Evaluating the Catalytic Efficiency of the Human Membrane-type 1 Matrix Metalloproteinase (MMP-14) Using AuNP-Peptide Conjugates.

Interactions of plasmonic nanocolloids such as gold nanoparticles and nanorods with proximal dye emitters result in efficient quenching of the dye photoluminescence (PL). This has become a popular strategy for developing analytical biosensors relying on this quenching process for signal transduction. Here, we report on the use of stable PEGylated gold nanoparticles, covalently coupled to dye-labeled peptides, as sensitive optically addressable sensors for determining the catalytic efficiency of the human matrix metalloproteinase-14 (MMP-14), a cancer biomarker.

Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides.

Characterizing the Brownian Diffusion of Nanocolloids and Molecular Solutions: Diffusion-Ordered NMR Spectroscopy vs Dynamic Light Scattering.

Hydrodynamic size is a characteristic dimension that reflects the Brownian diffusion of objects, such as proteins, macromolecules, and various colloids when dissolved/dispersed in fluid phases. This property is crucial when investigating the utility of colloidal nanocrystals and polymeric materials in biology. Dynamic light scattering (DLS) has been widely used to measure the diffusion coefficient and hydrodynamic size of such systems.

Elucidating the Role of Surface Coating in the Promotion or Prevention of Protein Corona around Quantum Dots.

Nonspecific interactions in biological media can lead to the formation of a protein corona around nanocolloids, which tends to alter their behavior and limit their effectiveness when used as probes for imaging or sensing applications. Yet, understanding the corona buildup has been challenging. We hereby investigate these interactions using luminescent quantum dots (QDs) as a model nanocolloid system, where we carefully vary the nature of the hydrophilic block in the surface coating, while maintaining the same dihydrolipoic acid (DHLA) bidentate coordinating motif.

Modification of Poly(maleic anhydride)-Based Polymers with HN-R Nucleophiles: Addition or Substitution Reaction?

Reacting poly(maleic anhydride)-based polymers with HN-R nucleophiles is a flexible and highly effective approach for preparing a variety of multifunctional, multicoordinating, and multireactive polymers. The exact transformation of the anhydride ring during this addition reaction is still an open question. In this report, we characterize the transformation of a representative block copolymer, poly(isobutylene- alt-maleic anhydride), with a few HN-R nucleophiles.

Enhanced Uptake of Luminescent Quantum Dots by Live Cells Mediated by a Membrane-Active Peptide.

The steady progress made over the past three decades in growing a variety of inorganic nanomaterials, with discreet control over their size and photophysical properties, has been exploited to develop several imaging and sensing applications. However, full integration of these materials into biology has been hampered by the complexity of delivering them into cells. In this report, we demonstrate the effectiveness of a chemically synthesized anticancer peptide to facilitate the rapid delivery of luminescent quantum dots (QDs) into live cells.

Intracellular Delivery of Gold Nanocolloids Promoted by a Chemically Conjugated Anticancer Peptide.

We report on the ability of a chemically synthesized anticancer peptide, SVS-1, to promote the rapid uptake of gold nanorods (AuNRs) and gold nanoparticles (AuNPs) by live HeLa cells. For this, AuNPs and AuNRs, surface ligated with a multicoordinating polymer that presents several amine groups per ligand, are simultaneously reacted with SVS-1 and Texas-Red dye; the latter allows fluorescence visualization of the nanocrystals. Using epifluorescence microscopy, we find that incubation of the SVS-1-conjugated AuNPs and AuNRs with a model cancer cell line yields extended staining throughout the cell cytoplasm, even at low conjugate concentrations (∼0.

Efficient Assembly of Quantum Dots with Homogenous Glycans Derived from Natural N-Linked Glycoproteins.

