Sarit S. Agasti

Gold Nanoparticles in Chemical and Biological Sensing

Detection of chemical and biological agents plays a fundamental role in biomedical, forensic and environmental sciences1–4 as well as in anti bioterrorism applications.5–7 The development of highly sensitive, cost effective, miniature sensors is therefore in high demand which requires advanced technology coupled with fundamental knowledge in chemistry, biology and material sciences.8–13 In general, sensors feature two functional components: a recognition element to provide selective/specific binding with the target analytes and a transducer component for signaling the binding event. An efficient sensor relies heavily on these two essential components for the recognition process in terms of response time, signal to noise (S/N) ratio, selectivity and limits of detection (LOD).14,15 Therefore, designing sensors with higher efficacy depends on the development of novel materials to improve both the recognition and transduction processes. Nanomaterials feature unique physicochemical properties that can be of great utility in creating new recognition and transduction processes for chemical and biological sensors15–27 as well as improving the S/N ratio by miniaturization of the sensor elements.28 Gold nanoparticles (AuNPs) possess distinct physical and chemical attributes that make them excellent scaffolds for the fabrication of novel chemical and biological sensors (Figure 1).29–36 First, AuNPs can be synthesized in a straightforward manner and can be made highly stable. Second, they possess unique optoelectronic properties. Third, they provide high surface-to-volume ratio with excellent biocompatibility using appropriate ligands.30 Fourth, these properties of AuNPs can be readily tuned varying their size, shape and the surrounding chemical environment. For example, the binding event between recognition element and the analyte can alter physicochemical properties of transducer AuNPs, such as plasmon resonance absorption, conductivity, redox behavior, etc. that in turn can generate a detectable response signal. Finally, AuNPs offer a suitable platform for multi-functionalization with a wide range of organic or biological ligands for the selective binding and detection of small molecules and biological targets.30–32,36 Each of these attributes of AuNPs has allowed researchers to develop novel sensing strategies with improved sensitivity, stability and selectivity. In the last decade of research, the advent of AuNP as a sensory element provided us a broad spectrum of innovative approaches for the detection of metal ions, small molecules, proteins, nucleic acids, malignant cells, etc. in a rapid and efficient manner.37 Figure 1 Physical properties of AuNPs and schematic illustration of an AuNP-based detection system. In this current review, we have highlighted the several synthetic routes and properties of AuNPs that make them excellent probes for different sensing strategies. Furthermore, we will discuss various sensing strategies and major advances in the last two decades of research utilizing AuNPs in the detection of variety of target analytes including metal ions, organic molecules, proteins, nucleic acids, and microorganisms.

Photoregulated Release of Caged Anticancer Drugs from Gold Nanoparticles

An anticancer drug (5-fluorouracil) was conjugated to the surface of gold nanoparticles through a photocleavable o-nitrobenzyl linkage. In this system, the particle serves as both cage and carrier for the therapeutic, providing a nontoxic conjugate that effectively releases the payload upon long wavelength UV irradiation.

Recognition-mediated activation of therapeutic gold nanoparticles inside living cells

Supramolecular chemistry provides a versatile tool for the organization of molecular systems into functional structures and the actuation of these assemblies for applications through the reversible association between complementary components. Application of this methodology in living systems represents a significant challenge due to the chemical complexity of cellular environments and lack of selectivity of conventional supramolecular interactions. Herein, we present a host-guest system featuring diaminohexane-terminated gold nanoparticles (AuNP-NH2) and complementary cucurbit[7]uril (CB[7]). In this system, threading of CB[7] on the particle surface reduces the cytotoxicity of AuNP-NH2 through sequestration of the particle in endosomes. Intracellular triggering of the therapeutic effect of AuNP-NH2 was then achieved via the administration of 1-adamantylamine (ADA), removing CB[7] from the nanoparticle surface and triggering the endosomal release and concomitant in situ cytotoxicity of AuNP-NH2. This supramolecular strategy for intracellular activation provides a new tool for potential therapeutic applications.

