated from their ultrafine dimensions. In general, nanomaterials are separately applied to either enhance detection by producing strong signals upon target recognition or to specifically extract analytes taking advantage of their high specific surface area. Herein, we report a dual-functional nanomaterial-based platform that can simultaneously enrich and enable sensitive detection of multiple metal ions. The macroporous graphene foam (GF) we prepared displays abundant phosphate groups on the surface and can extract divalent metal ions via metal-phosphate coordination. The enriched metal ions then activate the metal-responsive DNAzymes and produce the fluorescently labeled single-stranded DNAs that are adsorbed and quenched by the GF. The resultant fluorescence reduction can be used for metal quantitation. The present work demonstrated duplexed detection of Pb2+ and Cu2+ using the Pb- and Cu-responsive DNAzymes, achieving a low detection limit of 50 pM and 0.6 nM, respectively. Successful quantification of Pb2+ and Cu2+ in human serum and river water were achieved with high metal recovery. Since the phosphate-decorated GF can enrich diverse types of divalent metal cations, this dual-functional GF-DNAzyme platform can serve as a simple and cost-effective tool for rapid and accurate metal quantification in determination of human metal exposure and inspection of environmental contamination.
Project description:To facilitate the development of new nanomaterials, especially nanomedicines, a novel computational approach was developed to precisely predict the hydrophobicity of gold nanoparticles (GNPs). The core of this study was to develop a large virtual gold nanoparticle (vGNP) library with computational nanostructure simulations. Based on the vGNP library, a nanohydrophobicity model was developed and then validated against externally synthesized and tested GNPs. This approach and resulted model is an efficient and effective universal tool to visualize and predict critical physicochemical properties of new nanomaterials before synthesis, guiding nanomaterial design.
Project description:The challenge of optimizing both performance and safety in nanomaterials hinges on our ability to resolve which structural features lead to desired properties. It has been difficult to draw meaningful conclusions about biological impacts from many studies of nanomaterials due to the lack of nanomaterial characterization, unknown purity, and/or alteration of the nanomaterials by the biological environment. To investigate the relative influence of core size, surface chemistry, and charge on nanomaterial toxicity, we tested the biological response of whole animals exposed to a matrix of nine structurally diverse, precision-engineered gold nanoparticles (AuNPs) of high purity and known composition. Members of the matrix include three core sizes and four unique surface coatings that include positively and negatively charged headgroups. Mortality, malformations, uptake, and elimination of AuNPs were all dependent on these parameters, showing the need for tightly controlled experimental design and nanomaterial characterization. Results presented herein illustrate the value of an integrated approach to identify design rules that minimize potential hazard.
Project description:This review paper comprehensively summarizes advances made in the design of glycan nanobiosensors using diverse forms of nanomaterials. In particular, the paper covers the application of gold nanoparticles, quantum dots, magnetic nanoparticles, carbon nanoparticles, hybrid types of nanoparticles, proteins as nanoscaffolds and various nanoscale-based approaches to designing such nanoscale probes. The article covers innovative immobilization strategies for the conjugation of glycans on nanoparticles. Summaries of the detection schemes applied, the analytes detected and the key operational characteristics of such nanobiosensors are provided in the form of tables for each particular type of nanomaterial.
Project description:Antimicrobial action of nanomaterials is typically assigned to the nanomaterial composition, size and/or shape, whereas influence of complex corona stabilizing the nanoparticle surface is often neglected. We demonstrate sequential surface functionalization of tyrosine-reduced gold nanoparticles (AuNPs(Tyr)) with polyoxometalates (POMs) and lysine to explore controlled chemical functionality-driven antimicrobial activity. Our investigations reveal that highly biocompatible gold nanoparticles can be tuned to be a strong antibacterial agent by fine-tuning their surface properties in a controllable manner. The observation from the antimicrobial studies on a gram negative bacterium Escherichia coli were further validated by investigating the anticancer properties of these step-wise surface-controlled materials against A549 human lung carcinoma cells, which showed a similar toxicity pattern. These studies highlight that the nanomaterial toxicity and biological applicability are strongly governed by their surface corona.
Project description:The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods.This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane.The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.
Project description:DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes in vivo pertains to nontoxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme "10-23" gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10-23 DNAzyme was asymmetrically sensitive to the gold surface and when anchored through the 5' terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at ? = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for in vivo work.
Project description:Nanoparticles have been making their way in biomedical applications and personalized medicine, allowing for the coupling of diagnostics and therapeutics into a single nanomaterial-nanotheranostics. Gold nanoparticles, in particular, have unique features that make them excellent nanomaterials for theranostics, enabling the integration of targeting, imaging and therapeutics in a single platform, with proven applicability in the management of heterogeneous diseases, such as cancer. In this review, we focus on gold nanoparticle-based theranostics at the lab bench, through pre-clinical and clinical stages. With few products facing clinical trials, much remains to be done to effectively assess the real benefits of nanotheranostics at the clinical level. Hence, we also discuss the efforts currently being made to translate nanotheranostics into the market, as well as their commercial impact.
Project description:The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by benchtop centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro.