Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

Controlled Grafting Expansion Microscopy.

Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM.

A Cyanine-Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation.

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. 68 Ga-DOTATATE, a well-characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2-targeting and imaging properties of the resulting probe 68 Ga-DOTA-ICC-TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2-overexpressing tumors in the positron emission tomography (PET) scan and histological examination.

Polyglycerol for Half-Life Extension of Proteins-Alternative to PEGylation?

Since several decades, PEGylation is known to be the clinical standard to enhance pharmacokinetics of biotherapeutics. In this study, we introduce polyglycerol (PG) of different lengths and architectures (linear and hyperbranched) as an alternative polymer platform to poly(ethylene glycol) (PEG) for half-life extension (HLE). We designed site-selective N-terminally modified PG-protein conjugates of the therapeutic protein anakinra (IL-1ra, Kineret) and compared them systematically with PEG analogues of similar molecular weights. Linear PG and PEG conjugates showed comparable hydrodynamic sizes and retained their secondary structure, whereas binding affinity to IL-1 receptor 1 decreased with increasing polymer length, yet remained in the low nanomolar range for all conjugates. The terminal half-life of a 40 kDa linear PG-modified anakinra was extended 4-fold compared to the unmodified protein, close to its PEG analogue. Our results demonstrate similar performances of PEG- and PG-anakinra conjugates and therefore highlight the outstanding potential of polyglycerol as a PEG alternative for half-life extension of biotherapeutics.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

Expanding the Scope of Reporting Nanoparticles: Sensing of Lipid Phase Transitions and Nanoviscosities in Lipid Membranes.

Biological membrane fluidity and thus the local viscosity in lipid membranes are of vital importance for many life processes and implicated in various diseases. Here, we introduce a novel viscosity sensor design for lipid membranes based on a reporting nanoparticle, a sulfated dendritic polyglycerol (dPGS), conjugated to a fluorescent molecular rotor, indocarbocyanine (ICC). We show that dPGS-ICC provides high affinity to lipid bilayers, enabling viscosity sensing in the lipid tail region. The systematic characterization of viscosity- and temperature-dependent photoisomerization properties of ICC and dPGS-ICC allowed us to determine membrane viscosities in different model systems and in living cells using fluorescence lifetime imaging (FLIM). dPGS-ICC distinguishes between ordered lipids and the onset of membrane defects in small unilamellar single lipid vesicles and is highly sensitive in the fluid phase to small changes in viscosity introduced by cholesterol. In microscopy-based viscosity measurements of large multilamellar vesicles, we observed an order of magnitude more viscous environments by dPGS-ICC, lending support to the hypothesis of heterogeneous nanoviscosity environments even in single lipid bilayers. The existence of such complex viscosity structures could explain the large variation in the apparent membrane viscosity values found in the literature, depending on technique and probe, both for model membranes and live cells. In HeLa cells, a tumor-derived cell line, our nanoparticle-based viscosity sensor detects a membrane viscosity of ∼190 cP and is able to discriminate between cell membrane and intracellular vesicle localization. Thus, our results show the versatility of the dPGS-ICC nano-conjugate in physicochemical and biomedical applications by adding a new analytical functionality to its medical properties.

Fullerene Polyglycerol Amphiphiles as Unimolecular Transporters.

Due to their unique structure and properties, water-soluble fullerene derivatives are of great interest for various biomedical purposes. In this work, solution behavior, encapsulation and release properties, biocompatibility, and cellular uptake pathways of fullerene-polyglycerol amphiphiles (FPAs) with defined structures are investigated. The number of polyglycerol branches attached to the surface of fullerene affects the physicochemical properties of FPAs dramatically but not their cellular uptake. Release of encapsulated hydrophobic dyes from FPAs strongly depends on the number of their branches. Conjugation of a pH-sensitive dye to the FPAs as a probe showed that their self-assemblies are taken up through endocytotic pathways. It was observed that FPAs are able to transfer small molecules into cells both above and below their critical aggregation concentration. Taking advantage of the water solubility, biocompatibility, and transfer-ability of FPAs, they might find use as unimolecular carriers for future biomedical applications.

Dendritic polyglycerol sulfate attenuates murine graft-versus-host disease.