Nanoparticles for detection and diagnosis

Nanoparticle based platforms for identification of chemical and biological agents offer substantial benefits to biomedical and environmental science. These platforms benefit from the availability of a wide variety of core materials as well as the unique physical and chemical properties of these nanoscale materials. This review surveys some of the emerging approaches in the field of nanoparticle based detection systems, highlighting the nanoparticle based screening methods for metal ions, proteins, nucleic acids, and biologically relevant small molecules.

Intracellular Delivery of a Membrane-Impermeable Enzyme in Active Form using Functionalized Gold Nanoparticles

Gold nanoparticles were coated with a short peptide to promote intracellular delivery of membrane-impermeable proteins. Through microscopy and enzyme assays, we demonstrated the particles were able to transport functional enzymes into a variety of cell lines. Significantly, the transported proteins were able to escape from endosomes. Moreover, these particles showed no apparent cytotoxicity.

Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

Barcoding technology enabled measurement of hundreds of cellular proteins from cancer patients with single-cell resolution. Fine-Tuning Single Cancer Cell Protein Analysis Fine-needle aspirates (FNAs) use thin needles to obtain cells from tumor masses. FNAs can give tremendous insight into malignancy, but the number of cells is so small that current technologies for protein analysis, such as immunohistochemistry, are insufficient. To address this technological gap, Ullal and colleagues developed the antibody barcoding with photocleavable DNA (ABCD) platform that allows for simultaneous analysis of many surface proteins on cells from cancer patients. The authors first isolated cancer cells within the FNAs of patients. These cells were then exposed to a cocktail of 90 antibodies, covering the hallmark processes in cancer (for example, apoptosis and DNA damage), and each containing a unique “barcode”—a single strand of DNA that could be released by light (photocleaved) and quantified using fluorescent complementary probes. After validating protein expression on human cancer cell lines with known protein composition, Ullal et al. moved to FNA samples from patients with lung adenocarcinoma. The protein profiles of 11 single cells taken from one patient showed low correlation with the bulk tumor sample, indicating high intratumor heterogeneity. The authors also noted high intertumor heterogeneity, because six patients with tumors that looked identical under a microscope had different proteomic profiles. By clustering the protein expression results and comparing to the patients’ genetic makeup, the authors suggest that therapies could be better personalized. The ABCD platform could help researchers to better understand tumor heterogeneity, as well as allow clinicians to personalize therapies and perhaps even track therapeutic response less invasively, as the authors demonstrated in vitro and in four patients taking kinase inhibitors. Technological challenges remain in making this platform validated and ready for the bedside, but successful early demonstrations in patients with lung cancer suggest that larger-scale clinical trials are in the near future. Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

Drug delivery using nanoparticle-stabilized nanocapsules.

Microcapsules (MCs) are versatile systems with applications in areas as diverse as microreactors, catalysis,[1] diagnostics and drug delivery.[2] In these systems self-assembly of lipids and/or polymers can be used to generate several types of nano- and micro- capsules. These include vesicular structures such as liposomes,[3] polymerosomes,[4] colloidosomes,[5] and polyelectrolyte capsules that feature aqueous interiors and exteriors.[6] An alternate motif is provided by emulsions, where additives are used to stabilize the interface between immiscible fluids to produce e.g. oil-in-water emulsions.[7] Through tailoring of the composition and structure of the building blocks MCs of both types can be engineered with well-defined structures, functions and stability.[8] MCs provide excellent delivery vehicles for biomedical applications, featuring high payload-to-carrier ratios and protection of encapsulated materials from degradation.

Dendronized gold nanoparticles for siRNA delivery.

