Colorimetric Biosensors Based on Polymer/Gold Hybrid Nanoparticles: Topological Effects of the Polymer Coating.

Gold nanoparticles decorated with analyte recognition units can form the basis of colorimetric (bio)sensors. The presentation of those recognition units may play a critical role in determining sensor sensitivity. Herein, we use a model system to investigate the effect of the architecture of a polymeric linker that connects gold nanoparticles with the recognition units. Our results show that the number of the latter that can be adsorbed during the assembly of the colorimetric sensors depends on the linker topology. We also show that this may lead to substantial differences in colorimetric sensor performance, particularly in situations in which the interactions with the analyte are comparably weak. Finally, we discuss design principles for efficient colorimetric sensor materials based on our findings.

Tuning the network charge of biohybrid hydrogel matrices to modulate the release of SDF-1.

The delivery of chemotactic signaling molecules via customized biomaterials can effectively guide the migration of cells to improve the regeneration of damaged or diseased tissues. Here, we present a novel biohybrid hydrogel system containing two different sulfated glycosaminoglycans (sGAG)/sGAG derivatives, namely either a mixture of short heparin polymers (Hep-Mal) or structurally defined nona-sulfated tetrahyaluronans (9s-HA4-SH), to precisely control the release of charged signaling molecules. The polymer networks are described in terms of their negative charge, i.e. the anionic sulfate groups on the saccharides, using two parameters, the integral density of negative charge and the local charge distribution (clustering) within the network. The modulation of both parameters was shown to govern the release characteristics of the chemotactic signaling molecule SDF-1 and allows for seamless transitions between burst and sustained release conditions as well as the precise control over the total amount of delivered protein. The obtained hydrogels with well-adjusted release profiles effectively promote MSC migration in vitro and emerge as promising candidates for new treatment modalities in the context of bone repair and wound healing.

Thermodynamic Analysis of the Interaction of Heparin with Lysozyme.

Glycosaminoglycan (GAG)-protein binding governs critically important signaling events in living matter. Aiming at a quantitative analysis of the involved processes, we herein present a thermodynamic study of the interaction of the model GAG heparin and lysozyme in aqueous solution. Heparin is a highly charged linear polyelectrolyte with a charge parameter of 2.9 (37 °C). The binding constant Kb was determined by ITC as a function of the temperature and ionic strength adjusted through the concentration cs of added salt. The dependence on salt concentration cs was used to determine the net number of released counterions. Moreover, the binding constant at a reference salt concentration of 1 M Kb(1 M) was determined by extrapolation. The dependence on temperature of Kb was used to dissect the binding free energy ΔGb into the respective enthalpies ΔHb and entropies ΔSb together with the specific heat Δcp. A strong enthalpy-entropy cancelation was found similar to the results for many other systems. The binding free energy ΔGb could furthermore be split up into a part ΔGci due to counterion release and a residual part ΔGres. The latter quantity reflects specific contributions as, e.g., salt bridges, van der Waals interactions, or hydrogen bonds. The entire analysis shows that heparin-lysozyme interactions are mainly caused by counterion release; that is, ca. three counterions are being released upon binding one lysozyme molecule. Our reported approach of quantifying interactions between glycosaminoglycans and proteins is generally applicable and suitable to provide new insights in the physical modulation of biomolecular signals.

Charge-tuning of glycosaminoglycan-based hydrogels to program cytokine sequestration.

Glycosaminoglycan (GAG)-based biohybrid hydrogels of varied GAG content and GAG sulfation pattern were prepared and applied to sequester cytokines. The binding of strongly acidic and basic cytokines correlated with the integral space charge density of the hydrogel, while the binding of weakly charged cytokines was governed by the GAG sulfation pattern.

Functional Interference in the Bone Marrow Microenvironment by Disseminated Breast Cancer Cells.

