Evaluation of diverse peptidyl motifs for cellular delivery of semiconductor quantum dots Optical Nanosensing in Cells

Cell-penetrating peptides (CPPs) have rapidly become a mainstay technology for facilitating the delivery of a wide variety of nanomaterials to cells and tissues. Currently, the library of CPPs to choose from is still limited, with the HIV TAT-derived motif still being the most used. Among the many materials routinely delivered by CPPs, nanoparticles are of particular interest for a plethora of labeling, imaging, sensing, diagnostic, and therapeutic applications. The development of nanoparticle-based technologies for many of these uses will require access to a much larger number of functional peptide motifs that can both facilitate cellular delivery of different types of nanoparticles to cells and be used interchangeably in the presence of other peptides and proteins on the same surface. Here, we evaluate the utility of four peptidyl motifs for their ability to facilitate delivery of luminescent semiconductor quantum dots (QDs) in a model cell culture system. We find that an LAH4 motif, derived from a membrane-inserting antimicrobial peptide, and a chimeric sequence that combines a sweet arrow peptide with a portion originating from the superoxide dismutase enzyme provide effective cellular delivery of QDs. Interestingly, a derivative of the latter sequence lacking just a methyl group was found to be quite inefficient, suggesting that even small changes can have significant functional outcomes. Delivery was effected using 1 h incubation with cells, and fluorescent counterstaining strongly suggests an endosomal uptake process that requires a critical minimum number or ratio of peptides to be displayed on the QD surface. Concomitant cytoviability testing showed that the QD-peptide conjugates are minimally cytotoxic in the model COS-1 cell line tested. Potential applications of these peptides in the context of cellular delivery of nanoparticles and a variety of other (bio)molecules are discussed

Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnology

Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnolog

Cytotoxicity of Quantum Dots Used for <i>In Vitro</i> 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 <i>in vitro</i> 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 <i>in vitro</i> 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 <i>in vitro</i> cell labeling reagents that exhibit minimal impact on cellular viability

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

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 <i>via</i> 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

A New Family of Pyridine-Appended Multidentate Polymers As Hydrophilic Surface Ligands for Preparing Stable Biocompatible Quantum Dots

The growing utility of semiconductor quantum dots (QDs) in biochemical and cellular research necessitates, in turn, continuous development of surface functionalizing ligands to optimize their performance for ever more challenging and diverse biological applications. Here, we describe a new class of multifunctional polymeric ligands as a stable, compact and high affinity alternative to multidentate thiolated molecules. The polymeric ligands are designed with a poly­(acrylic acid) backbone where pyridines are used as anchoring groups that are not sensitive to degradation by air and light, along with short poly­(ethylene glycol) (PEG) pendant groups which are coincorporated for aqueous solubility, biocompatibility and colloidal stability. The percentages of each of the latter functional groups are controlled during initial synthesis along with incorporation of carboxyl groups which serve as chemical handles for subsequent covalent modification of the QD surface. A detailed physicochemical characterization indicates that the multiple pyridine groups are efficiently bound on the QD surface since they provide for relatively small overall hydrodynamic sizes along with good colloidal stability and strong fluorescence over a wide pH range, under high salt concentration and in extremely dilute conditions at room temperature under room light over extended timeframes. Covalent conjugation of dyes and metal-affinity coordination with functional enzymes to the QD surfaces were also demonstrated. Biocompatibility and long-term stability of the pyridine polymer coated QDs were then confirmed in a battery of relevant assays including cellular delivery by both microinjection and peptide facilitated uptake along with intracellular single QD tracking studies and cytotoxicity testing. Cumulatively, these results suggest this QD functionalization strategy is a viable alternative that provides some desirable properties of both compact, discrete ligands and large amphiphilic polymers

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

Examining the Polyproline Nanoscopic Ruler in the Context of Quantum Dots

The rigidity and defined length of the polyproline type II helix (PPII) have made it the structural basis of a nanoscopic ruler, which has been widely applied in Förster resonance energy transfer (FRET) studies. A growing body of data, however, has questioned the foundation for this and has provided evidence for structural perturbations to the PPII caused by temperature, salt content, solvent polarity, and even Pro repeat length. Here, we examine the polyproline ruler in the context of semiconductor quantum dots (QDs) and FRET. For this study, a series of polyproline peptides (Pro_<i>n</i>, <i>n</i> = 0, 3, 6, 9, 12, 15, 18) displaying a C-terminal hexahistidine sequence (His₆) and an N-terminal cysteine for site-specific labeling with Cy3 dye were synthesized. Peptide rigidity was first examined with ATTO 647 Ni²⁺-nitrilotriacetic acid acceptor dye coordinated to the His₆-termini of the Cy3 donor-labeled peptides. These conditions provided a steady-state fluorescent response that closely followed FRET predictions derived from the expected donor–acceptor distances; this confirmed the PPII conformation and nanoscopic ruler in the context of our sequences. Peptides were next assembled to negatively charged dihydrolipoic acid functionalized 530 nm emitting QDs, which then acted as a donor to the Cy3 acceptor. These data revealed decreases in FRET efficiency <i>E</i> but at significantly less than the magnitude predicted. Lastly, peptides were assembled to neutral poly­(ethylene glycol) or PEG-appended dihydrolipoic acid functionalized 530 nm QDs, and here FRET <i>E</i> did not change as peptide length increased. The latter observations were confirmed with excited-state lifetime measurements and single-pair FRET analysis. Circular dichroism spectroscopy was performed on select peptides both free in solution and as assembled to the PEGylated QDs along with physical characterization by dynamic light scattering and electrophoretic mobility. Overall, analysis confirms the initial validity of the rigid polyproline ruler, while it also suggests that peptide subpopulations adopt a different conformation when attached to QDs. Rather than a gross structural rearrangement, this change is consistent with a <i>trans</i> to <i>cis</i> bond reversion in some of the Pro–Pro peptidyl bond(s), which alters persistence length. This suggests that the PPII is highly context dependent and can be strongly influenced by microenvironments or interfacial effects and thus requires careful consideration of experimental format and related factors before being implemented with nanoparticles