James B. Delehanty

Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing

The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD-dopamine-peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O(2) at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD-dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamines inherent redox chemistry is presented.

Delivering quantum dots into cells: strategies, progress and remaining issues

The use of semiconductor quantum dots (QDs) in biological sensing and labeling continues to grow with each year. Current and projected applications include use as fluorescent labels for cellular labeling, intracellular sensors, deep-tissue and tumor imaging agents, sensitizers for photodynamic therapy, and more recently interest has been sparked in using them as vectors for studying nanoparticle-mediated drug delivery. Many of these applications will ultimately require the QDs to undergo targeted intracellular delivery, not only to specific cells, but also to a variety of subcellular compartments and organelles. It is apparent that this issue will be critical in determining the efficacy of using QDs, and indeed a variety of other nanoparticles, for these types of applications. In this review, we provide an overview of the current methods for delivering QDs into cells. Methods that are covered include facilitated techniques such as those that utilize specific peptide sequences or polymer delivery reagents and active methods such as electroporation and microinjection. We critically examine the benefits and liabilities of each strategy and illustrate them with selected examples from the literature. Several important related issues such as QD size and surface coating, methods for QD biofunctionalization, cellular physiology and toxicity are also discussed. Finally, we conclude by providing a perspective of how this field can be expected to develop in the future.

Cellular Uptake and Fate of PEGylated Gold Nanoparticles Is Dependent on Both Cell-Penetration Peptides and Particle Size

Numerous studies have examined how the cellular delivery of gold nanoparticles (AuNPs) is influenced by different physical and chemical characteristics; however, the complex relationship between AuNP size, uptake efficiency and intracellular localization remains only partially understood. Here we examine the cellular uptake of a series of AuNPs ranging in diameter from 2.4 to 89 nm that are synthesized and made soluble with poly(ethylene glycol)-functionalized dithiolane ligands terminating in either carboxyl or methoxy groups and covalently conjugated to cell penetrating peptides. Following synthesis, extensive physical characterization of the AuNPs was performed with UV-vis absorption, gel electrophoresis, zeta potential, dynamic light scattering, and high resolution transmission electron microscopy. Uptake efficiency and intracellular localization of the AuNP-peptide conjugates in a model COS-1 cell line were probed with a combination of silver staining, fluorescent counterstaining, and dual mode fluorescence coupled to nonfluorescent scattering. Our findings show that AuNP cellular uptake is directly dependent on the surface display of the cell-penetrating peptide and that the ultimate intracellular destination is further determined by AuNP diameter. The smallest 2.4 nm AuNPs were found to localize in the nucleus, while intermediate 5.5 and 8.2 nm particles were partially delivered into the cytoplasm, showing a primarily perinuclear fate along with a portion of the nanoparticles appearing to remain at the membrane. The 16 nm and larger AuNPs did not enter the cells and were located at the cellular periphery. A preliminary assessment of cytotoxicity demonstrated minimal effects on cellular viability following peptide-mediated uptake.

Semiconductor quantum dots in bioanalysis: crossing the valley of death.

Colloidal semiconductor quantum dots (QDs) have evolved beyond scientific novelties and are transitioning into bona fide analytical tools. We describe the burgeoning role of QDs in many different fields of bioanalyses and highlight the advantages afforded by their unique physical and optical properties.

Multifunctional Compact Zwitterionic Ligands for Preparing Robust Biocompatible Semiconductor Quantum Dots and Gold Nanoparticles

We describe the synthesis of a series of four different ligands which are used to prepare hydrophilic, biocompatible luminescent quantum dots (QDs) and gold nanoparticles (AuNPs). Overall, the ligands are designed to be compact while still imparting a zwitterionic character to the NPs. Ligands are synthesized appended to a bidentate dihydrolipoic acid- (DHLA) anchor group, allowing for high-affinity NP attachment, and simultaneously incorporate tertiary amines along with carboxyl and/or hydroxyl groups. These are placed in close proximity within the ligand structure and their capacity for joint ionization imparts the requisite zwitterionic nature to the nanocrystal. QDs functionalized with the four different compact ligands were subjected to extensive physical characterization including surface charge, wettability, hydrodynamic size, and tolerance to a wide pH range or high salt concentration over time. The utility of the compact ligand coated QDs was further examined by testing of direct conjugation to polyhistidine-appended protein and peptides, aqueous covalent-coupling chemistry, and the ability to engage in Förster resonance energy transfer (FRET). Conjugating cell penetrating peptides to the compact ligand coated QD series facilitated their rapid and efficient cellular uptake, while subsequent cytotoxicity tests showed no apparent decreases in cell viability. In vivo biocompatibility was also demonstrated by microinjecting the compact ligand coated QDs into cells and monitoring their stability over time. Inherent benefits of the ligand design could be extended beyond QDs as AuNPs functionalized with the same compact ligand series showed similar colloidal properties. The strong potential of these ligands to expand NP capabilities in many biological applications is highlighted.

