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.

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide’s ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably non-toxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semi-quantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

Combining the inherent scaffolding provided by DNA structure with spatial control over fluorophore positioning allows the creation of DNA-based photonic wires with the capacity to transfer excitation energy over distances greater than 150 Å. We demonstrate hybrid multifluorophore DNA-photonic wires that both self-assemble around semiconductor quantum dots (QDs) and exploit their unique photophysical properties. In this architecture, the QDs function as both central nanoscaffolds and ultraviolet energy harvesting donors that drive Förster resonance energy transfer (FRET) cascades through the DNA wires with emissions that approach the near-infrared. To assemble the wires, DNA fragments labeled with a series of increasingly red-shifted acceptor-dyes were hybridized in a predetermined linear arrangement to a complementary DNA template that was chemoselectively modified with a hexahistidine-appended peptide. The peptide portion facilitated metal-affinity coordination of multiple hybridized DNA-dye structures to a central QD completing the final nanocrystal-DNA photonic wire structure. We assembled several such hybrid structures where labeled-acceptor dyes were excited by the QDs and arranged to interact with each other via consecutive FRET processes. The inherently facile reconfiguration properties of this design allowed testing of alternate formats including the addition of an intercalating dye located in the template DNA or placement of multiple identical dye acceptors that engaged in homoFRET. Lastly, a photonic structure linking the central QD with multiple copies of DNA hybridized with 4-sequentially arranged acceptor dyes and demonstrating 4-consecutive energy transfer steps was examined. Step-by-step monitoring of energy transfer with both steady-state and time-resolved spectroscopy allowed efficiencies to be tracked through the structures and suggested that acceptor dye quantum yields are the predominant limiting factor. Integrating such DNA-based photonic structures with QDs can help create a new generation of biophotonic wire assemblies with widespread potential in nanotechnology.

semiconductors and noble metals continue to attract strong interest due to their unique nanoscale size-dependent properties. [ 2 ] Noble metal nanoparticles including those synthesized from gold, silver, platinium, and copper, with diameters below 2 nm, do not display a typical surface plasmon resonance (SPR), but rather discrete and size-tunable electronic transitions due to the strong quantum confi nement of free electrons in the particles; this leads to interesting molecule-like properties such as strong fl uorescence. [ 2,5,6 ] Ultrasmall gold nanoclusters (AuNCs) exhibiting UV, visible or near infrared (NIR) fl uorescence, in particular, display a combination of photonic and physical properties that make them especially attractive for biological utility. [5][6][7] AuNCs typically consist of gold clusters (Au n ) that range in discrete atomic sizes, for example n = 18, 21, 25, 28, 32, 39, 55, 102. [ 5-7 ] Their ultrasmall core size (<2.0 nm) makes them signifi cantly smaller than semiconductor quantum dots which are typically ≥5 nm for most core-shell structures. [ 4 ] In contrast to the use of elements such as Cd in semiconductor quantum dots, Au-based materials also have a long history of relatively non-toxic biological application. [ 1,2 ] AuNCs with NIR emission, in particular, can allow for imaging in the tissue transparency window, which, along with their multiphoton excitation properties, may permit Biocompatible, near-infrared luminescent gold nanoclusters (AuNCs) are synthesized directly in water using poly(ethylene glycol)-dithiolane ligands terminating in either a carboxyl, amine, azide, or methoxy group. The ≈1.5 nm diameter AuNCs fl uoresce at ≈820 nm with quantum yields that range from 4-8%, depending on the terminal functional group present, and display average luminescence lifetimes approaching 1.5 μ s. The two-photon absorption (TPA) cross-section and two-photon excited fl uorescence (TPEF) properties are also measured. Long-term testing shows the poly(ethylene glycol) stabilized AuNCs maintain colloidal stability in a variety of media ranging from saline to tissue culture growth medium along with tolerating storage of up to 2 years. DNA and dye-conjugation reactions confi rm that the carboxyl, amine, and azide groups can be utilized on the AuNCs for carbodiimide, succinimidyl ester, and Cu I -assisted cycloaddition chemistry, respectively. High signal-tonoise one-and two-photon cellular imaging is demonstrated. The AuNCs exhibit outstanding photophysical stability during continuous-extended imaging. Concomitant cellular viability testing shows that the AuNCs also elicit minimal cytotoxicity. Further biological applications for these luminescent nanoclustered materials are discussed.

Assembling DNA-based photonic wires around semiconductor quantum dots (QDs) creates optically active hybrid architectures that exploit the unique properties of both components. DNA hybridization allows positioning of multiple, carefully arranged fluorophores that can engage in sequential energy transfer steps while the QDs provide a superior energy harvesting antenna capacity that drives a Förster resonance energy transfer (FRET) cascade through the structures. Although the first generation of these composites demonstrated four-sequential energy transfer steps across a distance >150 Å, the exciton transfer efficiency reaching the final, terminal dye was estimated to be only ~0.7% with no concomitant sensitized emission observed. Had the terminal Cy7 dye utilized in that construct provided a sensitized emission, we estimate that this would have equated to an overall end-to-end ET efficiency of ≤ 0.1%. In this report, we demonstrate that overall energy flow through a second generation hybrid architecture can be significantly improved by reengineering four key aspects of the composite structure: (1) making the initial DNA modification chemistry smaller and more facile to implement, (2) optimizing donor-acceptor dye pairings, (3) varying donor-acceptor dye spacing as a function of the Förster distance R0, and (4) increasing the number of DNA wires displayed around each central QD donor. These cumulative changes lead to a 2 orders of magnitude improvement in the exciton transfer efficiency to the final terminal dye in comparison to the first-generation construct. The overall end-to-end efficiency through the optimized, five-fluorophore/four-step cascaded energy transfer system now approaches 10%. The results are analyzed using Förster theory with various sources of randomness accounted for by averaging over ensembles of modeled constructs. Fits to the spectra suggest near-ideal behavior when the photonic wires have two sequential acceptor dyes (Cy3 and Cy3.5) and exciton transfer efficiencies approaching 100% are seen when the dye spacings are 0.5 × R0. However, as additional dyes are included in each wire, strong nonidealities appear that are suspected to arise predominantly from the poor photophysical performance of the last two acceptor dyes (Cy5 and Cy5.5). The results are discussed in the context of improving exciton transfer efficiency along photonic wires and the contributions these architectures can make to understanding multistep FRET processes.

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.

We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP9GGH6 (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.