Graft-versus-host disease (GvHD) is a severe immune reaction commonly occurring after hematopoietic stem cell transplantation. The outcome of patients who do not respond to the currently used immunosuppressive drugs is poor, thus there is an urgent need for the evaluation of new therapies. Heparin has a well-known anti-inflammatory effect and heparin analogues with a low anticoagulant effect are interesting candidates as new anti-inflammatory drugs. We explored the therapeutic potential of dendritic polyglycerol sulfates (dPGS), a novel class of heparin derivatives, on murine acute GvHD in vivo. The therapeutic effect of dPGS on murine GvHD was more intense after intravenous application compared to subcutaneous injection. An increased survival rate and improved clinical scores were observed in mice treated with 5 mg/kg once a week. In these animals, there was a reduction in the percentage of CD4(+) and CD8(+) T cells, which are the main effectors of GvHD. In addition, dPGS treatment decreased the number of tumor necrosis factor alpha (TNFα)-producing T cells. Increasing the dose of dPGS reversed the positive effect on survival as well as the clinical score, which indicates a small therapeutic range. Here, we report for the first time that dPGS have a significant immunosuppressive in vivo effect in a mouse model of severe acute GvHD. Therefore, we propose to study dPGS as promising candidates for the development of potential new drugs in the treatment of steroid-refractory GvHD patients first in larger animals and later in humans.

Glycerol-based contrast agents: a novel series of dendronized pentamethine dyes.

The synthesis of water-soluble dyes, which absorb and emit in the range between 650 and 950 nm and display high extinction coefficients (ε) as well as high fluorescence quantum yields (Φf), is still a demand for optical imaging. We now present a synthetic route for the preparation of a new group of glycerol-substituted cyanine dyes from dendronized indole precursors that have been functionalized as N-hydroxysuccinimide (NHS) esters. High Φf values of up to 0.15 and extinction coefficients of up to 189 000 L mol(-1) cm(-1) were obtained for the pure dyes. Furthermore, conjugates of the new dendronized dyes with the antibody cetuximab (ctx) that were directed against the epidermal growth factor receptor (EGFR) of tumor cells could be prepared with dye to protein ratios between 0.3 and 2.2 to assess their potential as imaging probes. For the first time, ctx conjugates could be achieved without showing a decrease in Φf and with an increasing labeling degree that exceeded the value of the pure dye even at a labeling degree above 2. The incorporation of hydrophilically and sterically demanding dendrimers into cyanines prevented dimer formation after covalent conjugation to the antibody. The binding functionality of the resulting ctx conjugates to the EGFR was successfully demonstrated by cell microscopy studies using EGFR expressing cell lines. In summary, the combination of hydrophilic glycerol dendrons with reactive dye labels has been established for the first time and is a promising approach toward more powerful fluorescent labels with less dimerization.

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.

Effects of dendritic polyglycerol sulfate on articular chondrocytes.

INTRODUCTION: Inflammatory processes driven by cytokines play a crucial role during osteoarthritis (OA) progression. Dendritic polyglycerol sulfate (dPGS) was analyzed in vitro for its effects on articular chondrocytes, cartilage and cytokines involved in the OA process. METHODS: The metabolic activity of cultured human articular chondrocytes stimulated for 24 h with dPGS (10(-3)-10(-6) mol/L) was monitored using AlamarBlue(®) assay. The dPGS uptake was studied using fluorescence labeled nanoparticles. Further, chondrocytes were either treated with 10(-6) M dPGS, TNFα (10 ng/mL) alone or with a combination of both. The influence on extracellular matrix components, pro- and anti-inflammatory cytokines, matrix metalloproteinase (MMP)1 and the anaphylatoxin receptor C3aR was analyzed by RTD-PCR, flow cytometry and ELISA. RESULTS: Even at higher dosages (10(-3) mol/L), dPGS did not influence chondrocytes viability. Uptake of dPGS was successfully monitored in human articular chondrocytes and synovial fibroblasts, penetration into cartilage chips was up to ~50 µm. Cellular treatment with dPGS had no effect on synthesis of pro-inflammatory cytokines TNFα and IL-6, but expression of the anti-inflammatory IL-10 was upregulated. Cotreatment with TNFα and dPGS reduced the TNFα level, while IL-1ß, IL-6 and IL-10 expression did not change. Collagen type II gene expression was significantly reduced after preincubating cells with dPGS, but remained unaffected at the protein level. CONCLUSION: Results indicate that dPGS could play a role in regulation of cytokines associated with the inflammatory aspect of OA progression.

Influence of dendritic polyglycerol sulfates on knee osteoarthritis: an experimental study in the rat osteoarthritis model.