Combining chemoselective ligation with polyhistidine-driven self-assembly for the modular display of biomolecules on quantum dots.

One of the principle hurdles to wider incorporation of semiconductor quantum dots (QDs) in biology is the lack of facile linkage chemistries to create different types of functional QD--bioconjugates. A two-step modular strategy for the presentation of biomolecules on CdSe/ZnS core/shell QDs is described here which utilizes a chemoselective, aniline-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto peptides and DNA. This specifically provides them the ability to ratiometrically self-assemble to hydrophilic QDs. The versatility of this labeling approach was highlighted by ligating proteolytic substrate peptides, an oligoarginine cell-penetrating peptide, or a DNA-probe to cognate hexahistidine peptidyl sequences. The modularity allowed subsequently self-assembled QD constructs to engage in different types of targeted bioassays. The self-assembly and photophysical properties of individual QD conjugates were first confirmed by gel electrophoresis and Forster resonance energy transfer analysis. QD-dye-labeled peptide conjugates were then used as biosensors to quantitatively monitor the proteolytic activity of caspase-3 or elastase enzymes from different species. These sensors allowed the determination of the corresponding kinetic parameters, including the Michaelis constant (K(M)) and the maximum proteolytic activity (V(max)). QDs decorated with cell-penetrating peptides were shown to be successfully internalized by HEK 293T/17 cells, while nanocrystals displaying peptide--DNA conjugates were utilized as fluorescent probes in hybridization microarray assays. This modular approach for displaying peptides or DNA on QDs may be extended to other more complex biomolecules such as proteins or utilized with different types of nanoparticle materials.

Peptides for specifically targeting nanoparticles to cellular organelles: quo vadis?

The interfacing of nanomaterials and especially nanoparticles within all aspects of biological research continues to grow at a nearly unabated pace with projected applications focusing on powerful new tools for cellular labeling, imaging, and sensing, theranostic materials, and drug delivery. At the most fundamental level, many of these nanoparticles are meant to target not only very specific cell-types, regardless of whether they are in a culture, tissue, an animal model, or ultimately a patient, but also in many cases a specific subcellular organelle. During this process, these materials will undergo a complex journey that must first find the target cell of interest, then be taken up by those cells across the extracellular membrane, and ultimately localize to a desired subcellular organelle, which may include the nucleus, plasma membrane, endolysosomal system, mitochondria, cytosol, or endoplasmic reticulum. To accomplish these complex tasks in the correct sequence, researchers are increasingly interested in selecting for and exploiting targeting peptides that can impart the requisite capabilities to a given nanoparticle construct. There are also a number of related criteria that need careful consideration for this undertaking centering on the nature and properties of the peptide vector itself, the peptide-nanoparticle conjugate characteristics, and the target cell. Here, we highlight some important issues and key research areas related to this burgeoning field. We begin by providing a brief overview of some criteria for optimal attachment of peptides to nanoparticles, the predominant methods by which nanoparticles enter cells, and some of the peptide sequences that have been utilized to facilitate nanoparticle delivery to cells focusing on those that engender the initial targeting and uptake. Because almost all materials delivered to cells by peptides utilize the endosomal system of vesicular transport and in many cases remain sequestered within the vesicles, we critically evaluate the issue of endosomal escape in the context of some recently reported successes in this regard. Following from this, peptides that have been reported to deliver nanoparticles to specific subcellular compartments are examined with a focus on what they delivered and the putative mechanisms by which they were able to accomplish this. The last section focuses on two areas that are critical to realizing this overall approach in the long term. The first is how to select for peptidyl sequences capable of improved or more specific cellular or subcellular targeting based upon principles commonly associated with drug discovery. The second looks at what has been done to create modular peptides that incorporate multiple desirable functionalities within a single, contiguous sequence. This provides a viable alternative to either the almost insurmountable challenge of finding one sequence capable of all functions or, alternatively, attaching different peptides with different functionalities to the same nanoparticle in different ratios when trying to orchestrate their net effects. Finally, we conclude with a brief perspective on the future evolution and broader impact of this growing area of bionanoscience.