Small interfering RNA (siRNA) is a versatile tool for regulation of gene expression.[1] The ability of siRNA to silence translation and protein expression of cognate cellular mRNA provides the potential to regulate a wide array of therapeutically-relevant processes.[2] Despite the therapeutic promise of siRNA, there are challenges to its effective clinical and laboratory use.[3] Naked siRNA has low stability against nuclease activity and exhibits poor transfection efficacy due to its relatively large molecular weight and anionic character.[4] These challenges have been addressed using viral and synthetic delivery vehicles.[5] Viral delivery systems feature high transfection efficiencies,[6] however, these vectors can face challenges arising from immunogenicity[7] and mutagenicity.[8] Non-viral siRNA delivery systems have the potential to provide improved safety and predictability relative to viral vectors,[9] and can be broken down into two major groups: covalently conjugated particles[10] and assemblies formed through electrostatic assembly with cationic materials.[11] Both strategies are effective, with the supramolecular strategy allowing the release of genetic material from the surface of the delivery vehicle into the cytosol following uptake, an important requirement for some applications.[12] A variety of cationic materials have been used to condense siRNA for delivery, including cationic lipids[13] and polymers.[14, 15] Functionalized inorganic nanomaterials including carbon nanotubes,[16] iron oxide nanoparticles,[17] quantum dots,[18] and gold nanoparticles[19] provide alternative platforms for siRNA delivery, featuring diverse structures, sizes, core properties and ease of functionalization.[20] Additionally, these materials produce the structural properties of high molecular weight polymers using low molecular weight ligands, with the inorganic core functioning as a space-filling structural element for the presentation of the surface monolayer.[21] We hypothesized that reducing the bulk of the collapsible structure of a polymer to an inorganic nanoparticle would reduce the toxicity of the vehicle (by eliminating unnecessary exposable functionalities that are hidden during complexation), while retaining the favorable properties of polymer delivery vectors through proper functionalization of the surface monolayer. This is an important feature for siRNA delivery. Due to the small size of siRNA, the molecule experiences less efficient interactions with cationic delivery materials than plasmid-sized DNA. SiRNA therefore requires a greater number of vehicles per molecule, or a vehicle with a greater amount of cationic character. Both cases potentially lead to greater toxicity issues. We report here the use of gold nanoparticles featuring dendritic polyethylenimine-like ligands[22] to generate a supramolecular siRNA delivery vehicle featuring high knockdown efficiency and low toxicity. Three different particles featuring gold cores (2 nm diameter) and triethylenetetramine (TETA) terminated dendron ligands were generated for this study via Murray place-exchange reaction (Scheme 1a).[23] The dendronized ligands feature biodegradable glutamic acid scaffolds and cationic TETA moieties that interact electrostatically with negatively charged siRNA (Scheme 1b). All three particles are cationic and resist aggregation as indicated by their zeta potentials. (Figure 1 and Figure S1). Figure 1 Hydrodynamic diameter and zeta potential of G0-AuNP, G1-AuNP, and G2-AuNP. Gel retardation assay of AuNP/β-gal-siRNA complexation at different molar ratios showed a decrease in band intensity due to the fluorescence quenching by complexation with ... Scheme 1 a) Chemical structures of G0-AuNP, G1-AuNP, and G2-AuNP. b) Schematic illustration of AuNP/ β-gal-siRNA complexation and transfection into cells. The molar ratio of dendronized AuNPs to anionic siRNA required for efficient formation of condensed complexes was determined through an agarose gel retardation assay (Figure 1). All of the particles appeared to interact with siRNA, as band intensities associated with free siRNA fell with increasing concentrations of each. The G2 particle, however, appeared to be most efficient at retarding the electrophoretic mobility of siRNA, showing complete retardation at a NP/siRNA molar ratio of 2. Both the G0-AuNP and G1-AuNP still showed free siRNA bands at a NP/β-gal-siRNA ratio of 4 (Figure 1). In further experiments, we found that, when allowed to fully complex with siRNA, G0-AuNP/β-gal-siRNA and G1-AuNP/β-gal-siRNA complexes