Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials.

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 nontoxic 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, semiquantitative 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.

Quantum dots and fluorescent protein FRET-based biosensors.

There has been considerable recent interest in the creation of nanoparticle-biomolecule hybrid materials for uses such as in vitro and in vivo biosensing, biological imaging, and drug -delivery. Nanoparticles have a high surface to volume ratio, making them capable of being decorated with -various biomolecules on their surface which retain their biological activity. Techniques to bind these biomolecules to nanoparticle surfaces are also advancing rapidly. Here we demonstrate hybrid materials assembled around CdSe/ZnS core/shell semiconductor quantum dots (QDs). These intrinsically fluorescent materials are conjugated to the fluorescent proteins YFP, mCherry and the light harvesting complex b-phycoerythrin (b-PE). QDs have fluorescent properties that make them ideal as donor fluorophores for Förster resonance energy transfer (FRET) while the fluorescent proteins are able to act as FRET acceptors displaying many advantages over organic dyes. We examine FRET interactions between QDs and all three fluorescent proteins. Furthermore, we show QD-mCherry hybrid materials can be utilized for in vitro biosensing of caspase-3 enzymatic activity. We further show that QDs and fluorescent proteins can be conjugated together intracellularly with strong potential for live-cell imaging and biosensing applications.

Reactive semiconductor nanocrystals for chemoselective biolabeling and multiplexed analysis.

Effective biological application of nanocrystalline semiconductor quantum dots continues to be hampered by the lack of easily implemented and widely applicable labeling chemistries. Here, we introduce two new orthogonal nanocrystal bioconjugation chemistries that overcome many of the labeling issues associated with currently utilized approaches. These chemistries specifically target either (1) the ubiquitous amines found on proteins or (2) thiols present in either antibody hinge regions or recombinantly introduced into other proteins to facilitate site-specific labeling. The amine chemistry incorporates aniline-catalyzed hydrazone bond formation, while the sulfhydryl chemistry utilizes nanocrystals displaying surface activated maleimide groups. Both reactive chemistries are rapidly implemented, yielding purified nanocrystal-protein bioconjugates in as little as 3 h. Following initial characterization of the nanocrystal materials, the wide applicability and strong multiplexing potential of these chemistries are demonstrated in an array of applications including immunoassays, immunolabeling in both cellular and tissue samples, in vivo cellular uptake, and flow cytometry. Side-by-side comparison of the immunolabeled cells suggested a functional equivalence between results generated with the amine and thiol-labeled antibody-nanocrystal bioconjugates in that format. Three-color labeling was achieved in the cellular uptake format, with no significant toxicity observed while simultaneous five-color labeling of different epitopes was demonstrated for the immunolabeled tissue sample. Novel labeling applications are also facilitated by these chemistries, as highlighted by the ability to directly label cellular membranes in adherent cell cultures with the thiol-reactive chemistry.

Spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques.

Multicolor fluorescent labeling of both intra- and extracellular structures is a powerful technique for simultaneous monitoring of multiple complex biochemical processes. This approach remains extremely challenging, however, as it often necessitates the combinatorial use of numerous targeting probes (e.g., antibodies), multistep bioconjugation chemistries, different delivery strategies (e.g., electroporation or transfection reagents), cellular fixation coupled with membrane permeabilization, and complex spectral deconvolution. Here, we present a nanoparticle-based fluorescence labeling strategy for the multicolor labeling of distinct subcellular compartments within live cells without the need for antibody conjugation or cellular fixation/permeabilization. This multipronged approach incorporates an array of delivery strategies, which localize semiconductor quantum dots (QDs) to various subcellular structures. QD uptake is implemented in a spaciotemporal manner by staggering the delivery of QD-peptide composites and exploiting various innate (peptide-mediated endocytosis, peptide-membrane interaction, polymer-based transfection) along with physical (microinjection) cellular delivery modalities to live cells growing in culture over a 4 day period. Imaging of the different intracellular labels is simplified by the unique photophysical characteristics of the QDs in combination with Förster resonance energy transfer sensitization, which allow for multiple spectral windows to be accessed with one excitation wavelength. Using this overall approach, QDs were targeted to both early and late endosomes, the cellular cytosol, and the plasma membrane in live cells, ultimately allowing for simultaneous five-color fluorescent imaging.

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.

Monitoring botulinum neurotoxin a activity with peptide-functionalized quantum dot resonance energy transfer sensors.