Coating inorganic nanoparticles with polyethylene glycol (PEG)-appended ligands, as means to preserve their physical characteristics and promote steric interactions with biological systems, including enhanced aqueous solubility and reduced immunogenicity, has been explored by several groups. Conversely, macromolecules present in the human serum and on the surface of cells are densely coated with hydrophilic glycans that act to reduce nonspecific interactions, while facilitating specific binding and interactions. In particular, N-linked glycans are abundant on the surface of most serum proteins and are composed of a branched architecture that is typically characterized by a significant level of molecular heterogeneity.

Photochemical transformation of lipoic acid-based ligands: probing the effects of solvent, ligand structure, oxygen and pH.

We have combined optical absorption with the Ellman's test to identify the parameters that affect the transformation of the 5-membered dithiolanes to thiols in lipoic acid (LA) and its derivatives during UV-irradiation. We found that the nature and polarity of the solvent, the structure of the ligands, acidity of the medium and oxygen can drastically affect the amount of photogenerated thiols. These findings are highly relevant to the understanding of the photochemical transformation of this biologically relevant compound, and would benefit the increasing use of LA-based ligands for the surface functionalization of various nanomaterials.

The roles of surface chemistry, dissolution rate, and delivered dose in the cytotoxicity of copper nanoparticles.

The understanding of nanoparticle (NP) cytotoxicity is challenging because of incomplete information about physicochemical changes particles undergo once they come into contact with biological fluids. It is therefore essential to characterize changes in NP properties to better understand their biological fate and effects in mammalian cells. In this paper, we present a study on the effect of particle surface oxidation and dissolution rates of Cu NPs.

Aqueous Growth of Gold Clusters with Tunable Fluorescence Using Photochemically Modified Lipoic Acid-Based Ligands.

We report a one-phase aqueous growth of fluorescent gold nanoclusters (AuNCs) with tunable emission in the visible spectrum, using a ligand scaffold that is made of poly(ethylene glycol) segment appended with a metal coordinating lipoic acid at one end and a functional group at the other end. This synthetic scheme exploits the ability of the UV-induced photochemical transformation of LA-based ligands to provide DHLA and other thiol byproducts that exhibit great affinity to metal nanoparticles, obviating the need for chemical reduction of the dithiolane ring using classical reducing agents. The influence of various experimental conditions, including the photoirradiation time, gold precursor-to-ligand molar ratios, time of reaction, temperature, and the medium pH, on the growth of AuNCs has been systematically investigated.

Bio-orthogonal Coupling as a Means of Quantifying the Ligand Density on Hydrophilic Quantum Dots.

We describe the synthesis of two metal-coordinating ligands that present one or two lipoic acid (LA) anchors, a hydrophilic polyethylene glycol (PEG) segment and a terminal reactive group made of an azide or an aldehyde, two functionalities with great utility in bio-orthogonal coupling techniques. These ligands were introduced onto the QD surfaces using a combination of photochemical ligation and mixed cap exchange strategy, where control over the fraction of azide and aldehyde groups per nanocrystal can be easily achieved: LA-PEG-CHO, LA-PEG-N3, and bis(LA)-PEG-CHO. We then demonstrate the application of two novel bio-orthogonal coupling strategies directly on luminescent quantum dot (QD) surfaces that use click chemistry and hydrazone ligation under catalyst-free conditions.

Controlling the Architecture, Coordination, and Reactivity of Nanoparticle Coating Utilizing an Amino Acid Central Scaffold.

We have developed a versatile strategy to prepare a series of multicoordinating and multifunctional ligands optimized for the surface-functionalization of luminescent quantum dots (QDs) and gold nanoparticles (AuNPs) alike. Our chemical design relies on the modification of l-aspartic acid precursor to controllably combine, through simple peptide coupling chemistry, one or two lipoic acid (LA) groups and poly(ethylene glycol) (PEG) moieties in the same ligand. This route has provided two sets of modular ligands: (i) bis(LA)-PEG, which presents two lipoic acids (higher coordination) appended onto a single end-functionalized PEG, and (ii) LA-(PEG)2 made of two PEG moieties (higher branching, with various end reactive groups) appended onto a single lipoic acid.