Skeletal metastasis of breast cancer is associated with a poor prognosis and significant morbidity. Investigations in other solid tumors have revealed an impairment in hematopoietic function upon bone marrow invasion. However, the interaction between disseminated breast cancer cells and the bone marrow microenvironment which harbors them has not been addressed comprehensively. Employing advanced co-culture assays, proteomic studies, organotypic models as well as in vivo xenotransplant models, we define the consequences of this interaction on the stromal compartment of bone marrow, affected molecular pathways and subsequent effects on the hematopoietic stem and progenitor cells (HSPCs). The results showed a basic fibroblast growth factor (bFGF)-mediated, synergistic increase in proliferation of breast cancer cells and mesenchymal stromal cells (MSCs) in co-culture. The stromal induction was associated with elevated phosphoinositide-3 kinase (PI3K) signaling in the stroma, which coupled with elevated bFGF levels resulted in increased migration of breast cancer cells towards the MSCs. The perturbed cytokine profile in the stroma led to reduction in the osteogenic differentiation of MSCs via downregulation of platelet-derived growth factor-BB (PDGF-BB). Long term co-cultures of breast cancer cells, HSPCs, MSCs and in vivo studies in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl) /SzJ (NSG) mice showed a reduced support for HSPCs in the altered niche. The resultant non- conducive phenotype of the niche for HSPC support emphasizes the importance of the affected molecular pathways in the stroma as clinical targets. These findings can be a platform for further development of therapeutic strategies aiming at the blockade of bone marrow support to disseminated breast cancer cells. Stem Cells 2016;34:2224-2235.

Tackling Cell Transplantation Anoikis: An Injectable, Shape Memory Cryogel Microcarrier Platform Material for Stem Cell and Neuronal Cell Growth.

Highly macroporous semisynthetic cryogel microcarriers can be synthesized for culturing stem cells and neuronal type cells. Growth factors loaded to heparin-containing microcarriers show near zero-order release kinetics and cell-loaded microcarriers can be injected through a fine gauge cannula without negative effect on the cells. These carriers can be applied for cell transplantation applications.

Chemoselective peptide functionalization of starPEG-GAG hydrogels.

Glycosaminoglycan (GAG)-based hydrogels gain increasing interest in regenerative therapies. To support specific applications, the biomolecular functionality of gel matrices needs to be customized via conjugation of peptide sequences that mediate cell adhesion, expansion and differentiation. Herein, we present an orthogonal strategy for the formation and chemoselective functionalization of starPEG-GAG hydrogels, utilizing the uniform and specific conjugation of peptides and GAGs for customizing the resulting materials. The introduced approach was applied for the incorporation of three different types of RGD peptides to analyze the influence of peptide sequence and conformation on adhesion and morphogenesis of endothelial cells (ECs) grown on the peptide-containing starPEG-GAG hydrogels. The strongest cellular response was observed for hydrogels functionalized with cycloRGD followed by linear forms of RGDSP and RGD, showing that morphogenesis and growth rate of ECs is controlled by both type and quantity of the conjugated peptides.

Biohybrid networks of selectively desulfated glycosaminoglycans for tunable growth factor delivery.

Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on the network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine.

Sulfation of hyaluronic acid reconfigures the mechanistic pathway of bone morphogenetic protein-2 aggregation.

Bone morphogenetic protein-2 (BMP-2) is a critical growth factor of bone extracellular matrix (ECM), pivotal for osteogenesis. Glycosaminoglycans (GAGs), another vital ECM biomolecules, interact with growth factors, affecting signal transduction. Our study primarily focused on hyaluronic acid (HA), a prevalent GAG, and its sulfated derivative (SHA). We explored their impact on BMP-2's conformation, aggregation, and mechanistic pathways of aggregation using diverse optical and rheological methods. In the presence of HA and SHA, the secondary structure of BMP-2 underwent a structured transformation, characterized by a substantial increase in beta sheet content, and a detrimental alteration, manifesting as a shift towards unstructured content, respectively. Although both HA and SHA induced BMP-2 aggregation, their mechanisms differed. SHA led to rapid amorphous aggregates, while HA promoted amyloid fibrils with a lag phase and sigmoidal kinetics. Aggregate size and shape varied; HA produced larger structures, SHA smaller. Each aggregation type followed distinct pathways influenced by viscosity and excluded volume. Higher viscosity, low diffusivity of protein and higher excluded volume In the presence of HA promotes fibrillation having size in micrometer range. Low viscosity, high diffusivity of protein and lesser excluded volume leads to amorphous aggregate of size in nanometer range.

