Raghavendra Kikkeri

In Vitro Imaging and in Vivo Liver Targeting with Carbohydrate Capped Quantum Dots

PEGylated quantum dots (QDs) capped with d-mannose, d-galactose, and d-galactosamine have been synthesized. The stable, high quantum yield fluorescence of QDs was exploited to study specific carbohydrate-protein interactions in vitro and in vivo.

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria.

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Optimization of Localized Surface Plasmon Resonance Transducers for Studying Carbohydrate–Protein Interactions

Noble metal nanostructures supporting localized surface plasmons (SPs) have been widely applied to chemical and biological sensing. Changes in the refractive index near the nanostructures affect the SP extinction band, making localized surface plasmon resonance (LSPR) spectroscopy a convenient tool for studying biological interactions. Carbohydrate-protein interactions are of major importance in living organisms; their study is crucial for understanding of basic biological processes and for the construction of biosensors for diagnostics and drug development. Here LSPR transducers based on gold island films prepared by evaporation on glass and annealing were optimized for monitoring the specific interaction between Concanavalin A (Con A) and D-(+)-mannose. The sugar was modified with a PEG-thiol linker and immobilized on the Au islands. Sensing assays were performed under stationary and flow conditions, the latter providing kinetic parameters for protein binding and dissociation. Ellipsometry and Fourier transform-infrared (FT-IR) data, as well as scanning electron microscopy (SEM) imaging of fixated and stained samples, furnished independent evidence for the protein-sugar recognition. Enhanced response and visual detection of protein binding was demonstrated using Au nanoparticles stabilized with the linker-modified mannose molecules. Mannose-coated transducers display an excellent selectivity toward Con A in the presence of a large excess of bovine serum albumin (BSA).

Synthesis of Carbohydrate‐Functionalized Quantum Dots in Microreactors

Large quantities of monodisperse semiconductor nanocrystals, quantum dots (QDs), are needed for applications in electronics and the life sciences. For biological applications, the surface of QDs is often functionalized with carboxylic acids for the attachment of proteins or directly with carbohydrates. Traditional batch processes are of limited utility for the production of QDs on a larger scale owing to limited temperature control and lack of homogeneous mixing. Continuous-flow microreactors provide precise control over reaction conditions, including temperature, and the production time is independent of the process scale. The high surface-to-volume ratio of the microreactor channels enables precise temperature control as well as efficient mixing, allowing for the preparation of QDs with narrow size distribution. QDs have been prepared using microfabricated gas–liquid and liquid–liquid flow reactors. The preparation of surface-functionalized QDs under mild reaction conditions in the liquid phase remains challenging. Ideally, a continuous process would serve to both produce the quantum dots and to functionalize them. Herein we present a single-phase microfluidic system for the synthesis of highly luminescent, surface-functionalized CdSe and CdTe nanoparticles. In contrast to batch processes, which require temperatures of 250–300 8C, temperatures of 160 8C are sufficient in the flow process. Both the formation of the zinc sulfide shell and the functionalization of the nanoparticles with carboxy groups and carbohydrates were perfomed in a continuous-flow system (Figure 1). Differentsized quantum dots were obtained by simply varying the reaction time in the flow reactor. High reaction temperatures usually result in fast nucleation, and large nanocrystals are quickly obtained. At low temperatures, the size of the nanocrystals and the concentration of the unreacted precursors in the mixture can be balanced. Thus, continuous nucleation is suppressed, the residence time distribution (RTD) is narrowed, and homogeneous QD fractions are obtained by varying the reaction time. The homogenous reaction mixture and slow nucleation results in a mild process for the production of QDs using microreactors. CdSe and CdTe nanoparticles with different emission maxima were prepared by injection of a 1:1 mixture of Cd precursor and Se or Te precursor. The Cd precursor was prepared by the addition of oleic acid and oleylamine to a solution of cadmium oxide dissolved in lauric acid at 150 8C. The Se and Te precursors were prepared by dissolving elemental selenium or tellerium powder in tri-n-octylphosphine (TOP) in a Syrris microreactor. Reaction times ranged from 3 to 30 minutes. The CdSe and CdTe cores were purified by precipitation from methanol/chloroform/n-hexane and dried under vacuum. The average size distribution of each sample was calculated from the absorbance spectra (see Figure 1 in the Supporting Information). The optical properties of the QDs show a time-dependent bathochromic shift in the band-edge emission and enhanced intensity. The photoluminescence peaks of CdSe QDs are sharp, with fwhm (full width at half maximum) values of the band-edge luminescence between 40 and 50 nm (Figure 2), which indicates the narrow size distribution of the QDs. However, after 30 minutes of reaction time the fwhm increased from 40 to 90 nm, and a decrease in quantum yield indicated that saturated nucleation occurred after 20– Figure 1. Microreactor setup for the continuous-flow synthesis of functionalized QDs (OA: oleic acid; TOP: tri-n-octylphosphine).