Botulinum neurotoxins (BoNTs) are extremely potent bacterial toxins that contaminate food supplies along with having a high potential for exploitation as bioterrorism agents. There is a continuing need to rapidly and sensitively detect exposure to these toxins and to verify their active state, as the latter directly affects diagnosis and helps provide effective treatments. We investigate the use of semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA). A modular LcA peptide substrate was designed and optimized to contain a central LcA recognition/cleavage region, a unique residue to allow labeling with a Cy3 acceptor dye, an extended linker-spacer sequence, and a terminal oligohistidine that allows for final ratiometric peptide-QD-self-assembly. A number of different QD materials displaying charged or PEGylated surface-coatings were evaluated for their ability to self-assemble dye-labeled LcA peptide substrates by monitoring FRET interactions. Proteolytic assays were performed utilizing either a direct peptide-on-QD format or alternatively an indirect pre-exposure of peptide to LcA prior to QD assembly. Variable activities were obtained depending on QD materials and formats used with the most sensitive pre-exposure assay result demonstrating a 350 pM LcA limit of detection. Modeling the various QD-peptide sensor constructs provided insight into how the resulting assembly architecture influenced LcA recognition interactions and subsequent activity. These results also highlight the unique roles that both peptide design and QD features, especially surface-capping agents, contribute to overall sensor activity.

Self-assembled quantum dot-sensitized multivalent DNA photonic wires.

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.

Quantum dot DNA bioconjugates: attachment chemistry strongly influences the resulting composite architecture.

The unique properties provided by hybrid semiconductor quantum dot (QD) bioconjugates continue to stimulate interest for many applications ranging from biosensing to energy harvesting. Understanding both the structure and function of these composite materials is an important component in their development. Here, we compare the architecture that results from using two common self-assembly chemistries to attach DNA to QDs. DNA modified to display either a terminal biotin or an oligohistidine peptidyl sequence was assembled to streptavidin/amphiphilic polymer- or PEG-functionalized QDs, respectively. A series of complementary acceptor dye-labeled DNA were hybridized to different positions on the DNA in each QD configuration and the separation distances between the QD donor and each dye-acceptor probed with Förster resonance energy transfer (FRET). The polyhistidine self-assembly yielded QD-DNA bioconjugates where predicted and experimental separation distances matched reasonably well. Although displaying efficient FRET, data from QD-DNA bioconjugates assembled using biotin-streptavidin chemistry did not match any predicted separation distances. Modeling based upon known QD and DNA structures along with the linkage chemistry and FRET-derived distances was used to simulate each QD-DNA structure and provide insight into the underlying architecture. Although displaying some rotational freedom, the DNA modified with the polyhistidine assembles to the QD with its structure extended out from the QD-PEG surface as predicted. In contrast, the random orientation of streptavidin on the QD surface resulted in DNA with a wide variety of possible orientations relative to the QD which cannot be controlled during assembly. These results suggest that if a particular QD biocomposite structure is desired, for example, random versus oriented, the type of bioconjugation chemistry utilized will be a key influencing factor.

Delivering quantum dot-peptide bioconjugates to the cellular cytosol: escaping from the endolysosomal system.

For luminescent quantum dots (QDs) to realize their full potential as intracellular labeling, imaging and sensing reagents, robust noninvasive methods for their delivery to the cellular cytosol must be developed. Our aim in this study was to explore a range of methods aimed at delivering QDs to the cytosol. We have previously shown that QDs functionalized with a polyarginine 'Tat' cell-penetrating peptide (CPP) could be specifically delivered to cells via endocytic uptake with no adverse effects on cellular proliferation. We began by assessing the long-term intracellular fate and stability of these QD-peptide conjugates. We found that the QDs remained sequestered within acidic endolysosomal vesicles for at least three days after initial uptake while the CPP appeared to remain stably associated with the QD throughout this time. We next explored techniques designed to either actively deliver QDs directly to the cytosol or to combine endocytosis with subsequent endosomal escape to the cytosol in several eukaryotic cell lines. Active delivery methods such as electroporation and nucleofection delivered only modest amounts of QDs to the cytosol as aggregates. Delivery of QDs using a variety of transfection polymers also resulted in primarily endosomal sequestration of QDs. However, in one case the commercial PULSin reagent did facilitate a modest cytosolic dispersal of QDs, but only after several days in culture and with significant polymer-induced cytotoxicity. Finally, we demonstrated that an amphiphilic peptide designed to mediate cell penetration and vesicle membrane interactions could mediate rapid QD uptake by endocytosis followed by a slower efficient endosomal release which peaked at 48 h after initial delivery. Importantly, this QD-peptide bioconjugate elicited minimal cytotoxicity in the cell lines tested.

