Scientific publications

Limited drug loading capacity (LC), mostly below 5% w/w, is a significant drawback of nanoparticulate drug delivery systems (DDS). Squalenoylation technology, which employs bioconjugation of squalenyl moiety and drug, allows self-assemble of nanoparticles (NPs) in aqueous media with significantly high LC (>30% w/w). The synthesis and particle preparation of squalenoylated prodrugs are, however, not facile for molecules with multiple reactive groups. Taking a different approach, we describe the synthesis of amphiphilic squalenyl derivatives (SqDs) as well as the physicochemical and biopharmaceutical characterizations of their self-assembled NPs as DDSs. The SqDs included in this study are (i) cationic squalenyl diethanolamine (ii) PEGylated SqD (PEG 750 Da), (iii) PEGylated SqD (PEG 3,000 Da), and (iv) anionic squalenyl hydrogen sulfate. All four SqDs self-assemble into NPs in a size range from 100 to 200 nm in an aqueous solution. Furthermore, all NP derivatives demonstrate appropriate biocompatibility and adequate colloidal stability in physiological relevant pH environments. The mucoprotein binding of PEGylated NPs is reduced compared to the charged NPs. Most importantly, this technology allows excellent LC (at maximum of 45% w/w) of a wide range of multifunctional compounds, varying in physicochemical properties and molecular weight. Interestingly, the drug release profile can be tuned by different loading methods. In summary, the SqD-based NPs appear as versatile drug delivery platforms.

Abstract Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self-assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug-loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16-fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use.

Abstract Nanoparticles (NPs) are able to deliver a variety of substances into eukaryotic cells. However, their usage is often hampered by a lack of specificity, leading to the undesired uptake of NPs by virtually all cell types. In contrast to this, yeast is known to be specifically taken up into immune cells after entering the body. Therefore, we investigated the interaction of biodegradable surface-modified poly (lactic-co-glycolic acid) (PLGA) particles with yeast cells to overcome the unspecificity of the particulate carriers. Cells of different Saccharomyces cerevisiae strains were characterized regarding their interaction with PLGA-NPs under isotonic and hypotonic conditions. The particles were shown to efficiently interact with yeast cells leading to stable NP/yeast-complexes allowing to associate or even internalize compounds. Notably, applying those complexes to a co-culture model of HeLa cells and macrophages, the macrophages were specifically targeted. This novel nano-in-micro carrier system suggests itself as a promising tool for the delivery of biologically active agents into phagocytic cells combining specificity and efficiency. This article is protected by copyright. All rights reserved.

Extracellular vesicles are membranous structures shed by almost every living cell. Bacterial gram-negative outer membrane vesicles (OMVs) and gram-positive membrane vesicles (MVs) play important roles in adaptation to the surrounding environment, cellular components' exchange, transfer of antigens and virulence factors, and infection propagation. Streptococcus pneumoniae is considered one of the priority pathogens, with a global health impact due to the increase in infection burden and growing antibiotic resistance. We isolated MVs produced from the S. pneumoniae reference strain (R6) and purified them via size exclusion chromatography (SEC) to remove soluble protein impurities. We characterized the isolated MVs by nanoparticle tracking analysis (NTA) and measured their particle size distribution and concentration. Isolated MVs showed a mean particle size range of 130–160 nm and a particle yield of around 10¹² particles per milliliter. Cryogenic transmission electron microscopy (cryo-TEM) images revealed a very heterogeneous nature of isolated MVs with a broad size range and various morphologies, arrangements, and contents. We incubated streptococcal MVs with several mammalian somatic cells, namely, human lung epithelial A549 and human keratinocytes HaCaT cell lines, and immune cells including differentiated macrophage-like dTHP-1 and murine dendritic DC2.4 cell lines. All cell lines displayed excellent viability profile and negligible cytotoxicity after 24-h incubation with MVs at concentrations reaching 10⁶ MVs per cell (somatic cells) and 10⁵ MVs per cell (immune cells). We evaluated the uptake of fluorescently labeled MVs into these four cell lines, using flow cytometry and confocal microscopy. Dendritic cells demonstrated prompt uptake after 30-min incubation, whereas other cell lines showed increasing uptake after 2-h incubation and almost complete colocalization/internalization of MVs after only 4-h incubation. We assessed the influence of streptococcal MVs on antigen-presenting cells, e.g., dendritic cells, using enzyme-linked immunosorbent assay (ELISA) and observed enhanced release of tumor necrosis factor (TNF)-α, a slight increase of interleukin (IL)-10 secretion, and no detectable effect on IL-12. Our study provides a better understanding of gram-positive streptococcal MVs and shows their potential to elicit a protective immune response. Therefore, they could offer an innovative avenue for safe and effective cell-free vaccination against pneumococcal infections.

