Multidentate Zwitterionic Ligands Provide Compact and Highly Biocompatible Quantum Dots

Hydrophilic functional semiconductor nanocrystals that are also compact provide greatly promising platforms for use in bioinspired applications and are thus highly needed. To address this, we designed a set of metal coordinating ligands where we combined two lipoic acid groups, bis­(LA)-ZW, (as a multicoordinating anchor) with a zwitterion group for water compatibility. We further combined this ligand design with a new photoligation strategy, which relies on optical means instead of chemical reduction of the lipoic acid, to promote the transfer of CdSe-ZnS QDs to buffer media. In particular, we found that the QDs photoligated with this zwitterion-terminated bis­(lipoic) acid exhibit great colloidal stability over a wide range of pHs, to an excess of electrolytes, and in the presence of growth media and reducing agents, in addition to preserving their optical and spectroscopic properties. These QDs are also stable at nanomolar concentrations and under ambient conditions (room temperature and white light exposure), a very promising property for fluorescent labeling in biology. In addition, the compact ligands permitted metal–histidine self-assembly between QDs photoligated with bis­(LA)-ZW and two different His-tagged proteins, maltose binding protein and fluorescent mCherry protein. The remarkable stability of QDs capped with these multicoordinating and compact ligands over a broad range of conditions and at very small concentrations, combined with the compatibility with metal–histidine conjugation, could be very useful for a variety of applications, ranging from protein tracking and ligand–receptor binding to intracellular sensing using energy transfer interactions

Understanding the Self-Assembly of Proteins onto Gold Nanoparticles and Quantum Dots Driven by Metal-Histidine Coordination

Coupling of polyhistidine-appended biomolecules to inorganic nanocrystals driven by metal-affinity interactions is a greatly promising strategy to form hybrid bioconjugates. It is simple to implement and can take advantage of the fact that polyhistidine-appended proteins and peptides are routinely prepared using well established molecular engineering techniques. A few groups have shown its effectiveness for coupling proteins onto Zn- or Cd-rich semiconductor quantum dots (QDs). Expanding this conjugation scheme to other metal-rich nanoparticles (NPs) such as AuNPs would be of great interest to researchers actively seeking effective means for interfacing nanostructured materials with biology. In this report, we investigated the metal-affinity driven self-assembly between AuNPs and two engineered proteins, a His₇-appended maltose binding protein (MBP-His) and a fluorescent His₆-terminated mCherry protein. In particular, we investigated the influence of the capping ligand affinity to the nanoparticle surface, its density, and its lateral extension on the AuNP-protein self-assembly. Affinity gel chromatography was used to test the AuNP-MPB-His₇ self-assembly, while NP-to-mCherry-His₆ binding was evaluated using fluorescence measurements. We also assessed the kinetics of the self-assembly between AuNPs and proteins in solution, using time-dependent changes in the energy transfer quenching of mCherry fluorescent proteins as they immobilize onto the AuNP surface. This allowed determination of the dissociation rate constant, <i>K</i>_d^–1 ∼ 1–5 nM. Furthermore, a close comparison of the protein self-assembly onto AuNPs or QDs provided additional insights into which parameters control the interactions between imidazoles and metal ions in these systems

Intracellular Delivery of Luminescent Quantum Dots Mediated by a Virus-Derived Lytic Peptide

We describe a new quantum dot (QD)-conjugate prepared with a lytic peptide, derived from a nonenveloped virus capsid protein, capable of bypassing the endocytotic pathways and delivering large amounts of QDs to living cells. The polypeptide, derived from the Nudaurelia capensis Omega virus, was fused onto the C-terminus of maltose binding protein that contained a hexa-HIS tag at its N-terminus, allowing spontaneous self-assembly of controlled numbers of the fusion protein per QD via metal–HIS interactions. We found that the efficacy of uptake by several mammalian cell lines was substantial even for small concentrations (10–100 nM). Upon internalization the QDs were primarily distributed outside the endosomes/lysosomes. Moreover, when cells were incubated with the conjugates at 4 °C, or in the presence of chemical endocytic inhibitors, significant intracellular uptake continued to occur. These findings indicate an entry mechanism that does not involve endocytosis, but rather the perforation of the cell membrane by the lytic peptide on the QD surfaces

UV and Sunlight Driven Photoligation of Quantum Dots: Understanding the Photochemical Transformation of the Ligands

