Miao Wu

Mind your P's and Q's: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications.

Semiconductor quantum dots (QDs) and semiconducting polymer nanoparticles (Pdots) are brightly emissive materials that offer many advantages for bioanalysis and bioimaging, and are complementary to revolutionary advances in fluorescence technology. Within the context of biological applications, this review compares the evolution and different stages of development of these two types of nanoparticle, and addresses current perceptions about QDs. Although neither material is a wholesale replacement for fluorescent dyes, recent trends have demonstrated that both types of nanoparticle can excel in applications that are often too demanding for fluorescent dyes alone. Examples discussed in this review include single particle tracking and imaging, multicolor imaging and multiplexed detection, biosensing, point-of-care diagnostics, in vivo imaging and drug delivery.

Quantum Dot-Based Concentric FRET Configuration for the Parallel Detection of Protease Activity and Concentration

Protease expression, activity, and inhibition play crucial roles in a multitude of biological processes; however, these three aspects of their function are difficult for any one bioanalytical probe to measure. To help address this challenge, we report a multifunctional concentric Förster resonance energy transfer (FRET) configuration that combines two modes of biorecognition using aptamers and peptide substrates coassembled to a central semiconductor quantum dot (QD). The aptamer is sensitive to the concentration of protease and the peptide is sensitive to its hydrolytic activity. The role of the QD is to serve as a nanoscale scaffold and initial donor for energy transfer with both Cyanine 3 (Cy3) and Alexa Fluor 647 (A647) fluorescent dyes associated with the aptamer and peptide, respectively. Using thrombin as a model protease, we show that a ratiometric analysis of the emission from the QD, Cy3, and A647 permits discrimination between thrombin and thrombin-like activity, and distinguishes between active, reversibly inhibited, and irreversibly inhibited thrombin. Reliable quantitative results were obtained from a kinetic analysis of the changes in FRET. This concentric FRET format, which capitalizes on both the physical and optical properties of QDs, should be adaptable to other protease targets for which both peptide substrates and binding aptamers are known. It is thus expected to become valuable a tool for the real-time analysis of protease activity and regulation.

Acceleration of Proteolytic Activity Associated with Selection of Thiol Ligand Coatings on Quantum Dots

Nanoparticle bioconjugates are attractive probes for measuring the activity of hydrolytic enzymes. In these configurations, the localization of multiple copies of a hydrolase substrate to a nanoparticle scaffold has been reported to enhance apparent activity by factors of 2 to 3 compared to that for equivalent amounts of substrate in bulk solution. Here, we studied the effect of surface chemistry on protease activity using multivalent QD-peptide substrate conjugates as a model system. QDs were coated with cysteine (CYS), glutathione (GSH), dihydrolipoic acid (DHLA), or 3-mercaptopropionic acid (MPA) ligands, and thrombin and trypsin were used as model proteases. Proteolytic activity was measured for different combinations of ligand and protease using Förster resonance energy transfer (FRET)-based assays. The highest levels of activity were observed with CYS and GSH coatings, and the lowest levels of activity were observed with DHLA and MPA coatings. In all cases, proteolytic activity was accelerated compared to that for an equivalent amount of substrate in bulk solution, with up to 80- and 65-fold increases in the apparent specificity constants for thrombin and trypsin, respectively. Thrombin was more strongly affected by the QD surface chemistry, with up to a 50-fold variation in its apparent specificity constant between ligand coatings, whereas only a 5-fold variation was observed with trypsin. These trends were correlated to adsorption of the proteases on the QDs and are discussed in the context of the physicochemical properties of both components. This work clearly indicates a critical role for the nanoparticle interface in mediating substrate turnover and provides some of the strongest support to date for a so-called hopping model of activity.

Concentric Förster resonance energy transfer imaging.