Intracellular bioconjugation of targeted proteins with semiconductor quantum dots.

We demonstrate controlled in vivo bioconjugation of a targeted intracellular protein to semiconductor quantum dots (QDs). Metal-affinity driven coordination of oligohistidine-appended proteins for chelated divalent cations was exploited to facilitate this interaction. Monomeric mCherry red fluorescent protein recombinantly engineered to express an N-terminal hexahistidine sequence was expressed from a eukaryotic plasmid vector following transfection into COS-1 cells. QDs solubilized with a carboxylated polymeric ligand and pretreated with Ni(2+) were then microinjected into the mCherry-expressing COS-1 cells. Förster resonance energy transfer (FRET) between the central QD donors and mCherry acceptors specifically coordinated to their surface was utilized to probe and confirm intracellular conjugate formation. We unexpectedly found that mCherry attachment to the QDs also substantially improves its resistance to photobleaching. This proof-of-concept, highlighting targeted intracellular bioconjugation to QDs, suggests that many cytoplasmic proteins expressing the ubiquitous hexahistidine affinity handle can be specifically attached to QDs in vivo. This approach can facilitate long-term monitoring of their spatio-temporal activity or, alternatively, allow engineering and in situ assembly of designer chimeric QD-fluorescent protein sensors.

Polyvalent display and packing of peptides and proteins on semiconductor quantum dots: predicted versus experimental results.

Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to approximately 44 kDa. Conjugation to the surface of dihydrolipoic acid-functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye-labeled peptides and proteins are self-assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space-filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.

Peptides for specific intracellular delivery and targeting of nanoparticles: implications for developing nanoparticle-mediated drug delivery.

The use of peptides to mediate the delivery and uptake of nanoparticle (NP) materials by mammalian cells has grown significantly over the past 10 years. This area of research has important implications for the development of new therapeutic materials and for the emerging field of NP-mediated drug delivery. In this review, we highlight recent advances in the delivery of various NPs by some of the more commonly employed cellular delivery peptides and discuss important related factors such as NP-peptide bioconjugation, uptake efficiency, intracellular fate and toxicity. We also highlight various demonstrations of therapeutic applications of NP-peptide conjugates where appropriate. The paper concludes with a brief forward-looking perspective discussing what can be expected as this field develops in the coming years.

Quantum dots: a powerful tool for understanding the intricacies of nanoparticle-mediated drug delivery.

Nanoparticle-mediated drug delivery (NMDD) is an emerging research area that seeks to address many of the pharmacokinetic issues encountered with traditional systemically administered drug therapies. Although the field is still in its infancy, recent research has already highlighted the potential for improved drug delivery and targeted therapeutics; however, the real promise lies in combining drug therapy with diagnostic imaging, nucleic acid delivery/gene therapy and/or biosensing applications all in one engineered nanoparticle vector. In this review, the authors discuss the unique contributions that luminescent semiconductor nanocrystals or quantum dots (QDs) offer for NMDD, how they can function as a powerful nanoscale platform to understand this process at its most basic level, and even provide drug-related properties in certain circumstances. Selected examples from the current literature are utilized to describe both their potential and the contributions they have already made towards the design and implementation of NMDD vectors. Important related issues such as QD biofunctionalization and toxicity are also discussed. The paper concludes with a perspective of how this field can be expected to develop in the future.

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins. The library was screened for the ability to rescue dnaA(ts) mutants, and a candidate gfp-dnaA was used to replace the dnaA gene of wild-type cells. The resulting cells produce close to physiological levels of GFP-DnaA from the endogenous promoter as their only source of DnaA and somewhat under-initiate replication with moderate asynchrony. Visualization of GFP-tagged DnaA in living cells revealed that DnaA adopts a helical pattern that spirals along the long axis of the cell, a pattern also seen in wild-type cells by immunofluorescence with affinity purified anti-DnaA antibody. Although the DnaA helices closely resemble the helices of the actin analogue MreB, co-visualization of GFP-tagged DnaA and RFP-tagged MreB demonstrates that DnaA and MreB adopt discrete helical structures along the length of the longitudinal cell axis.