BACKGROUND: Anti-inflammatory nanoparticular compounds could represent a strategy to diminish osteoarthritis (OA) progression. The present study was undertaken to prove the uptake of nanoparticular dendritic polyglycerol sulfates (dPGS) by rat-derived articular chondrocytes and to answer the question of whether dPGS could modulate knee joint cartilage degradation in a rat OA model and whether complications could arise. METHODS: dPGS uptake and cytotoxicity was assessed in cultured primary rat-derived articular chondrocytes. Subsequently, OA was induced in the right knee joints of 12 male Wistar rats by medial collateral ligament and meniscus transection. Unoperated left knees remained as controls. Six weeks post surgery six rats were either treated daily (14 days) with 30 mg/kg dPGS (s.c.) or a similar volume of physiological saline. Animals were analyzed clinically for gait alterations. Explanted knee joints were studied histologically using OA scores according to Mankin (1971), Glasson et al., (2010) and the synovitis score according to Krenn et al., (2006). Liver, spleen and kidneys were analyzed for degenerative changes due to dPGS accumulation. RESULTS: dPGS was taken up after 2 hours by the chondrocytes. Whereas no significant clinical signs of OA could be detected, at the histological level, all operated rat knee joints revealed features of OA in the medial compartment. The values produced by both OA score systems were lower in rats treated with dPGS compared with saline-treated animals. Synovitis score did not significantly differ between the groups. The analyzed organs revealed no degenerative changes. CONCLUSIONS: dPGS presented overall cyto- and biocompatibility, no accumulation in metabolizing organs and chondroprotective properties in the osteoarthritic knee joint.

Detecting and Quantifying Biomolecular Interactions of a Dendritic Polyglycerol Sulfate Nanoparticle Using Fluorescence Lifetime Measurements.

Interactions of nanoparticles with biomaterials determine the biological activity that is key for the physiological response. Dendritic polyglycerol sulfates (dPGS) were found recently to act as an inhibitor of inflammation by blocking selectins. Systemic application of dPGS would present this nanoparticle to various biological molecules that rapidly adsorb to the nanoparticle surface or lead to adsorption of the nanoparticle to cellular structures such as lipid membranes. In the past, fluorescence lifetime measurements of fluorescently tagged nanoparticles at a molecular and cellular/tissue level have been proven to reveal valuable information on the local nanoparticle environment via characteristic fluorescent lifetime signatures of the nanoparticle bound dye. Here, we established fluorescence lifetime measurements as a tool to determine the binding affinity to fluorescently tagged dPGS (dPGS-ICC; ICC: indocarbocyanine). The binding to a cell adhesion molecule (L-selectin) and a human complement protein (C1q) to dPGS-ICC was evaluated by the concentration dependent change in the unique fluorescence lifetime signature of dPGS-ICC. The apparent binding affinity was found to be in the nanomolar range for both proteins (L-selectin: 87 ± 4 nM and C1q: 42 ± 12 nM). Furthermore, the effect of human serum on the unique fluorescence lifetime signature of dPGS-ICC was measured and found to be different from the interactions with the two proteins and lipid membranes. A comparison between the unique lifetime signatures of dPGS-ICC in different biological environments shows that fluorescence lifetime measurements of unique dPGS-ICC fluorescence lifetime signatures are a versatile tool to probe the microenvironment of dPGS in cells and tissue.

In vivo imaging with a fundus camera in a rat model of laser-induced choroidal neovascularization.

PURPOSE: To investigate the use of imaging and quantitative measurement capabilities of a modified fundus camera in a rat model of laser-induced choroidal neovascularization. METHODS: Following induction of experimental choroidal neovascularization, Dark Agouti rats underwent serial in vivo imaging with a fundus camera (FF450plus, Carl Zeiss MediTec, Jena, Germany), including color, reflectance and fluorescence imaging. RESULTS: A custom-made setting allowed high-resolution imaging. Change of fluorescence intensity following intravenous or intravitreal dye injection could be quantitatively monitored over time. Hardware binning resulted in an improved signal-to-noise ratio and a reduction of flash light intensity. Simultaneous fluorescence imaging following injection of two different dendritic polygylcerol sulfate dyes could be demonstrated. CONCLUSION: This study demonstrates the use and optimizations of a fundus camera for various in vivo imaging modalities in rats. Molecular imaging of the eye may allow for better insights into cellular dysfunction and optimization of therapeutic strategies.

An amphiphilic perylene imido diester for selective cellular imaging.