We have recently reported that photoinduced ligation of ZnS-overcoated quantum dots (QDs) offers a promising strategy to promote the phase transfer of these materials to polar and aqueous media using multidentate lipoic acid (LA)-modified ligands. In this study we investigate the importance of the underlying parameters that control this process, in particular, whether or not photoexcited QDs play a direct role in the photoinduced ligation. We find that irradiation of the ligand alone prior to mixing with hydrophobic QDs is sufficient to promote ligand exchange. Furthermore, photoligation onto QDs can also be carried out simply by using sunlight. Combining the use of Ellman’s test with matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, we probe the nature of the photochemical transformation of the ligands. We find that irradiation (using either a UV photoreactor or sunlight) alters the nature of the disulfide groups in the lipoic acid, yielding a different product mixture than what is observed for chemically reduced ligands. Irradiation of the ligand in solution generates a mixture of monomeric and oligomeric compounds. Ligation onto the QDs selectively favors oligomers, presumably due to their higher coordination onto the metal-rich QD surfaces. We also show that photoligation using mixed ligands allows the preparation of reactive nanocrystals. The resulting QDs are coupled to proteins and peptides and tested for cellular staining. This optically controlled ligation of QDs combined with the availability of a variety of multidentate and multifunctional LA-modified ligands open new opportunities for developing fluorescent platforms with great promises for use in imaging and sensor design

Functional-Group-Dependent Formation of Bioactive Fluorescent-Plasmonic Nanohybrids

We detail the assembly, driven by metal-affinity coordination, of fluorescent-plasmonic hybrid constructs that are also biologically active. The hybrid constructs are prepared by first assembling polymer-encapsulated luminescent quantum dots that present amine-, carboxy-, and lipoic acid-terminated groups (QD-FG) and plasmonic gold nanoparticles capped with rather low density of lipoic acid-appended zwitterion ligands (AuNP-LA-ZW). The dual QD-AuNP constructs were then coupled to polyhistidine-appended maltose binding proteins, yielding the final trifunctional assemblies. The coordination of amine-, carboxy-, and lipoic acid-terminated QDs with AuNP-LA-ZW was characterized using steady-state and time-resolved fluorescence quenching measurements. We measured rather different coordination affinities between the functional groups on the QDs and the AuNP surfaces. This assembly mode still allowed the partially exposed AuNPs in the inorganic/polymer hybrid to bind to polyhistidine-appended proteins. This protein assembly was confirmed using amylose affinity chromatography, which also confirmed the structural integrity of the hybrid and biological activity of the bound protein. Owing to the high colloidal stability of the surface-modified QDs and AuNP-LA-ZW, combined with flexible functionalization, we anticipate that this strategy could facilitate the integration of hybrid inorganic/polymer constructs with specific photophysical properties into biological systems

Self-Assembled Gold Nanoparticle–Fluorescent Protein Conjugates as Platforms for Sensing Thiolate Compounds via Modulation of Energy Transfer Quenching

The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal–histidine coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ∼95–97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction, and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (<i>K</i>_d^–1) for these compounds. These findings combined indicate that the AuNP–fluorescent protein conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures

Photoligation of an Amphiphilic Polymer with Mixed Coordination Provides Compact and Reactive Quantum Dots

We introduce a new set of multicoordinating polymers as ligands that combine two distinct metal-chelating groups, lipoic acid and imidazole, for the surface functionalization of QDs. These ligands combine the benefits of thiol and imidazole coordination to reduce issues of thiol oxidation and weak binding affinity of imidazole. The ligand design relies on the introduction of controllable numbers of lipoic acid and histamine anchors, along with hydrophilic moieties and reactive functionalities, onto a poly­(isobutylene-<i>alt</i>-maleic anhydride) chain via a one-step nucleophilic addition reaction. We further demonstrate that this design is fully compatible with a novel and mild photoligation strategy to promote the in situ ligand exchange and phase transfer of hydrophobic QDs to aqueous media under borohydride-free conditions. Ligation with these polymers provides highly fluorescent QDs that exhibit great long-term colloidal stability over a wide range of conditions, including a broad pH range (3–13), storage at nanomolar concentration, under ambient conditions, in 100% growth media, and in the presence of competing agents with strong reducing property. We further show that incorporating reactive groups in the ligands permits covalent conjugation of fluorescent dye and redox-active dopamine to the QDs, producing fluorescent platforms where emission is controlled/tuned by Förster Resonance Energy Transfer (FRET) or pH-dependent charge transfer (CT) interactions. Finally, the polymer-coated QDs have been coupled to cell-penetrating peptides to facilitate intracellular uptake, while subsequent cytotoxicity tests show no apparent decrease in cell viability