Concentric Förster resonance energy transfer (cFRET) configurations based on semiconductor quantum dots (QDs) are promising probes for biological sensing because they offer multiplexing capability in a single vector with robust ratiometric detection by exploiting a network of FRET pathways. To expand the scope and utility of cFRET probes, it is necessary to develop and validate cFRET imaging methodology. In this technical note, we present such a methodology using a protease-sensitive cFRET configuration that comprises a green-emitting QD, Alexa Fluor 555 (A555), and Alexa Fluor 647 (A647). Photoluminescence (PL) images were acquired with three filter-based emission channels to permit measurement of A555/QD and A647/QD PL ratios. With reference to calibration samples, these PL ratios were used to calculate quantitative progress curves for proteolytic activity in regions of interest in the acquired images. Importantly, the imaging methodology reproduces quantitative results obtained with a monochromator-based fluorescence plate reader. Spatiotemporal resolution is demonstrated by tracking the activity of two prototypical proteases, trypsin and chymotrypsin, as they diffuse down the length of a capillary. This methodology is expected to enable the future use of cFRET probes for cellular sensing and other imaging assays.

Quantitative measurement of proteolytic rates with quantum dot-peptide substrate conjugates and Förster resonance energy transfer.

An important challenge in biology is the development of probes for visualizing and quantitatively tracking enzyme activity. Proteases are an important class of enzyme with value as both diagnostic and therapeutic targets. In this chapter, we describe the preparation of quantum dot (QD)-peptide substrate conjugates as probes for measuring proteolytic activity. QDs have several highly advantageous optical properties that make these materials especially well suited for applications in bioanalysis and bioimaging. Further, peptide substrates for proteases can be controllably self-assembled to QDs and this capability, in combination with Förster resonance energy transfer (FRET), enables the design of quantitative in vitro assays capable of directly reporting on proteolytic activity. We present a detailed method for the preparation, calibration, and application of such QD probes, along with methods of analysis to generate progress curves for the proteolytic digestion of substrate. Representative data are illustrated for two different proteases and two different QD-fluorescent dye FRET pairs. The general methodology is likely to be applicable with other hydrolytic enzymes in addition to proteases. Overall, the method is straightforward to implement with commercially available materials and does not require specialized expertise.

Intracellularly Actuated Quantum Dot–Peptide–Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway

Nanoparticle (NP)-mediated drug delivery (NMDD) has emerged as a novel method to overcome the limitations of traditional systemic delivery of therapeutics, including the controlled release of the NP-associated drug cargo. Currently, our most advanced understanding of how to control NP-associated cargos is in the context of soft nanoparticles (e.g., liposomes), but less is known about controlling the release of cargos from the surface of hard NPs (e.g., gold NPs). Here we employ a semiconductor quantum dot (QD) as a prototypical hard NP platform and use intracellularly triggered actuation to achieve spatiotemporal control of drug release and modulation of drug efficacy. Conjugated to the QD are two peptides: (1) a cell-penetrating peptide (CPP) that facilitates uptake of the conjugate into the endocytic pathway and (2) a display peptide conjugated to doxorubicin (DOX) via three different linkages (ester, disulfide, and hydrazone) that are responsive to enzymatic cleavage, reducing conditions, and low pH, respectively. Formation of the QD-[peptide-DOX]-CPP complex is driven by self-assembly that allows control over both the ratio of each peptide species conjugated to the QD and the eventual drug dose delivered to cells. Förster resonance energy transfer assays confirmed successful assembly of the QD-peptide complexes and functionality of the linkages. Confocal microscopy was employed to visualize residence of the QD-[peptide-DOX]-CPP complexes in the endocytic pathway, and distinct differences in DOX localization were noted for the ester linkage, which showed clear signs of nuclear delivery versus the hydrazone, disulfide, and amide control. Finally, delivery of the QD-[peptide-DOX]-CPP conjugate resulted in cytotoxicity for the ester linkage that was comparable to free DOX. Attachment of DOX via the hydrazone linkage facilitated intermediary toxicity, while the disulfide and amide control linkages showed minimal toxicity. Our data demonstrate the utility of hard NP-peptide bioconjugates to function as multifunctional scaffolds for simultaneous control over cellular drug uptake and toxicity and the vital role played by the nature of the chemical linkage that appends the drug to the NP carrier.