A new amphiphilic membrane marker based on a water-soluble dendritic polyglycerol perylene imido dialkylester has been designed, synthesized, and its optical properties characterized. In water it forms fluorescently quenched micellar self-aggregates, but when incorporated into a lipophilic environment, it monomerizes, and the highly fluorescent properties of the perylene core are recovered. These properties make it an ideal candidate for the imaging of artificial and cellular membranes as demonstrated by biophysical studies.

Synthesis and biological evaluation of radio and dye labeled amino functionalized dendritic polyglycerol sulfates as multivalent anti-inflammatory compounds.

Herein we describe a platform technology for the synthesis and characterization of partially aminated, (35)S-labeled, dendritic polyglycerol sulfate (dPG(35)S amine) and fluorescent dPGS indocarbocyanine (ICC) dye conjugates. These polymer conjugates, based on a biocompatible dendritic polyglycerol scaffold, exhibit a high affinity to inflamed tissue in vivo and represent promising candidates for therapeutic and diagnostic applications. By utilizing a one-step sequential copolymerization approach, dendritic polyglycerol (Mn ≈ 4.5 kDa) containing 9.4% N-phthalimide protected amine functionalities was prepared on a large scale. Sulfation and simultaneous radio labeling with (35)SO3 pyridine complex, followed by cleavage of the N-phthalimide protecting groups, yielded dPG(35)S amine as a beta emitting, inflammation specific probe with free amino functionalities for conjugation. Furthermore, efficient labeling procedures with ICC via iminothiolane modification and subsequent "Michael" addition of the maleimide functionalized ICC dye, as well as by amide formation via NHS derivatized ICC on a dPGS amine scaffold, are described. The dPGS-ICC conjugates were investigated with respect to their photophysical properties, and both the radiolabeled and fluorescent compounds were comparatively visualized in histological tissue sections (radio detection and fluorescence microscopy) of animals treated with dPGS. Furthermore, cellular uptake of dPGS-ICC was found in endothelial cord blood (HUVEC) and the epithelial lung cells (A549). The presented synthetic routes allow a reproducible, controlled synthesis of dPGS amine on kilogram scale applying a one-pot batch reaction process. dPGS amine can be used for analysis via radioactivity or fluorescence, thereby creating a new platform for inflammation specific, multimodal imaging purposes using other attachable probes or contrast agents.

New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging.

The rational design of bright optical probes and dye-biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye-protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye-dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M(-1) cm(-1) and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS.

Targeted delivery of dendritic polyglycerol-doxorubicin conjugates by scFv-SNAP fusion protein suppresses EGFR+ cancer cell growth.

Development of effective polymer-based nanocarriers for the successful application in cancer therapy still remains a great challenge in current research. In the present study we present a dendritic polyglycerol-based multifunctional drug immunoconjugate that specifically targets and kills cancer cell lines expressing epidermal growth factor receptor (EGFR). The nanocarrier was provided with a dendritic core as a multifunctional anchoring point, doxorubicin (Doxo) coupled through a pH-sensitive linker, a fluorescence marker, poly(ethylene glycol), as solubilizing and shielding moiety, and a scFv antibody conjugated through the SNAP-Tag technology. The study provides the proof of principle that SNAP-tag technology can be used to generate drug-carrying nanoparticles efficiently modified with single-chain antibodies to specifically target and destroy cancer cells.

Optical imaging.

Non-invasive optical imaging techniques, such as fluorescence imaging (FI) or bioluminescence imaging (BLI) have emerged as important tools in biomedical research. As demonstrated in different animal disease models, they enable visualization of physiological and pathophysiological processes at the cellular and molecular level in vivo with high specificity. Optical techniques are easy to use, fast, and affordable. Furthermore, they are characterized by their high sensitivity. In FI, very low amounts of the imaging agent (nano- to femtomol or even less) can be detected. Due to the absorption and scattering of light in tissue, optical techniques exhibit a comparably low spatial resolution in the millimeter range and a depth limit of a few centimeters. However, non-invasive imaging of biological processes in small animals and in outer or inner surfaces as well as during surgery even in humans is feasible. Currently two agents for fluorescence imaging are clinically approved, namely indocyanine green (ICG) and 5-aminolevulinic acid (5-ALA). In the past years, a number of new optical imaging agents for FI and reporter systems for BLI have been developed and successfully tested in animal models. Some of the FI agents might promise the application in clinical oncology. In this chapter, we describe the basic principles of non-invasive optical imaging techniques, give examples for the visualization of biological processes in animal models of cancer, and discuss potential clinical applications in oncology.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation.

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.