Design of a Multi-Dopamine-Modified Polymer Ligand Optimally Suited for Interfacing Magnetic Nanoparticles with Biological Systems

We have designed a set of multifunctional and multicoordinating polymer ligands that are optimally suited for surface functionalizing iron oxide and potentially other magnetic nanoparticles (NPs) and promoting their integration into biological systems. The amphiphilic polymers are prepared by coupling (via nucleophilic addition) several amine-terminated dopamine anchoring groups, poly­(ethylene glycol) moieties, and reactive groups onto a poly­(isobutylene-<i>alt</i>-maleic anhydride) (PIMA) chain. This design greatly benefits from the highly efficient and reagent-free one-step reaction of maleic anhydride groups with amine-containing molecules. The availability of several dopamine groups in the same ligand greatly enhances the ligand affinity, via multiple coordination, to the magnetic NPs, while the hydrophilic and reactive groups promote colloidal stability in buffer media and allow subsequent conjugation with target biomolecules. Iron oxide nanoparticles ligand exchanged with these polymer ligands have a compact hydrodynamic size and exhibit enhanced long-term colloidal stability over the pH range of 4–12 and in the presence of excess electrolytes. Nanoparticles ligated with terminally reactive polymers have been easily coupled to target dyes and tested in live cell imaging with no measurable cytotoxicity. Finally, the resulting hydrophilic nanoparticles exhibit large and size-dependent <i>r</i>₂ relaxivity values

Interactions between Magnetic Nanowires and Living Cells: Uptake, Toxicity, and Degradation

We report on the uptake, toxicity, and degradation of magnetic nanowires by NIH/3T3 mouse fibroblasts. Magnetic nanowires of diameters 200 nm and lengths between 1 and 40 μm are fabricated by controlled assembly of iron oxide (γ-Fe2O3) nanoparticles. Using optical and electron microscopy, we show that after 24 h incubation the wires are internalized by the cells and located either in membrane-bound compartments or dispersed in the cytosol. Using fluorescence microscopy, the membrane-bound compartments were identified as late endosomal/lysosomal endosomes labeled with lysosomal associated membrane protein (Lamp1). Toxicity assays evaluating the mitochondrial activity, cell proliferation, and production of reactive oxygen species show that the wires do not display acute short-term (<100 h) toxicity toward the cells. Interestingly, the cells are able to degrade the wires and to transform them into smaller aggregates, even in short time periods (days). This degradation is likely to occur as a consequence of the internal structure of the wires, which is that of a noncovalently bound aggregate. We anticipate that this degradation should prevent long-term asbestos-like toxicity effects related to high aspect ratio morphologies and that these wires represent a promising class of nanomaterials for cell manipulation and microrheology

Interactions between Magnetic Nanowires and Living Cells: Uptake, Toxicity, and Degradation

We report on the uptake, toxicity, and degradation of magnetic nanowires by NIH/3T3 mouse fibroblasts. Magnetic nanowires of diameters 200 nm and lengths between 1 and 40 μm are fabricated by controlled assembly of iron oxide (γ-Fe2O3) nanoparticles. Using optical and electron microscopy, we show that after 24 h incubation the wires are internalized by the cells and located either in membrane-bound compartments or dispersed in the cytosol. Using fluorescence microscopy, the membrane-bound compartments were identified as late endosomal/lysosomal endosomes labeled with lysosomal associated membrane protein (Lamp1). Toxicity assays evaluating the mitochondrial activity, cell proliferation, and production of reactive oxygen species show that the wires do not display acute short-term (<100 h) toxicity toward the cells. Interestingly, the cells are able to degrade the wires and to transform them into smaller aggregates, even in short time periods (days). This degradation is likely to occur as a consequence of the internal structure of the wires, which is that of a noncovalently bound aggregate. We anticipate that this degradation should prevent long-term asbestos-like toxicity effects related to high aspect ratio morphologies and that these wires represent a promising class of nanomaterials for cell manipulation and microrheology