The Influence of Sulfation Degree of Glycosaminoglycan-Functionalized 3D Collagen I Networks on Cytokine Profiles of In Vitro Macrophage-Fibroblast Cocultures.

Cell-cell interactions between fibroblasts and immune cells, like macrophages, are influenced by interaction with the surrounding extracellular matrix during wound healing. In vitro hydrogel models that mimic and modulate these interactions, especially of soluble mediators like cytokines, may allow for a more detailed investigation of immunomodulatory processes. In the present study, a biomimetic extracellular matrix model based on fibrillar 3D collagen I networks with a functionalization with heparin or 6-ON-desulfated heparin, as mimics of naturally occurring heparan sulfate, was developed to modulate cytokine binding effects with the hydrogel matrix. The constitution and microstructure of the collagen I network were found to be stable throughout the 7-day culture period. A coculture study of primary human fibroblasts/myofibroblasts and M-CSF-stimulated macrophages was used to show its applicability to simulate processes of progressed wound healing. The quantification of secreted cytokines (IL-8, IL-10, IL-6, FGF-2) in the cell culture supernatant demonstrated the differential impact of glycosaminoglycan functionalization of the collagen I network. Most prominently, IL-6 and FGF-2 were shown to be regulated by the cell culture condition and network constitution, indicating changes in paracrine and autocrine cell-cell communication of the fibroblast-macrophage coculture. From this perspective, we consider our newly established in vitro hydrogel model suitable for mechanistic coculture analyses of primary human cells to unravel the role of extracellular matrix factors in key events of tissue regeneration and beyond.

A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4ß1 integrin binding motif, are differently influenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

Hydrogel-based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo. However, due to their complex multifactorial regulation, adapting these tissue and disease models for high-throughput screening workflows remains challenging. In this study, a new precision culture scaling (PCS-X) methodology combines statistical techniques (design of experiment and multiple linear regression) with automated, parallelized experiments and analyses to customize hydrogel-based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation, and media composition are systematically explored to induce vasculogenesis in endothelial mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384-well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound- and dose-dependent manner, demonstrating the scope and power of PCS-X in creating parallelized tissue and disease models for drug discovery and individualized therapies.

Minimal peptide motif for non-covalent peptide-heparin hydrogels.

Reduction of complexity of the extracellular matrix (ECM) to a non-covalent structure with minimal chemically defined components represents an attractive avenue for understanding the biology of the ECM. The resulting system could lead to the design of tailor-made biomaterials that incorporate varying functionalities. Negatively charged glycosaminoglycans are the major components of the ECM. Their interaction with positively charged proteins is important for dynamic three-dimensional scaffold formation and function. We designed and screened minimal peptide motifs whose conjugates with polyethylene glycol interact with heparin to form non-covalent hydrogels. Here we show the structure/function relationship of the (RA)(n) and (KA)(n) motifs and determined that both basic residues and the heparin-induced α-helix formation are important for the assembly process. Simple rules allowed us to tune various aspects of the matrix system such as the gelation rates, biodegradability, rheological properties, and biofunctionality. The hydrogels can encapsulate cells and support cell survival.

Characterization of polymer membranes by MALDI mass-spectrometric imaging techniques.

For physical and chemical characterization of polymers, a wide range of analytical methods is available. Techniques like NMR and X-ray are often combined for a detailed characterization of polymers used in medical applications. Over the past few years, MALDI mass-spectrometry has been developed as a powerful tool for space-resolved analysis, not least because of its mass accuracy and high sensitivity. MALDI imaging techniques combine the potential of mass-spectrometric analysis with imaging as additional spatial information. MALDI imaging enables the visualization of localization and distribution of biomolecules, chemical compounds, and other molecules on different surfaces. In this study, surfaces of polymeric dialyzer membranes, consisting of polysulfone (PS) and polyvinylpyrrolidone (PVP), were investigated, regarding chemical structure and the compound's distribution. Flat membranes as well as hollow fiber membranes were analyzed by MALDI imaging. First, analysis parameters like laser intensity and laser raster step size (spatial resolution in resulting image) were established in accordance with polymer's characteristics. According to the manufacturing process, luminal and abluminal membrane surfaces are characterized by differences in chemical composition and physical characteristics. The MALDI imaging demonstrated that the abluminal membrane surface consists more of polysulfone than polyvinylpyrrolidone, and the luminal membrane surface displayed more PVP than PS. The addition of PVP as hydrophilic modifier to polysulfone-based membranes increases the biocompatibility of the dialysis membranes. The analysis of polymer distribution is a relevant feature for characterization of dialysis membranes. In conclusion, MALDI imaging is a powerful technique for polymer membrane analysis, regarding not only detection and identification of polymers but also localization and distribution in membrane surfaces.