Hydrogel-based bio-inks have recently attracted more attention for 3D printing applications in tissue engineering due to their remarkable intrinsic properties, such as a cell supporting environment. However, their usually weak mechanical properties lead to poor printability and low stability of the obtained structures. To obtain good shape fidelity, current approaches based on extrusion printing use high viscosity solutions, which can compromise cell viability. This paper presents a novel bio-printing methodology based on a dual-syringe system with a static mixing tool that allows in situ crosslinking of a two-component hydrogel-based ink in the presence of living cells. The reactive hydrogel system consists of carboxymethyl chitosan (CMCh) and partially oxidized hyaluronic acid (HAox) that undergo fast self-covalent crosslinking via Schiff base formation. This new approach allows us to use low viscosity solutions since in situ gelation provides the appropriate structural integrity to maintain the printed shape. The proposed bio-ink formulation was optimized to match crosslinking kinetics with the printing process and multi-layered 3D bio-printed scaffolds were successfully obtained. Printed scaffolds showed moderate swelling, good biocompatibility with embedded cells, and were mechanically stable after 14 days of the cell culture. We envision that this straightforward, powerful, and generalizable printing approach can be used for a wide range of materials, growth factors, or cell types, to be employed for soft tissue regeneration.

Cooperative action of biochemical and biomechanical signals regulates the interactions between cells and the supporting matrix in natural tissues. Herein, we describe a hydrogel for 4D cell culture which allows user-defined stiffening of the cellular environment and presentation of bioadhesive cues in an orthogonal manner using light of different wavelengths. Stiffening of the gel is initiated by VIS light, while activation of the biochemical function is triggered by UV light. We demonstrate the versatility of this system by triggering, directing and/or hindering cell migration from spheroids based on photoactivated stiffening or integrin-binding to the hydrogels. This material allows in situ and independent manipulation of the physicochemical cues in the cellular microenvironment in vitro, and could eventually be extended to in vivo.

The etherate of (Ph2SiO)8[Al(O)OH]4 can be transformed into the pyrazine adduct (Ph2SiO)8[Al(O)OH]4·3N(C2H2)2N (1), the ethyl acetate adduct (Ph2SiO)8[Al(O)OH]4·3H3C-C(O)OC2H5 (2), the 1,6-hexane diol adduct (Ph2SiO)8[Al(O)OH]4·2HO–CH2(CH2)4CH2–OH (3) and the 1,4-cyclohexane diol adduct (Ph2SiO)8[Al(O)OH]4·4HO–CH(CH2CH2)2CH–OH (4). In all compounds the OH groups of the starting material bind to the bases through O–H···N (1) or O–H···O hydrogen bonds (2, 3, 4) as found from single-crystal X-ray diffraction analyses. Whereas in 1 only three of the central OH groups bind to the pyrazines, in 2 two of them bind to the same carbonyl oxygen atom of the ethyl acetate resulting in an unprecedented O–H···O···H–O double hydrogen bridge. The hexane diol adduct 3 in the crystal forms a one-dimensional coordination polymer with an intramolecularly to two OH groups grafted hexane diol loop, while the second hexane diol is connecting intermolecularly. In the cyclohexane diol adduct 4 all OH groups of the central Al4(OH)4 ring bind to different diols, leaving one alcohol group per diol uncoordinated. These “free” OH groups form an (O-H···)4 assembly creating a three-dimensional overall structure. When reacting with (Ph2SiO)8[Al(O)OH]4 lysine loses water, turns into the cyclic 3-amino-2-azepanone, and transforms through chelation of one of the aluminum atoms the starting material into a new polycycle. The isolated compound has the composition (Ph2SiO)12[Al(O)OH]4[Al2O3]2·4 C6H12N2O·6(CH2)4O (5).