On the quest for the elusive mechanism of action of daptomycin: Binding, fusion, and oligomerization.

Daptomycin, sold under the trade name CUBICIN, is the first lipopeptide antibiotic to be approved for use against Gram-positive organisms, including a number of highly resistant species. Over the last few decades, a number of studies have tried to pinpoint the mechanism of action of daptomycin. These proposed modes of action often have points in common (e.g. the requirement for Ca2+ and lipid membranes containing a high proportion of phosphatidylglycerol (PG) headgroups), but also points of divergence (e.g. oligomerization in solution and in membranes, membrane perturbation vs. inhibition of cell envelope synthesis). In this study, we investigate how concentration effects may have an impact on the interpretation of the biophysical data used to support a given mechanism of action. Results obtained from small angle neutron scattering (SANS) experiments and molecular dynamics (MD) simulations show that daptomycin oligomerizes at high concentrations (both with and without Ca2+) in solution, but that this oligomer readily falls apart. Photon correlation spectroscopy (PCS) experiments demonstrate that daptomycin causes fusion more readily in DMPC/PG membranes than in POPC/PG, suggesting that the latter may be a better model system. Finally, fluorescence and Förster resonance energy transfer (FRET) experiments reveal that daptomycin binds strongly to the lipid membrane and that oligomerization occurs in a concentration-dependent manner. The combined experiments provide an improved framework for more general and rigorous biophysical studies toward understanding the elusive mechanism of action of daptomycin. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Nanoparticle bioconjugate for controlled cellular delivery of doxorubicin

Nanoparticle (NP)-mediated drug delivery offers the potential to overcome limitations of systemic delivery, including the ability to specifically target cargo and control release of NP-associated drug cargo. Doxorubicin (DOX) is a widely used FDA-approved cancer therapeutic; however, multiple side effects limit its utility. Thus, there is wide interest in modulating toxicity after cell delivery. Our goal here was to realize a NP-based DOX-delivery system that can modulate drug toxicity by controlling the release kinetics of DOX from the surface of a hard NP carrier. To achieve this, we employed a quantum dot (QD) as a central scaffold which DOX was appended via three different peptidyl linkages (ester, disulfide, hydrazone) that are cleavable in response to various intracellular conditions. Attachment of a cell penetrating peptide (CPP) containing a positively charged polyarginine sequence facilitates endocytosis of the ensemble. Polyhistidine-driven metal affinity coordination was used to self-assemble both peptides to the QD surface, allowing for fine control over both the ratio of peptides attached to the QD as well as DOX dose delivered to cells. Microplate-based Förster resonance energy transfer assays confirmed the successful ratiometric assembly of the conjugates and functionality of the linkages. Cell delivery experiments and cytotoxicity assays were performed to compare the various cleavable linkages to a control peptide where DOX is attached through an amide bond. The role played by various attachment chemistries used in QD-peptide-drug assemblies and their implications for the rationale in design of NPbased constructs for drug delivery is described here.

Semiconductor Quantum Dots and Energy Transfer for Optical Sensing and Bioanalysis: Applications

Semiconductor quantum dots (QDs) are very promising materials for optical sensing and bioanalysis. This chapter builds on Chap. 10, which reviewed the optical properties of QDs and their benefits for energy transfer, by illustrating the utility of QDs and energy transfer for optical sensing and bioanalysis. Representative examples of different in vitro assays and cellular probes from the literature are described. Energy transfer mechanisms including Forster resonance energy transfer (FRET ), bioluminescence and chemiluminescence resonance energy transfer (BRET and CRET ), nanosurface energy transfer (NSET ), and charge transfer can be used for optical signal generation in homogeneous assays, single-particle assays, and heterogeneous assays targeting bioanalytes as diverse as nucleic acids, proteins, small molecules, ions, and the activity of enzymes such as proteases, kinases, and nucleases. The importance and versatility of QDs in optical sensing and bioanalysis has been growing steadily since their introduction and will continue to grow in the near future as QD-based assays are optimized and applied to new problems, and new capabilities are developed.