Design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting†

Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized β-cyclodextrins (βCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded βCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal D-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that βCDs and liposomes displaying terminal D-Gal/D-GalNAc residues were preferentially endocytosed. In contrast, uptake of βCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized βCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized βCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.

Lectin Biosensing Using Digital Analysis of Ru(II)-Glycodendrimers

A novel, digital, single-operation analytical method to study glycodendrimer-lectin interactions is described. Robust, highly fluorescent derivatives of tris(bipyridine)ruthenieum(II) ([Ru(bipy)(3)](2+)) bearing 2, 4, 6, or 18 mannose or galactose units were designed to perform molecular logic operations. Inputs for these systems were pH, N,N-4,4-bis(benzyl-2-boronic acid)bipyridinium dibromide, and different lectins (concanavalin A, Galantus nivalis agglutinin, and asialoglycoprotein). The relative change in fluorescence quantum yield of the Ru(II)-glycodendrimers served as output. Together, the fluorescent emission readout, the logic analysis of the photoinduced electron transfer, and the optical behavior provide a single-step method to quickly screen a glycodendrimer library and select the best dendrimer model for studying carbohydrate-lectin interactions.

Facile synthesis of size dependent Ru(II)-carbohydrate dendrimers via click chemistry.

A facile and flexible approach for the preparation of Ru(II) complexes containing different carbohydrates based on the Cu(II)-catalyzed Huisgen-[3+2] cycloaddition is described.

Detection of bacteria using glyco-dendronized polylysine prepared by continuous flow photofunctionalization.

Biocompatible glyco-dendronized poly-l-lysine (PLL) polymers carry either three or nine mannose- or galactose-bearing dendrons that selectively bind, and thus can be used to detect, bacteria. Central to the synthesis of glyco-dendronized polymers was the development of a continuous flow [2 + 2] photocycloaddition reaction to connect the dendrons and PLL. Glycodendronized polymers cluster bacteria by binding to cell-surface carbohydrate receptors and thereby result in an easy read-out using microscopic analyses.

What Happens to Hydrophobic Interactions during Transfer from the Solution to the Gas Phase? The Case of Electrospray-Based Soft Ionization Methods

The disappearance of the hydrophobic effect in the gas phase due to the absence of an aqueous surrounding raises a long-standing question: can noncovalent complexes that are exclusively bound by hydrophobic interactions in solution be preserved in the gas phase? Some reports of successful detection by mass spectrometry of complexes largely stabilized by hydrophobic effect are questionable by the presence of electrostatic forces that hold them together in the gas phase. Here, we report on the MS-based analysis of model supramolecular complexes with a purely hydrophobic association in solution, β-cyclodextrin, and synthetic adamantyl-containing ligands with several binding sites. The stability of these complexes in the gas phase is investigated by quantum chemical methods (DFT-M06). Compared with the free interaction partners, the inclusion complex between β-cyclodextrin and adamantyl-containing ligand is shown to be stabilized in the gas phase by ΔG = 9.6xa0kcal mol–1. The host–guest association is mainly enthalpy-driven due to strong dispersion interactions caused by a large nonpolar interface and a high steric complementarity of the binding partners. Interference from other types of noncovalent binding forces is virtually absent. The complexes are successfully detected via electrospray ionization mass spectrometry, although a high dissociation yield is also observed. We attribute this pronounced dissociation of the complexes to the collisional activation of ions in the atmospheric interface of mass spectrometer. The comparison of several electrospray-based ionization methods reveals that cold spray ionization provides the softest ion generation conditions for these complexes.

Ru(II) Glycodendrimers as Probes to Study Lectin-Carbohydrate Interactions and Electrochemically Measure Monosaccharide and Oligosaccharide Concentrations

We describe a novel platform on which to study carbohydrate-protein interactions based on ruthenium(II) glycodendrimers as optical and electrochemical probes. Using the prototypical concanavalin A (ConA)-mannose lectin-carbohydrate interaction as an example, oligosaccharide concentrations were electrochemically monitored. The displacement of the Ru(II) complex from lectin-functionalized gold surfaces was repeatedly regenerated. This new platform presents a method to monitor many different complex sugars in parallel.