The response of cultured cells to the mechanical properties of hydrogel substrates depends ultimately on the response of single crosslinks to external forces exerted at cell attachment points. We prepared hydrogels by co-polymerization of poly(ethylene glycol diacrylate) (PEGDA) and carboxy poly(ethylene glycol) acrylate (ACPEG-COOH) and confirmed fibroblast spreading on the hydrogel after the ACPEG linker was functionalized with the RGD cell adhesive motif. We performed specific force spectroscopy experiments on the same ACPEG linkers in order to probe the mechanics of single cross-links which mediate the cell attachment and spreading. Measurements were performed with tips of an atomic force microscope (AFM) functionalized with streptavidin and ACPEG linkers functionalized with biotin. We compared hydrogels of varying elastic modulus between 4 and 41 kPa which exhibited significant differences in cell spreading. An effective spring constant for the displacement of single cross-links at the hydrogel surface was derived from the distributions of rupture force and molecular stiffness. A factor of ten in the elastic modulus E of the hydrogel corresponded to a factor of five in the effective spring constant k of single crosslinks, indicating a transition in scaling with the mesh size ξ from the macroscopic E ∝ ξ−3 to the molecular k ∝ ξ−2. The quantification of stiffness and deformation at the molecular length scale contributes to the discussion of mechanisms in force-regulated phenomena in cell biology.

Abstract A substrate mimicking the surface topography and temperature sensitivity of skin goosebumps is fabricated. Close-packed arrays of thermoresponsive microgel particles undergo topographical changes in response to temperature changes between 25 and 37 °C, resembling the goosebump structure that human skin develops in response to temperature changes or other circumstances. Specifically, positively charged poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) brushes serve as an anchoring substrate for negatively charged poly(NIPAm-co-AA) microgels. The packing density and particle morphology can be tuned by brush layer thickness and pH of the microgel suspension. For brush layer thickness below 50 nm, particle monolayers are observed, with slightly flattened particle morphology at pH 3 and highly collapsed particles at pH above 7. Polymer brush films with thickness above 50 nm lead to the formation of particle multilayers. The temperature responsiveness of the monolayer assemblies allows reversible changes in the film morphology, which in turn affects underwater adhesion and friction at 25 and 37 °C. These results are promising for the design of new functional materials and may also serve as a model for biological structures and processes.

Abstract Transition metal dichalcogenides are attractive two-dimensional electrode materials for electrochemical energy storage devices due to their high reversible charge storage capacity. Hybridization of these materials with carbon promises enhanced performance by facilitating the access to reactive sites and extended mechanical stabilization. Herein, we introduce a NbS2/C hybrid material exhibiting a gyroidal microstructure synthesized through macromolecular co-assembly of a tailored block copolymer and an organometallic niobium precursor and subsequent sulfidation. Our synthesis allows the preparation of mechanically stable monoliths with NbS2 nanocrystals engulfed in a highly porous carbon shell. Due to the curvature of the gyroidal structure, abundant reactive sites are exposed that lead to an attractive performance in a lithium-containing electrolyte with a capacity of up to 400 mAh/g.