Kelly Boeneman Gemmill

Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnology

Chemistries that Facilitate Nanotechnology Kim E. Sapsford,† W. Russ Algar, Lorenzo Berti, Kelly Boeneman Gemmill,‡ Brendan J. Casey,† Eunkeu Oh, Michael H. Stewart, and Igor L. Medintz*,‡ †Division of Biology, Department of Chemistry and Materials Science, Office of Science and Engineering Laboratories, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, United States ‡Center for Bio/Molecular Science and Engineering Code 6900 and Division of Optical Sciences Code 5611, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States College of Science, George Mason University, 4400 University Drive, Fairfax, Virginia 22030, United States Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Sacramento, California 95817, United States Sotera Defense Solutions, Crofton, Maryland 21114, United States

Cytotoxicity of Quantum Dots Used for In Vitro Cellular Labeling: Role of QD Surface Ligand, Delivery Modality, Cell Type, and Direct Comparison to Organic Fluorophores

Interest in taking advantage of the unique spectral properties of semiconductor quantum dots (QDs) has driven their widespread use in biological applications such as in vitro cellular labeling/imaging and sensing. Despite their demonstrated utility, concerns over the potential toxic effects of QD core materials on cellular proliferation and homeostasis have persisted, leaving in question the suitability of QDs as alternatives for more traditional fluorescent materials (e.g., organic dyes, fluorescent proteins) for in vitro cellular applications. Surprisingly, direct comparative studies examining the cytotoxic potential of QDs versus these more traditional cellular labeling fluorophores remain limited. Here, using CdSe/ZnS (core/shell) QDs as a prototypical assay material, we present a comprehensive study in which we characterize the influence of QD dose (concentration and incubation time), QD surface capping ligand, and delivery modality (peptide or cationic amphiphile transfection reagent) on cellular viability in three human cell lines representing various morphological lineages (epithelial, endothelial, monocytic). We further compare the effects of QD cellular labeling on cellular proliferation relative to those associated with a panel of traditionally employed organic cell labeling fluorophores that span a broad spectral range. Our results demonstrate the important role played by QD dose, capping ligand structure, and delivery agent in modulating cellular toxicity. Further, the results show that at the concentrations and time regimes required for robust QD-based cellular labeling, the impact of our in-house synthesized QD materials on cellular proliferation is comparable to that of six commercial cell labeling fluorophores. Cumulatively, our results demonstrate that the proper tuning of QD dose, surface ligand, and delivery modality can provide robust in vitro cell labeling reagents that exhibit minimal impact on cellular viability.

Complex Förster Energy Transfer Interactions between Semiconductor Quantum Dots and a Redox-Active Osmium Assembly

The ability of luminescent semiconductor quantum dots (QDs) to engage in diverse energy transfer processes with organic dyes, light-harvesting proteins, metal complexes, and redox-active labels continues to stimulate interest in developing them for biosensing and light-harvesting applications. Within biosensing configurations, changes in the rate of energy transfer between the QD and the proximal donor, or acceptor, based upon some external (biological) event form the principle basis for signal transduction. However, designing QD sensors to function optimally is predicated on a full understanding of all relevant energy transfer mechanisms. In this report, we examine energy transfer between a range of CdSe-ZnS core-shell QDs and a redox-active osmium(II) polypyridyl complex. To facilitate this, the Os complex was synthesized as a reactive isothiocyanate and used to label a hexahistidine-terminated peptide. The Os-labeled peptide was ratiometrically self-assembled to the QDs via metal affinity coordination, bringing the Os complex into close proximity of the nanocrystal surface. QDs displaying different emission maxima were assembled with increasing ratios of Os-peptide complex and subjected to detailed steady-state, ultrafast transient absorption, and luminescence lifetime decay analyses. Although the possibility exists for charge transfer quenching interactions, we find that the QD donors engage in relatively efficient Förster resonance energy transfer with the Os complex acceptor despite relatively low overall spectral overlap. These results are in contrast to other similar QD donor-redox-active acceptor systems with similar separation distances, but displaying far higher spectral overlap, where charge transfer processes were reported to be the dominant QD quenching mechanism.

Optimizing protein coordination to quantum dots with designer peptidyl linkers.

Semiconductor quantum dots (QDs) demonstrate select optical properties that make them of particular use in biological imaging and biosensing. Controlled attachment of biomolecules such as proteins to the QD surface is thus critically necessary for development of these functional nanobiomaterials. QD surface coatings such as poly(ethylene glycol) impart colloidal stability to the QDs, making them usable in physiological environments, but can impede attachment of proteins due to steric interactions. While this problem is being partially addressed through the development of more compact QD ligands, here we present an alternative and complementary approach to this issue by engineering rigid peptidyl linkers that can be appended onto almost all expressed proteins. The linkers are specifically designed to extend a terminal polyhistidine sequence out from the globular protein structure and penetrate the QD ligand coating to enhance binding by metal-affinity driven coordination. α-Helical linkers of two lengths terminating in either a single or triple hexahistidine motif were fused onto a single-domain antibody; these were then self-assembled onto QDs to create a model immunosensor system targeted against the biothreat agent ricin. We utilized this system to systematically evaluate the peptidyl linker design in functional assays using QDs stabilized with four different types of coating ligands including poly(ethylene glycol). We show that increased linker length, but surprisingly not added histidines, can improve protein to QD attachment and sensor performance despite the surface ligand size with both custom and commercial QD preparations. Implications for these findings on the development of QD-based biosensors are discussed.

Peptide-Functionalized Quantum Dot Biosensors

Quantum dot (QD) nanomaterials have a number of electro-optical properties that make them ideal for biosensing applications. QDs combined with peptides have been used for both targeting and sensing applications, however this review will focus specifically on peptide-functionalized QD biosensors, whose signal transduction occurs through active modulation of the QD photoluminescent properties.