Preparation of compact biocompatible quantum dots using multicoordinating molecular-scale ligands based on a zwitterionic hydrophilic motif and lipoic acid anchors.

Luminescent quantum dots (QDs) can potentially be used for many biological experiments, provided that they are constructed in such a way as to be stable in biological matrices. Furthermore, QDs that are compact in size and easy to couple to biomolecules can be readily used for applications ranging from protein tracking to vasculature imaging. In this protocol, we describe the preparation of ligands comprising either one or two lipoic acid (LA) groups chemically linked to a zwitterion moiety.

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-alt-maleic anhydride) chain via a one-step nucleophilic addition reaction.

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.

Photoligation combined with zwitterion-modified lipoic acid ligands provides compact and biocompatible quantum dots.

We describe the design and synthesis of a series of compact ligands made of lipoic acid (LA)-based coordinating anchors and hydrophilic zwitterion groups. This ligand design is combined with a novel photoligation strategy to promote the transfer of QDs to polar and buffer media. This approach has provided hydrophilic QDs that exhibit great colloidal stability over a broad range of pHs and in the presence of cell culture media.

Strategies for interfacing inorganic nanocrystals with biological systems based on polymer-coating.

Interfacing inorganic nanoparticles and biological systems with the aim of developing novel imaging and sensing platforms has generated great interest and much activity. However, the effectiveness of this approach hinges on the ability of the surface ligands to promote water-dispersion of the nanoparticles with long term colloidal stability in buffer media. These surface ligands protect the nanostructures from the harsh biological environment, while allowing coupling to target molecules, which can be biological in nature (e.

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-alt-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.

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).

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.

Combining ligand design with photoligation to provide compact, colloidally stable, and easy to conjugate quantum dots.

We describe the design and synthesis of two compact multicoordinating (lipoic acid-appended) zwitterion ligands for the capping of luminescent quantum dots, QDs. This design is combined with a novel and easy to implement photoligation strategy to promote the in situ ligand exchange and transfer of the QDs to buffer media. This method involves the irradiation of the native hydrophobic nanocrystals in the presence of the ligands, which promotes in situ cap exchange and phase transfer of the QDs, eliminating the need for a chemical reduction of the dithiolane groups.

Growth of highly fluorescent polyethylene glycol- and zwitterion-functionalized gold nanoclusters.

We have prepared and characterized a new set of highly fluorescent gold nanoclusters (AuNCs) using one-step aqueous reduction of a gold precursor in the presence of bidentate ligands made of lipoic acid anchoring groups, appended with either a poly(ethylene glycol) short chain or a zwitterion group. The AuNCs fluoresce in the red to near-infrared region of the optical spectrum with emission centered at ∼750 nm and a quantum yield of ∼10-14%, and they exhibit long fluorescence lifetimes (up to ∼300 ns). Dispersions of these AuNCs exhibit great long-term colloidal stability, over a wide range of pHs (2-13) and in the presence of high electrolyte concentrations, and a strong resistance to reducing agents such as glutathione.

Growth of in situ functionalized luminescent silver nanoclusters by direct reduction and size focusing.

We have used one phase growth reaction to prepare a series of silver nanoparticles (NPs) and luminescent nanoclusters (NCs) using sodium borohydride (NaBH(4)) reduction of silver nitrate in the presence of molecular scale ligands made of polyethylene glycol (PEG) appended with lipoic acid (LA) groups at one end and reactive (-COOH/-NH(2)) or inert (-OCH(3)) functional groups at the other end. The PEG segment in the ligand promotes solubility in a variety of solvents including water, while LAs provide multidentate coordinating groups that promote Ag-ligand complex formation and strong anchoring onto the NP/NC surface. The particle size and properties were primarily controlled by varying the Ag-to-ligand (Ag:L) molar ratios and the molar amount of NaBH(4) used.