Stefanie Wedepohl

A microgel construction kit for bioorthogonal encapsulation and pH-controlled release of living cells.

pH-Cleavable cell-laden microgels with excellent long-term viabilities were fabricated by combining bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) and droplet-based microfluidics. Poly(ethylene glycol)dicyclooctyne and dendritic poly(glycerol azide) served as bioinert hydrogel precursors. Azide conjugation was performed using different substituted acid-labile benzacetal linkers that allowed precise control of the microgel degradation kinetics in the interesting pH range between 4.5 and 7.4. By this means, a pH-controlled release of the encapsulated cells was achieved upon demand with no effect on cell viability and spreading. As a result, the microgel particles can be used for temporary cell encapsulation, allowing the cells to be studied and manipulated during the encapsulation and then be isolated and harvested by decomposition of the microgel scaffolds.

l-Selectin -A dynamic regulator of leukocyte migration

The leukocytic cell adhesion receptor L-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance. Distinct domains of L-selectin contribute to proper leukocyte migration. In this review, we discuss the contributions of these domains with respect to L-selectin function: the regulation by serine phosphorylation of the cytoplasmic tail, the role of the transmembrane domain in receptor positioning on the cell surface as well as the N-glycosylation of the extracellular part and the identification of novel binding partners.

N-glycan analysis of recombinant L-Selectin reveals sulfated GalNAc and GalNAc-GalNAc motifs.

The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants (LEHis). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. alpha1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc-GalNAc or SO(4)-GalNAc-GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function.

Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA120), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLeX), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA120 was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA120 affinity (KD=4 μM). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLeX ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.

The effect of polyglycerol sulfate branching on inflammatory processes.

In this study, the extent to which the scaffold architecture of polyglycerol sulfates affects inflammatory processes and hemocompatibility is investigated. Competitive L-selectin binding assays, cellular uptake studies, and blood compatibility readouts are done to evaluate distinct biological properties. Fully glycerol based hyperbranched polyglycerol architectures are obtained by either homopolymerization of glycidol (60% branching) or a new copolymerization strategy of glycidol with ethoxyethyl glycidyl ether. Two polyglycerols with 24 and 42% degree of branching (DB) are synthesized by using different monomer feed ratios. A perfectly branched polyglycerol dendrimer is synthesized according to an iterative two-step protocol based on allylation of the alcohol and subsequent catalytic dihydroxylation. All the polyglycerol sulfates are synthesized with a comparable molecular weight and degree of sulfation. The DB make the different polymer conjugates perform different ways. The optimal DB is 60% in all biological assays.

Immobilization of Stimuli-Responsive Nanogels onto Honeycomb Porous Surfaces and Controlled Release of Proteins

In this article, we describe the formation of functional honeycomb-like porous surfaces fabricated by the breath figures technique using blends of either amino-terminated poly(styrene) or a poly(styrene)-b-poly(acrylic acid) block copolymer with homopoly(styrene). Thus, the porous interfaces exhibited either amino or acid groups selectively located inside of the holes, which were subsequently employed to anchor stimuli-responsive nanogels by electrostatic interactions. These nanogels were prepared from poly(N-isopropylacrylamide) (PNIPAM) cross-linked with dendritic polyglycerol (dPG) and semi-interpenetrated with either 2-(dimethylamino)ethyl methacrylate (DMAEMA) or 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) to produce positively and negatively charged nanogel surfaces, respectively. The immobilization of these semi-interpenetrated networks onto the surfaces allowed us to have unique stimuli-responsive surfaces with both controlled topography and composition. More interestingly, the surfaces exhibited stimuli-responsive behavior by variations on the pH or temperature. Finally, the surfaces were evaluated regarding their capacity to induce a thermally triggered protein release at temperatures above the cloud point temperature (T(cp)) of the nanogels.

