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- ItemOne-Pot Synthesis of Copper Iodide-Polypyrrole Nanocomposites(Basel : MDPI, 2021) Konakov, Artem O.; Dremova, Nadejda N.; Khodos, Igor I.; Koch, Marcus; Zolotukhina, Ekaterina V.; Silina, YuliyaA novel one-pot chemical synthesis of functional copper iodide-polypyrrole composites, CuI-PPy, has been proposed. The fabrication process allows the formation of nanodimensional metal salt/polymer hybrid structures in a fully controlled time- and concentration-dependent manner. The impact of certain experimental conditions, viz., duration of synthesis, sequence of component addition and concentrations of the intact reagents on the structure, dimensionality and yield of the end-product was evaluated in detail. More specifically, the amount of marshite CuI within the hybrid composite can be ranged from 60 to 90 wt.%, depending on synthetic conditions (type and concentration of components, process duration). In addition, the conditions allowing the synthesis of nano-sized CuI distributed inside the polypyrrole matrix were found. A high morphological stability and reproducibility of the synthesized nanodimensional metal-polymer hybrid materials were approved. Finally, the electrochemical activity of the formed composites was verified by cyclic voltammetry studies. The stability of CuI-PPy composite deposited on the electrodes was strongly affected by the applied anodic limit. The proposed one-pot synthesis of the hybrid nanodimensional copper iodide-polypyrrole composites is highly innovative, meets the requirements of Green Chemistry and is potentially useful for future biosensor development. In addition, this study is expected to generally contribute to the knowledge on the hybrid nano-based composites with tailored properties.
- ItemMyxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections(Basel : MDPI, 2020) Goes, Adriely; Lapuhs, Philipp; Kuhn, Thomas; Schulz, Eilien; Richter, Robert; Panter, Fabian; Dahlem, Charlotte; Koch, Marcus; Garcia, Ronald; Kiemer, Alexandra K.; Müller, Rolf; Fuhrmann, GregorIn 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
- ItemQuantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy(Basel : MDPI, 2021) Peckys, Diana B.; Gaa, Daniel; de Jonge, NielsCurrently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.
- ItemIntegrating Biophysics in Toxicology(Basel : MDPI, 2020) Del Favero, G.; Kraegeloh, A.Integration of biophysical stimulation in test systems is established in diverse branches of biomedical sciences including toxicology. This is largely motivated by the need to create novel experimental setups capable of reproducing more closely in vivo physiological conditions. Indeed, we face the need to increase predictive power and experimental output, albeit reducing the use of animals in toxicity testing. In vivo, mechanical stimulation is essential for cellular homeostasis. In vitro, diverse strategies can be used to model this crucial component. The compliance of the extracellular matrix can be tuned by modifying the stiffness or through the deformation of substrates hosting the cells via static or dynamic strain. Moreover, cells can be cultivated under shear stress deriving from the movement of the extracellular fluids. In turn, introduction of physical cues in the cell culture environment modulates differentiation, functional properties, and metabolic competence, thus influencing cellular capability to cope with toxic insults. This review summarizes the state of the art of integration of biophysical stimuli in model systems for toxicity testing, discusses future challenges, and provides perspectives for the further advancement of in vitro cytotoxicity studies.
- ItemStructural and Chemical Hierarchy in Hydroxyapatite Coatings(Basel : MDPI, 2020) Gross, Karlis A.; Petzold, Christiane; Pluduma-LaFarge, Liene; Kumermanis, Maris; HAugen, Håvard J.Hydroxyapatite coatings need similarly shaped splats as building blocks and then a homogeneous microstructure to unravel the structural and chemical hierarchy for more refined improvements to implant surfaces. Coatings were thermally sprayed with differently sized powders (20–40, 40–63 and 63–80 µm) to produce flattened homogeneous splats. The surface was characterized for splat shape by profilometry and Atomic force microscopy (AFM), crystal size by AFM, crystal orientation by X-ray diffraction (XRD) and structural variations by XRD. Chemical composition was assessed by phase analysis, but variations in chemistry were detected by XRD and Raman spectroscopy. The resulting surface electrical potential was measured by Kelvin probe AFM. Five levels of structural hierarchy were suggested: the coating, the splat, oriented crystals, alternate layers of oxyapatite and hydroxyapatite (HAp) and the suggested anion orientation. Chemical hierarchy was present over a lower range of order for smaller splats. Coatings made from smaller splats exhibited a greater electrical potential, inferred to arise from oxyapatite, and supplemented by ordered OH− ions in a rehydroxylated surface layer. A model has been proposed to show the influence of structural hierarchy on the electrical surface potential. Structural hierarchy is proposed as a means to further refine the properties of implant surfaces.
