Juan B. Blanco-Canosa

The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.

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.

An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation

The straightforward C-terminal modification of peptides assembled on a solid support remains a significant challenge in peptide and protein chemistry. In particular, C-terminal thioester peptides are important intermediates for the generation of active esters, amides and hydrazides[1,2] and are an essential component of many synthetic strategies for protein synthesis.[3] Currently, the most effective approach for the synthesis of peptidyl thioesters is the in situ neutralization protocol for Boc solid phase peptide synthesis (Boc-SPPS)[4] using thioester linkers.[2,5] However, many laboratories use Fmoc-SPPS exclusively and such protocols are favored when synthesizing glyco- and phosphopeptides. The thioester linkers used for Boc-SPPS have limited utility for Fmoc-SPPS due to the requirement for repeated Fmoc removal under basic conditions. Considerable effort has been applied to address this challenge[6] including optimized Fmoc deprotection cocktails,[7] thiol labile safety catch linkers,[8] activation of protected peptides in solution,[9] and recently thioesters have been generated using O to S[10] or N to S[11] acyl transfer. Despite these notable advances, the synthesis of thioester peptides by Fmoc-SPPS remains significantly more challenging than the synthesis of the corresponding acid or amide peptide.

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.

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.

Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing

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.

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.

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QDs surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis-Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide-alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor-acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.

Interactions between Redox Complexes and Semiconductor Quantum Dots Coupled via a Peptide Bridge

Colloidal quantum dots (QDs) have a large fraction of their atoms arrayed on their surfaces and are capped with bifunctional ligands, which make their photoluminescence highly sensitive to potential charge transfer to or from the surrounding environment. In this report, we used peptides as bridges between CdSe-ZnS QDs and metal complexes to promote charge transfer between the metal complexes and QDs. We found that quenching of the QD emission is highly dependent on the relative position of the oxidation levels of QDs and metal complex used; it also traces the number of metal complexes brought in close proximity of the nanocrystal surface. In addition, partial bleaching of the absorption was measured for the QD-metal complex assemblies. These proximity driven interactions were further used to construct sensing assemblies to detect proteolytic enzyme activity.