Dominic Gröger

The Role of Dimension in Multivalent Binding Events: Structure–Activity Relationship of Dendritic Polyglycerol Sulfate Binding to L-Selectin in Correlation with Size and Surface Charge Density

L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin-ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC(50) values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure-affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles.

Synthesis and biodistribution studies of 3H- and 64Cu-labeled dendritic polyglycerol and dendritic polyglycerol sulfate

Dendritic polyglycerol sulfate (dPGS) is a biocompatible, bioactive polymer which exhibits anti-inflammatory activity in vivo and thus represents a promising candidate for therapeutic and diagnostic applications. To investigate the in vivo pharmacokinetics in detail, dPGS with a molecular weight of approx. 10 kDa was radiolabeled with (3)H and (64)Cu, and evaluated by performing biodistribution studies and small animal positron emission tomography (PET). (3)H-labeling was accomplished by an oxidation-reduction process with sodium periodate and [(3)H]-borohydride. (64)Cu-labeling was achieved by conjugation of isothiocyanate- or maleimide-functionalized copper(II)-chelating ligands based on 1,4-bis(2-pyridinylmethyl)-1,4,7-triazacyclononane (DMPTACN) to an amino functionalized dPGS scaffold, followed by reaction with an aqueous solution containing (64)CuCl2. Independent biodistribution by radioimaging and PET imaging studies with healthy mice and rats showed that the neutral dPG was quantitatively renally eliminated, whereas the polysulfated analogues accumulated mainly in the liver and spleen. Small amounts of the dPGS derivatives were slowly excreted via the kidneys. The degree of uptake by the reticuloendothelial system (RES) was similar for dPGS with 40% or 85% sulfation, and surface modification of the scaffold with the DMPTACN chelator did not appear to significantly affect the biodistribution profile. On the basis of our data, the applicability of bioactive dPGS as a therapeutic agent might be limited due to organ accumulation even after 3 weeks. The inert characteristics and clearance of the neutral polymer, however, emphasizes the potential of dPG as a multifunctional scaffold for various nanomedical applications.

Dendritic Polyglycerol Sulfate Inhibits Microglial Activation and Reduces Hippocampal CA1 Dendritic Spine Morphology Deficits.

Hyperactivity of microglia and loss of functional circuitry is a common feature of many neurological disorders including those induced or exacerbated by inflammation. Herein, we investigate the response of microglia and changes in hippocampal dendritic postsynaptic spines by dendritic polyglycerol sulfate (dPGS) treatment. Mouse microglia and organotypic hippocampal slices were exposed to dPGS and an inflammogen (lipopolysaccharides). Measurements of intracellular fluorescence and confocal microscopic analyses revealed that dPGS is avidly internalized by microglia but not CA1 pyramidal neurons. Concentration and time-dependent response studies consistently showed no obvious toxicity of dPGS. The adverse effects induced by proinflammogen LPS exposure were reduced and dendritic spine morphology was normalized with the addition of dPGS. This was accompanied by a significant reduction in nitrite and proinflammatory cytokines (TNF-α and IL-6) from hyperactive microglia suggesting normalized circuitry function with dPGS treatment. Collectively, these results suggest that dPGS acts anti-inflammatory, inhibits inflammation-induced degenerative changes in microglia phenotype and rescues dendritic spine morphology.

Selectivity in bone targeting with multivalent dendritic polyanion dye conjugates.

Targeting bone with anionic macromolecules is a potent approach for the development of novel diagnostics and therapeutics for bone related diseases. A highly efficient modular synthesis of dendritic polyglycerol (dPG) polyanion dye conjugates, namely, sulfates, sulfonates, carboxylates, phosphates, phosphonates, and bisphosphonates via click chemistry is presented. By investigating the microarchitecture of stained bone sections with confocal laser scanning microscopy, the bisphosphonate, phosphonate, and phosphate functionalized polymers are identified as strongly penetrating compounds, whereas sulfates, sulfonates, and carboxylates reveal a weaker binding to hydroxyapatite (HA) but a more pronounced affinity toward collagen. In a quantitative HA binding assay, the affinity of the dPG sulfonate, sulfate, and carboxylate toward collagen and the exceptional high HA affinity of the phosphorous containing polyelectrolytes are validated. This shows the potential of dendritic polyphosphates and phosphonates as alternatives to the commonly employed bisphosphonate modification. In cytotoxicity studies with murine fibroblasts, the conjugates have no significant effect on the cell viability at 10(-5) m. All polyanions are taken up into the cells within 24 h. The presented synthetic approach allows versatile extensions for preparing conjugates for selective bone imaging applications, tissue engineering, and drug delivery.

Shell Cleavable Dendritic Polyglycerol Sulfates Show High Anti‐Inflammatory Properties by Inhibiting L‐Selectin Binding and Complement Activation

A new class of fully synthetic shell cleavable multivalent polysulfates is prepared by introducing degradable linkers into a stable biocompatible dendritic polyglycerol scaffold and subsequent sulfation. The sulfated polymers show different degradation profiles, low anticoagulant and high anti-inflammatory properties, are able to efficiently bind to L-selectin and inhibit the complement activation at very low concentrations in vitro.

Tissue and cellular localization of nanoparticles using 35S labeling and light microscopic autoradiography

UNLABELLED Microscopical visualization of nanoparticles in tissues is essential for assessing their distribution in whole organisms and their interaction with the cellular microenvironment, including possible toxic effects. However, labeling of nanoparticles with fluorescent dyes may affect their physicochemical properties. Moreover, the detection of organic nanoparticles in their tissue context often poses a particular challenge due to their closer similarities with biomolecules. As part of a biodistribution and toxicity study on organic anti-inflammatory nanoscaled dendritic polyglycerol sulfate amine (dPGS amine) we have established light microscopic autoradiography (LMA) for the tracking of (35)S labeled dPGS in standard histopathological tissue samples following intravenous injection in mice. The dPG(35)S amine was specifically localized in hepatic Kupffer cells with no histopathologic evidence of toxic, degenerate or inflammatory side effects. The combination of radiolabeling of organic nanoparticles with LMA offers a novel approach for their localization in microscopical slides, also allowing for a simultaneous standard toxicopathology analysis. FROM THE CLINICAL EDITOR In this study, a novel light microscopic autoradiography utilizing (35)S isotope demonstrates a combined approach to visualize nanoparticle locations in microscopic slides with no obvious toxicity to the studied cells and with minimal external hazard.

Anionic Dendritic Polymers for Biomedical Applications

Among dendritic architectures, polyanionic dendritic polymers have emerged as novel versatile platforms for applications in nanomedicine. Their appealing properties, such as high functional surface, low polydispersity, hydrophilicity, enhanced circulation time in the blood stream, and superior biocompatibility compared to their cationic counterparts, make anionic dendritic polymers ideal candidates for the biomedical field. In this book chapter, the biomedical applications of anionic dendritic polymers are discussed, with a focus on recent examples ranging from drug delivery to anti‐viral and anti‐inflammatory drugs.