- ItemTofacitinib Loaded Squalenyl Nanoparticles for Targeted Follicular Delivery in Inflammatory Skin Diseases(Basel : MDPI, 2020) Christmann, Rebekka; Ho, Duy-Khiet; Wilzopolski, Jenny; Lee, Sangeun; Koch, Marcus; Loretz, Brigitta; Vogt, Thomas; Bäumer, Wolfgang; Schaefer, Ulrich F.; Lehr, Claus-MichaelTofacitinib (TFB), a Janus kinase inhibitor, has shown excellent success off-label in treating various dermatological diseases, especially alopecia areata (AA). However, TFB’s safe and targeted delivery into hair follicles (HFs) is highly desirable due to its systemic adverse effects. Nanoparticles (NPs) can enhance targeted follicular drug delivery and minimize interfollicular permeation and thereby reduce systemic drug exposure. In this study, we report a facile method to assemble the stable and uniform 240 nm TFB loaded squalenyl derivative (SqD) nanoparticles (TFB SqD NPs) in aqueous solution, which allowed an excellent loading capacity (LC) of 20%. The SqD NPs showed an enhanced TFB delivery into HFs compared to the aqueous formulations of plain drug in an ex vivo pig ear model. Furthermore, the therapeutic efficacy of the TFB SqD NPs was studied in a mouse model of allergic dermatitis by ear swelling reduction and compared to TFB dissolved in a non-aqueous mixture of acetone and DMSO (7:1 v/v). Whereas such formulation would not be acceptable for use in the clinic, the TFB SqD NPs dispersed in water illustrated a better reduction in inflammatory effects than plain TFB’s aqueous formulation, implying both encouraging good in vivo efficacy and safety. These findings support the potential of TFB SqD NPs for developing a long-term topical therapy of AA.
- ItemLipid–Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media(Basel : MDPI, 2022) Kliesch, Lena; Delandre, Simon; Gabelmann, Aljoscha; Koch, Marcus; Schulze, Kai; Guzmán, Carlos A.; Loretz, Brigitta; Lehr, Claus-MichaelTo combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid–polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20–40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)–LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles’ performance in supplemented media.
- ItemCylindrical Microparticles Composed of Mesoporous Silica Nanoparticles for the Targeted Delivery of a Small Molecule and a Macromolecular Drug to the Lungs: Exemplified with Curcumin and siRNA(Basel : MDPI, 2021) Fischer, Thorben; Winter, Inga; Drumm, Robert; Schneider, MarcThe transport of macromolecular drugs such as oligonucleotides into the lungs has become increasingly relevant in recent years due to their high potency. However, the chemical structure of this group of drugs poses a hurdle to their delivery, caused by the negative charge, membrane impermeability and instability. For example, siRNA to reduce tumour necrosis factor alpha (TNF-α) secretion to reduce inflammatory signals has been successfully delivered by inhalation. In order to increase the effect of the treatment, a co-transport of another anti-inflammatory ingredient was applied. Combining curcumin-loaded mesoporous silica nanoparticles in nanostructured cylindrical microparticles stabilized by the layer-by-layer technique using polyanionic siRNA against TNF-α was used for demonstration. This system showed aerodynamic properties suited for lung deposition (mass median aerodynamic diameter of 2.85 ± 0.44 µm). Furthermore, these inhalable carriers showed no acute in vitro toxicity tested in both alveolar epithelial cells and macrophages up to 48 h incubation. Ultimately, TNF-α release was significantly reduced by the particles, showing an improved activity co-delivering both drugs using such a drug-delivery system for specific inhibition of TNF-α in the lungs.
- ItemEGFR Expression in HER2-Driven Breast Cancer Cells(Basel : MDPI, 2020) Weinberg, Florian; Peckys, Diana B.; de Jonge, NielsThe epidermal growth factor receptor HER2 is overexpressed in 20% of breast cancer cases. HER2 is an orphan receptor that is activated ligand-independently by homodimerization. In addition, HER2 is able to heterodimerize with EGFR, HER3, and HER4. Heterodimerization has been proposed as a mechanism of resistance to therapy for HER2 overexpressing breast cancer. Here, a method is presented for the simultaneous detection of individual EGFR and HER2 receptors in the plasma membrane of breast cancer cells via specific labeling with quantum dot nanoparticles (QDs). Correlative fluorescence microscopy and liquid phase electron microscopy were used to analyze the plasma membrane expression levels of both receptors in individual intact cells. Fluorescent single-cell analysis of SKBR3 breast cancer cells dual-labeled for EGFR and HER2 revealed a heterogeneous expression for receptors within both the cell population as well as within individual cells. Subsequent electron microscopy of individual cells allowed the determination of individual receptors label distributions. QD-labeled EGFR was observed with a surface density of (0.5–5) × 101 QDs/µm2, whereas labeled HER2 expression was higher ranging from (2–10) × 102 QDs/µm2. Although most SKBR3 cells expressed low levels of EGFR, an enrichment was observed at large plasma membrane protrusions, and amongst a newly discovered cellular subpopulation termed EGFR-enriched cells.
- ItemEmerging Biofabrication Techniques : A Review on Natural Polymers for Biomedical Applications(Basel : MDPI, 2021) Puertas-Bartolomé, María; Mora-Boza, Ana; García-Fernández, LuisNatural polymers have been widely used for biomedical applications in recent decades. They offer the advantages of resembling the extracellular matrix of native tissues and retaining biochemical cues and properties necessary to enhance their biocompatibility, so they usually improve the cellular attachment and behavior and avoid immunological reactions. Moreover, they offer a rapid degradability through natural enzymatic or chemical processes. However, natural polymers present poor mechanical strength, which frequently makes the manipulation processes difficult. Recent advances in biofabrication, 3D printing, microfluidics, and cell-electrospinning allow the manufacturing of complex natural polymer matrixes with biophysical and structural properties similar to those of the extracellular matrix. In addition, these techniques offer the possibility of incorporating different cell lines into the fabrication process, a revolutionary strategy broadly explored in recent years to produce cell-laden scaffolds that can better mimic the properties of functional tissues. In this review, the use of 3D printing, microfluidics, and electrospinning approaches has been extensively investigated for the biofabrication of naturally derived polymer scaffolds with encapsulated cells intended for biomedical applications (e.g., cell therapies, bone and dental grafts, cardiovascular or musculoskeletal tissue regeneration, and wound healing).