Sensing caspase 3 activity with quantum dot-fluorescent protein assemblies.

We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically self-assembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescence resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD-mCherry conjugates specifically cleaved the engineered mCherry linker sequence, altering the energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.

Resonance Energy Transfer Between Luminescent Quantum Dots and Diverse Fluorescent Protein Acceptors.

We characterized the resonance energy transfer interactions for conjugates consisting of QD donors self-assembled with three distinct fluorescent protein acceptors: two monomeric fluorescent proteins, the dsRed derivative mCherry or yellow fluorescent protein and the multi-chromophore b-phycoerythrin light harvesting complex. Using steady-state and time-resolved fluorescence, we showed that nonradiative transfer of excitation energy in these conjugates can be described within the Förster dipole-dipole formalism, with transfer efficiencies that vary with the degree of spectral overlap, donor-acceptor separation distance and the number of acceptors per QD. Comparison between the quenching data and simulation of the conjugate structures indicated that while energy transfer to monomeric proteins was identical to what was measured for QD-dye pairs, interactions with b-phycoerythrin were more complex. For the latter, the overall transfer efficiency results from the cumulative contribution of individual channels between the central QD and the chromophores distributed throughout the protein structure. Due to the biocompatible nature of fluorescent proteins, these QD-assemblies may have great potential for use in intracellular imaging and sensing.

Chromosomal replication and the cell membrane.

The importance of the cell membrane in bacterial chromosomal replication continues to emerge. Recent advances include better definition of the biochemical interaction between membrane acidic phospholipids and the replication initiator, DnaA protein, the physiological impact that an altered membrane lipid composition has on chromosomal replication and the identification and characterization of recently identified membrane-associated proteins that regulate replication and participate in chromosomal segregation.

Restoration of growth to acidic phospholipid-deficient cells by DnaA(L366K) is independent of its capacity for nucleotide binding and exchange and requires DnaA.

In the absence of adequate levels of cellular acidic phospholipids, Escherichia coli remain viable but are arrested for growth. Expression of a DnaA protein that contains a single amino acid substitution in the membrane-binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth. DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP. Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids. DnaA(L366K) behaved like wild-type DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding. Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wild-type DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells.

Amplified centrosomes in breast cancer: a potential indicator of tumor aggressiveness.

Molecular mechanisms leading to genomic instability and phenotypic variation during tumor development and progression are poorly understood. Such instability represents a major problem in the management of breast cancer because of its contribution to more aggressive phenotypes as well as chemoresistance. In this study we analyzed breast carcinomas and tumor-derived cell lines to determine the relationship between centrosome amplification and established prognostic factors. Our results show that centrosome amplification can arise independent of ER or p53 status and is a common feature of aneuploid breast tumors. Centrosome amplification is associated with mitotic spindle abnormalities in breast carcinomas and thus may contribute to genomic instability and the development of more aggressive phenotypes during tumor progression.

Centrosome amplification drives chromosomal instability in breast tumor development.

Earlier studies of invasive breast tumors have shown that 60-80% are aneuploid and approximately 80% exhibit amplified centrosomes. In this study, we investigated the relationship of centrosome amplification with aneuploidy, chromosomal instability, p53 mutation, and loss of differentiation in human breast tumors. Twenty invasive breast tumors and seven normal breast tissues were analyzed by fluorescence in situ hybridization with centromeric probes to chromosomes 3, 7, and 17. We analyzed these tumors for both aneuploidy and unstable karyotypes as determined by chromosomal instability. The results were then tested for correlation with three measures of centrosome amplification: centrosome size, centrosome number, and centrosome microtubule nucleation capacity. Centrosome size and centrosome number both showed a positive, significant, linear correlation with aneuploidy and chromosomal instability. Microtubule nucleation capacity showed no such correlation, but did correlate significantly with loss of tissue differentiation. Centrosome amplification was detected in in situ ductal carcinomas, suggesting that centrosome amplification is an early event in these lesions. Centrosome amplification and chromosomal instability occurred independently of p53 mutation, whereas p53 mutation was associated with a significant increase in centrosome microtubule nucleation capacity. Together, these results demonstrate that independent aspects of centrosome amplification correlate with chromosomal instability and loss of tissue differentiation and may be involved in tumor development and progression. These results further suggest that aspects of centrosome amplification may have clinical diagnostic and/or prognostic value and that the centrosome may be a potential target for cancer therapy.