Analysis of the Binding of Cytokines to Highly Charged Polymer Networks.

A model describing the binding of biological signaling proteins to highly charged polymer networks is presented. The networks are formed by polyelectrolyte chains for which the distance between two charges at the chain is smaller than the Bjerrum length. Counterion condensation on such highly charged chains immobilizes a part of the counterions. The Donnan-equilibrium between the polymer network and the aqueous solution with salt concentration c s b $c_s^b$ is used to calculate the salt concentration of the co- and counterions c s g $c_s^g$ entering the network. Two factors are decisive: i) The electrostatic interaction between the network and the protein is given by the Donnan-potential of the network and the net charge of the protein. In addition to this leading term, a second term describes the change in the Born-energy of the proteins when entering the network. ii) The interaction of the protein with the highly charged chains within the network is governed by counterion release: Patches of positive charge at the protein become multivalent counterions of the polyelectrolyte chains thus releasing a concomitant number of condensed counterions. The model compares favorably to experimental data obtained on a set of biohybrid polymer networks composed of crosslinked glycosaminoglycan chains that interact with a mixture of key signaling proteins.

Three-Dimensional Biohybrid StarPEG-Heparin Hydrogel Cultures for Modeling Human Neuronal Development and Alzheimer's Disease Pathology.

In this chapter, we present the methodology currently used in our laboratory to generate a starPEG-MMP (starPEG)- and heparin maleimide HM06 (heparin)-based 3D cell culture system, in a hydrogel, that can be used to study human neuronal development and Alzheimer's disease (AD) pathology. A 3D cell culture system can mimic the in vivo cellular environment better than a 2D format, in which these cells exhibit neural network formation, electrophysiological activity, tissue-specific extracellular matrix (ECM) deposition, and neurotransmitter responsiveness. When treated with amyloid beta-42 (Aß42) peptides, this system recapitulates many of the pathological effects of AD, including reduced neural stem cell proliferation, impaired neuronal network formation, dystrophic axonal ends, synaptic loss, failure to deposit ECM, elevated tau hyperphosphorylation, and formation of neurofibrillary tangles. Culturing human primary cortical astrocyte (pHA)- or induced pluripotent stem cell (iPSC)-derived human neural stem cells in this biohybrid hydrogel system has led to the discovery of novel regulatory pathways underlying neurodegenerative pathology in different phases of AD.

Discriminant Principal Component Analysis of ToF-SIMS Spectra for Deciphering Compositional Differences of MSC-Secreted Extracellular Matrices.

Identifying characteristic extracellular matrix (ECM) variants is a key challenge in mechanistic biology, bioengineering, and medical diagnostics. The reported study demonstrates the potential of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect subtle differences between human mesenchymal stromal cell (MSC)-secreted ECM types as induced by exogenous stimulation or emerging pathology. ToF-SIMS spectra of decellularized ECM samples are evaluated by discriminant principal component analysis (DPCA), an advanced multivariate analysis technique, to decipher characteristic compositional features. To establish the approach, signatures of major ECM proteins are determined from samples of pre-defined mixtures. Based on that, sets of ECM variants produced by MSCs in vitro are analyzed. Differences in the content of collagen, fibronectin, and laminin in the ECM resulting from the combined supplementation of MSC cultures with polymers that induce macromolecular crowding and with ascorbic acid are detected from the DPCA of ToF-SIMS spectra. The results are verified by immunostaining. Finally, the comparative ToF-SIMS analysis of ECM produced by MSCs of healthy donors and patients suffering from myelodysplastic syndrome display the potential of the novel methodology to reveal disease-associated alterations of the ECM composition.