Elucidating Surface Ligand-Dependent Kinetic Enhancement of Proteolytic Activity at Surface-Modified Quantum Dots

Combining biomolecules such as enzymes with nanoparticles has much to offer for creating next generation synergistically functional bionanomaterials. However, almost nothing is known about how these two disparate components interact at this critical biomolecular-materials interface to give rise to improved activity and emergent properties. Here we examine how the nanoparticle surface can influence and increase localized enzyme activity using a designer experimental system consisting of trypsin proteolysis acting on peptide-substrates displayed around semiconductor quantum dots (QDs). To minimize the complexity of analyzing this system, only the chemical nature of the QD surface functionalizing ligands were modified. This was accomplished by synthesizing a series of QD ligands that were either positively or negatively charged, zwitterionic, neutral, and with differing lengths. The QDs were then assembled with different ratios of dye-labeled peptide substrates and exposed to trypsin giving rise to progress curves that were monitored by Förster resonance energy transfer (FRET). The resulting trypsin activity profiles were analyzed in the context of detailed molecular dynamics simulations of key interactions occurring at this interface. Overall, we find that a combination of factors can give rise to a localized activity that was 35-fold higher than comparable freely diffusing enzyme-substrate interactions. Contributing factors include the peptide substrate being prominently displayed extending from the QD surface and not sterically hindered by the longer surface ligands in conjunction with the presence of electrostatic and other productive attractive forces between the enzyme and the QD surface. An intimate understanding of such critical interactions at this interface can produce a set of guidelines that will allow the rational design of next generation high-activity bionanocomposites and theranostics.

Peptide-mediated cellular delivery of semiconductor quantum dots

CdSe/ZnS semiconductor quantum dots (QDs) are ideal materials for biological sensing and cellular imaging applications due to their superior photophysical properties in comparison to fluorescent proteins or dyes and their ease of conjugation to biological materials. We have previously developed a number of in vitro FRET based biosensors in the laboratory for detection of proteases and biological and chemical agents. We would like to expand these biosensing capabilities into cellular systems, requiring development of QD cellular delivery techniques. Peptide mediated cellular delivery of QDs is ideal as peptides are small, easily conjugated to QDs, easily manipulated and synthesized, and can be designed with “handles” for further chemical conjugation with other cargo. Here we discuss four cell delivery peptides that facilitate QD uptake in live cells. Understanding these peptides will help us design better nanoparticle cellular delivery systems and advance our capabilities for in vivo biosensing.

Recent development of dihydrolipoic acid appended ligands for robust and biocompatible quantum dots

Biocompatible nanoparticles have recently attracted significant attention due to increasing interest in their use for biological sensing, cellular labeling and in vivo imaging. Semiconductor quantum dots (QDs) with good colloidal stability as well as small hydrodynamic sizes are particularly useful within these applications. We have developed a series of dihydrolipoic acid (DHLA) based surface ligands to enhance the colloidal stability and biocompatibility of water soluble QDs. Modification of DHLA with poly(ethylene glycol) derivatives provided the QDs with extended colloidal stability over a wide pH range and under high salt concentrations, which contrasts with the limited colloidal stability provided by DHLA alone. Functionalization of the PEG termini enabled one to have easy access to the QD surface and construct a variety of stable QD-biomolecules conjugates. A series of DHLA-based compact ligands with zwitterionic character has also been explored to develop compact sized QDs without sacrificing the colloidal stability. Despite their smaller sizes than the PEG analogs, the QDs coated with the zwitterionic ligands still have excellent colloidal stability and minimize nonspecific interactions in biological environments. Recent studies of thiol-based multidentate ligands and ligand exchange methods further improved the colloidal stability and fluorescence quantum yields.

Membrane-targeting peptides for nanoparticle-facilitated cellular imaging and analysis

The controlled delivery of nanomaterials to the plasma membrane is critical for the development of nanoscale probes that can eventually enable cellular imaging and analysis of membrane processes. Chief among the requisite criteria are delivery/targeting modalities that result in the long-term residence (e.g., days) of the nanoparticles on the plasma membrane while simultaneously not interfering with regular cellular physiology and homeostasis. Our laboratory has developed a suite of peptidyl motifs that target semiconductor nanocrystals (quantum dots (QDs) to the plasma membrane where they remain resident for up to three days. Notably, only small a percentage of the QDs are endocytosed over this time course and cellular viability is maintained. This talk will highlight the utility of these peptide-QD constructs for cellular imaging and analysis.

Characterizing Functionalized DNA for Use in Nanomedicine

DNA as a structural nanomaterial demonstrates great potential as both an in vivo and in vitro designer platform for diagnostic and therapeutic medical use. Much of this work hinges on the ability of DNA to assemble into discrete, controlled structures that interact with, or bind to, other inorganic materials such as nanoparticles or biological molecules which include, for example, drugs and proteins. For these functionalized structures to be most effective, the spatial accuracy of their assembly must be precisely monitored and controlled. Clearly, to design and implement all forms of these functionalized DNA structures, a full characterization will ultimately be a critical necessity. With the current array of characterization techniques available, it can be difficult to choose one specific method especially considering that the efficacy can depend on the type of structure and the final application and environment in which the structure will be used. A review of current methods used for the characterization of complex DNA nanostructures can provide us with a greater understanding of which structures and applications will benefit from specific techniques. More importantly, it can also yield an understanding of which characterization methods can be used in concert to provide a more in depth and integrated understanding of a particular construct as a whole. Comparative characterization may also provide information on the many subtleties and nuances that are to be expected in these complex systems. In this critical overview of available characterization methods, we examine the techniques currently in use for these purposes.