precipitated at these higher NP/β-gal-siRNA ratios, thus limiting their utility. These results indicate that tuning the degree of multivalency is important for generating AuNP/siRNA complexes that are useful for delivery applications. For this reason, G2-AuNP, capable of generating the most stable self-assembled nanoplexes with siRNA, was used for all subsequent studies. To confirm the condensation ratio between G2-AuNP and siRNA, an ethidium bromide (EtBr) fluorescence exclusion assay, in which the quenching of EtBr upon displacement by G2-AuNP, was performed (Figure S2). The corrected fluorescence curve likewise demonstrated the minimum binding ratio for G2-AuNP/β-gal-siRNA complexation was 2:1. The G2-AuNP/β-gal-siRNA complexes were characterized by dynamic light scattering (DLS). Nanoplexes in ddH2O (double distilled water, MilliQ) were ~110 nm diameter (Figure S3). The assembly size increased to ~700 nm in a serum-containing media (OptiMEM®). The size (~825 nm) did not respond to significant changes even in the presence of media composed of 40% serum (Figure S4). Such changes are typical of supramolecular systems, which are easily affected by changes in pH, ionic strength (which can shield charges and alter the protonation state of surface ligands) and proteins (which can adsorb to the surfaces of nanoparticles and form supramolecular “coronas”).[24] SiRNA mediated knockdown of β-galactosidase (β-gal) expression was studied using an enzyme activity assay in SVR-bag4 endothelial cells. To determine the optimal concentration of siRNA, SVR-bag4 cells were treated with G2-AuNP/β-gal siRNA at different concentrations (NP/siRNA = 2). β-gal silencing appeared to be saturated at G2-AuNP/β-gal-siRNA complex concentrations above 0.042 μM (Figure 2a). To investigate the correlation between gene silencing efficiency and NP/siRNA ratios, SVR-bag4 cells were also treated with a series of G2-AuNP/β-gal-siRNA complexes with molar ratios of 0.5, 1, and 2 (Figure 2b). Gene silencing was found to increase with NP/siRNA ratio, with maximum gene silencing (48%) observed in the case of completely complexed siRNA (NP/siRNA = 2). The knockdown efficiency observed for G2-AuNP/β-gal-siRNA at NP/siRNA 2 was similar to Lipofectamine™ (Invitrogen), a commercially available vehicle with a validated protocol. (Figure 2b). In the absence of either AuNP, siRNA, or a correct siRNA sequence, no significant suppression of β-gal was observed (Figure 2b). Figure 2 β-gal gene silencing in SVR-bag4 cell. a) Gene silencing effect of G2-AuNP/β-gal-siRNA with different concentration from 0.021 to 0.084 μM (NP/siRNA=2). b) Gene silencing effect of naked β-gal siRNA, naked nonsense siRNA, ... Cytotoxicity of both G2-AuNP and G2-AuNP/β-gal-siRNA complexes was evaluated by Alamar blue® assay. Again, SVR bag4 cells were used. Exposure of cells to the G2-AuNP alone for 2 days led to a slight decrease in cell viability (Figure 3a). Exposure of cells to G2-AuNP/β-gal-siRNA complexes for an identical period of time resulted in no significant toxicity (Figure 3b), likely through the charge-quenching effects of siRNA in the nanoparticle complex. Figure 3 Cell viability determined by Alamar blue® assay. Cell viability was determined after the treatment of a) G2-AuNP and b) G2-AuNP/β-gal-siRNA complex at various molar ratios (β-gal siRNA=75 pmol). In summary, we have demonstrated the utility of dendronized AuNPs as a vector for siRNA delivery. In these studies, G2-AuNP suppressed β-gal expression by ~ 50% with minimal toxicity. These nanoparticle vectors possess the benefits of polymeric delivery vehicles such as PEI, while minimizing toxicity through the use of non-toxic core functionality. The application of these systems in biotechnology and biomedicine are currently being explored and will be reported in due course.

Quantitative super-resolution imaging with qPAINT

Counting molecules in complexes is challenging, even with super-resolution microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative points accumulation in nanoscale topography (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the number of single-molecule FISH probes bound to an mRNA target.

Self-assembly and cross-linking of FePt nanoparticles at planar and colloidal liquid-liquid interfaces

Terpyridine thiol functionalized FePt and Au NPs were self-assembled and cross-linked at the liquid-liquid interfaces using Fe(II) metal ion. Complexation of terpyridine with Fe(II) metal ion leads to NP network and affords stable membranes and colloidal shells at the liquid-liquid interfaces.