Modular poly(ethylene glycol) ligands for biocompatible semiconductor and gold nanocrystals with extended pH and ionic stability

We describe the design of new ligands made by coupling commercially available poly(ethylene glycol) methyl ether (mPEG, HO-PEG-OCH3) and thioctic acid (TA) via a stable amide bond to form TA-PEG-OCH3 molecules. The ligands were obtained by a simple transformation of the hydroxyl group on the mPEG into an amine group, followed by attachment of TA viaN,N′-dicyclohexylcarbodiimide (DCC) coupling. Following ring opening of the 1,2-dithiolane on the TA-PEG-OCH3 to form a dihydrolipoic acid (DHLA) group, DHLA-PEG-OCH3 was obtained. Cap exchange of nanoparticles with DHLA-PEG-OCH3 provided dispersions in buffer solutions that were stable over a broad pH range (from 3 to 13 for CdSe-ZnS QDs and 2–13 for Au nanoparticles). Using DHLA-PEG-OCH3 either neat or mixed with amine- or carboxyl-terminated ligands (DHLA-PEG-NH2 or DHLA-PEG-COOH) allowed tuning of the surface functionalities of these nanoparticles. Microinjection of the ligand-exchanged QDs into live cells indicated that the newly capped QDs were stable and well dispersed in the cell cytosol for up to 32 h following delivery. The fluorescence distribution and its evolution over time of these DHLA-PEG-OCH3-QDs indicate improved intracellular stability and reduced non-specific interactions compared to nanocrystals capped with DHLA-PEG-OH.

Multidentate Poly(ethylene glycol) Ligands Provide Colloidal Stability to Semiconductor and Metallic Nanocrystals in Extreme Conditions

We present the design and synthesis of a new set of poly(ethylene glycol) (PEG)-based ligands appended with multidentate anchoring groups and test their ability to provide colloidal stability to semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs) in extreme buffer conditions. The ligands are made of a PEG segment appended with two thioctic acid (TA) or two dihydrolipoic acid (DHLA) anchoring groups, bis(TA)-PEG-OCH(3) or bis(DHLA)-PEG-OCH(3). The synthesis utilizes Michael addition to create a branch point at the end of a PEG chain combined with carbodiimide-coupling to attach two TA groups per PEG chain. Dispersions of CdSe-ZnS core-shell QDs and AuNPs with remarkable long-term colloidal stability at pHs ranging from 1.1 to 13.9 and in the presence of 2 M NaCl have been prepared and tested using these ligands. AuNPs with strong resistance to competition from dithiothreitol (as high as 1.5 M) have also been prepared. This opens up possibilities for using them as stable probes in a variety of bio-related studies where resistance to degradation at extreme pHs, at high electrolyte concentration, and in thiol-rich environments is highly desirable. The improved colloidal stability of nanocrystals afforded by the tetradentate ligands was further demonstrated via the assembly of stable QD-nuclear localization signal peptide bioconjugates that promoted intracellular uptake.

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

Luminescent semiconductor quantum dots (QDs) are one of the more popular nanomaterials currently utilized within biological applications. However, what is not widely appreciated is their growing role as versatile energy transfer (ET) donors and acceptors within a similar biological context. The progress made on integrating QDs and ET in biological configurations and applications is reviewed in detail here. The goal is to provide the reader with (1) an appreciation for what QDs are capable of in this context, (2) how this field has grown over a relatively short time span, and, in particular, (3) how QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active nanomaterial-based bioconjugates. An initial discussion of QD materials along with key concepts surrounding their preparation and bioconjugation is provided given the defining role these aspects play in the QDs ability to succeed in subsequent ET applications. The discussion is then divided around the specific roles that QDs provide as either Förster resonance energy transfer (FRET) or charge/electron transfer donor and/or acceptor. For each QD-ET mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining their biosensing and related ET utility. Other configurations such as incorporation of QDs into multistep ET processes or use of initial chemical and bioluminescent excitation are treated similarly. ET processes that are still not fully understood such as QD interactions with gold and other metal nanoparticles along with carbon allotropes are also covered. Given their maturity, some specific applications ranging from in vitro sensing assays to cellular imaging are separated and discussed in more detail. Finally a perspective on how this field will continue to evolve is provided.