In vivo comparative study of distinct polymeric architectures bearing a combination of paclitaxel and doxorubicin at a synergistic ratio

ABSTRACT Nowadays, combination therapy became a standard in oncology. In this study, we compare the activity of two polymeric carriers bearing a combination of the anticancer drugs paclitaxel (PTX) and doxorubicin (DOX), which differ mainly in their architecture and supramolecular assembly. Drugs were covalently bound to a linear polymer, polyglutamic acid (PGA) or to a dendritic scaffold, polyglycerol (PG) decorated with poly(ethylene glycol) (PEG), forming PGA‐PTX‐DOX and PG‐PTX‐bz‐DOX‐PEG, respectively. We explored the relationship between the polymeric architectures and their performance with the aim to augment the pharmacological benefits of releasing both drugs simultaneously at the tumor site at a synergistic ratio. We recently designed and characterized a PGA‐PTX‐DOX conjugate. Here, we describe the synthesis and characterization of PG dendritic scaffold bearing the combination of PTX and DOX. The performance of both conjugates was evaluated in a murine model of mammary adenocarcinoma in immunocompetent mice, to investigate whether the activity of the treatments is affected by the immune system. Drug conjugation to a nano‐sized polymer enabled preferred tumor accumulation by extravasation‐dependent targeting, making use of the enhanced permeability and retention (EPR) effect. Both PGA‐PTX‐DOX and PG‐PTX‐bz‐DOX‐PEG nano‐sized conjugates exhibited superior anti‐tumor efficacy and safety compared to the combination of the free drugs, at equivalent concentrations. However, while PGA‐PTX‐DOX was more efficient than a mixture of each drug conjugated to a separate PGA chain, as was previously shown, PG‐PTX‐bz‐DOX‐PEG had similar activity to the mixture of the PG‐PTX‐bz‐PEG and PG‐DOX‐PEG conjugates. Our results show that both conjugates are potential candidates as precision combination nanomedicines for the treatment of breast cancer.

Synthesis and Evaluation of Nonsulfated and Sulfated Glycopolymers as L- and P-selectin Inhibitors

Selectins are carbohydrate-binding proteins and responsible for leukocyte extravasation in inflammation. Here we demonstrate the potential of synthetic glycocompounds as inhibitors for the selectin-ligand interaction. Pentaerythritol derivatives showed distinct selectin-binding properties with IC50 values up to 1.5 μM. Multivalent hyperbranched polyglycerol (hPG) derivatives did not lead to a substantial increase in inhibition, but a more than 1000-fold enhancement was realized when sulfated glyco-hPGs were tested. IC50 values in the high picomolar to low nanomolar range were obtained for selectin inhibition, which highlights the relevance of sulfate groups that seem to dominate the binding mode.

Near Infrared Dye Conjugated Nanogels for Combined Photodynamic and Photothermal Therapies

There is a need for new and smart formulations that will help overcome the limitations of organic dyes used in photodynamic (PDT) and photothermal (PTT) therapy and significantly accelerate their clinical translation. Therefore the aim of this work was to create a responsive nanogel scaffold as a smart vehicle for dye administration. We developed a methodology that enables the conjugation of organic dyes to thermoresponsive nanogels and yields biocompatible, nanometer-sized products with low polydispersity. The potential of the dye-nanogel conjugate as a photothermal and photodynamic agent has been demonstrated by an in vitro evaluation with a model human carcinoma cell line. Additionally, confocal cell images showed their cellular uptake profile and their potential for bioimaging and intracellular drug delivery. These conjugates are a promising scaffold as a theranostic agents and will enable further applications in combination with controlled drug release.

One-pot synthesis of doxorubicin-loaded multiresponsive nanogels based on hyperbranched polyglycerol

Doxorubicin-loaded nanogels with multiresponsive properties are prepared using hyperbranched polyglycerol as a biocompatible scaffold. The nanogels are synthesized in a single step combining free-radical polymerization and a mild nanoprecipitation technique. The nanogels respond to different biological stimuli such as low pH and reductive environments, resulting in a more efficient cell proliferation inhibition in A549 cells.