Sustained Release of Human Adipose Tissue Stem Cell Secretome from Star-Shaped Poly(ethylene glycol) Glycosaminoglycan Hydrogels Promotes Motor Improvements after Complete Transection in Spinal Cord Injury Rat Model.

Adipose tissue-derived stem cells (ASCs) have been shown to assist regenerative processes after spinal cord injury (SCI) through their secretome, which promotes several regenerative mechanisms, such as inducing axonal growth, reducing inflammation, promoting cell survival, and vascular remodeling, thus ultimately leading to functional recovery. However, while systemic delivery (e.g., i.v. [intravenous]) may cause off-target effects in different organs, the local administration has low efficiency due to fast clearance by body fluids. Herein, a delivery system for human ASCs secretome based on a hydrogel formed of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan heparin (Hep) that is suitable to continuously release pro-regenerative signaling mediators such as interleukin (IL)-4, IL-6, brain-derived neurotrophic factor, glial-cell neurotrophic factor, and beta-nerve growth factor over 10 days, is reported. The released secretome is shown to induce differentiation of human neural progenitor cells and neurite outgrowth in organotypic spinal cord slices. In a complete transection SCI rat model, the secretome-loaded hydrogel significantly improves motor function by reducing the percentage of ameboid microglia and systemically elevates levels of anti-inflammatory cytokines. Delivery of ASC-derived secretome from starPEG-Hep hydrogels may therefore offer unprecedented options for regenerative therapy of SCI.

Nerve growth factor receptor (Ngfr) induces neurogenic plasticity by suppressing reactive astroglial Lcn2/Slc22a17 signaling in Alzheimer's disease.

Neurogenesis, crucial for brain resilience, is reduced in Alzheimer's disease (AD) that induces astroglial reactivity at the expense of the pro-neurogenic potential, and restoring neurogenesis could counteract neurodegenerative pathology. However, the molecular mechanisms promoting pro-neurogenic astroglial fate despite AD pathology are unknown. In this study, we used APP/PS1dE9 mouse model and induced Nerve growth factor receptor (Ngfr) expression in the hippocampus. Ngfr, which promotes neurogenic fate of astroglia during the amyloid pathology-induced neuroregeneration in zebrafish brain, stimulated proliferative and neurogenic outcomes. Histological analyses of the changes in proliferation and neurogenesis, single-cell transcriptomics, spatial proteomics, and functional knockdown studies showed that the induced expression of Ngfr reduced the reactive astrocyte marker Lipocalin-2 (Lcn2), which we found was sufficient to reduce neurogenesis in astroglia. Anti-neurogenic effects of Lcn2 was mediated by Slc22a17, blockage of which recapitulated the pro-neurogenicity by Ngfr. Long-term Ngfr expression reduced amyloid plaques and Tau phosphorylation. Postmortem human AD hippocampi and 3D human astroglial cultures showed elevated LCN2 levels correlate with reactive gliosis and reduced neurogenesis. Comparing transcriptional changes in mouse, zebrafish, and human AD brains for cell intrinsic differential gene expression and weighted gene co-expression networks revealed common altered downstream effectors of NGFR signaling, such as PFKP, which can enhance proliferation and neurogenesis in vitro when blocked. Our study suggests that the reactive non-neurogenic astroglia in AD can be coaxed to a pro-neurogenic fate and AD pathology can be alleviated with Ngfr. We suggest that enhancing pro-neurogenic astroglial fate may have therapeutic ramifications in AD.

Electrokinetic analysis to reveal composition and structure of biohybrid hydrogels.

Biohybrid hydrogels combining electrically neutral synthetic polymers and highly anionic glycosaminoglycans (GAGs) offer exciting options for regenerative therapies as they allow for the electrostatic conjugation of various growth factors. Unraveling details of ionization and structure within such networks defines an important analytical challenge that requires the extension of current methodologies. Here, we present a mean-field approach to quantify the density of ionizable groups, GAG concentration, and cross-linking degree of such hydrogels based on experimental data from microslit electrokinetics and ellipsometry. An exemplary poly(ethylene glycol)-heparin system was analyzed to demonstrate how electrostatic fingerprints of hydrogels obtained by the introduced strategy can sensitively display composition and structure of the polymer networks.