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- ItemDefect-free Naphthalene Diimide Bithiophene Copolymers with Controlled Molar Mass and High Performance via Direct Arylation Polycondensation(Washington, DC : ACS Publications, 2015) Matsidik, Rukiya; Komber, Hartmut; Luzio, Alessandro; Caironi, Mario; Sommer, MichaelA highly efficient, simple, and environmentally friendly protocol for the synthesis of an alternating naphthalene diimide bithiophene copolymer (PNDIT2) via direct arylation polycondensation (DAP) is presented. High molecular weight (MW) PNDIT2 can be obtained in quantitative yield using aromatic solvents. Most critical is the suppression of two major termination reactions of NDIBr end groups: nucleophilic substitution and solvent end-capping by aromatic solvents via C–H activation. In situ solvent end-capping can be used to control MW by varying monomer concentration, whereby end-capping is efficient and MW is low for low concentration and vice versa. Reducing C–H reactivity of the solvent at optimized conditions further increases MW. Chain perfection of PNDIT2 is demonstrated in detail by NMR spectroscopy, which reveals PNDIT2 chains to be fully linear and alternating. This is further confirmed by investigating the optical and thermal properties as a function of MW, which saturate at Mn ≈ 20 kDa, in agreement with controls made by Stille coupling. Field-effect transistor (FET) electron mobilities μsat up to 3 cm2/(V·s) are measured using off-center spin-coating, with FET devices made from DAP PNDIT2 exhibiting better reproducibility compared to Stille controls.
- ItemMonoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)(San Francisco, California, US : PLOS, 2016) Thamm, Kristina; Graupner, Sylvi; Werner, Carsten; Huttner, Wieland B.; Corbeil, Denis; Nabi, Ivan RThe pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.
- ItemInteraction between immobilized polyelectrolyte complex nanoparticles and human mesenchymal stromal cells(Auckland : DOVE Medical Press, 2014) Woltmann, B.; Torger, B.; Müller, M.; Hempel, U.Background: Implant loosening or deficient osseointegration is a major problem in patients with systemic bone diseases (eg, osteoporosis). For this reason, the stimulation of the regional cell population by local and sustained drug delivery at the bone/implant interface to induce the formation of a mechanical stable bone is promising. The purpose of this study was to investigate the interaction of polymer-based nanoparticles with human bone marrow-derived cells, considering nanoparticles' composition and surface net charge. Materials and methods: Polyelectrolyte complex nanoparticles (PECNPs) composed of the polycations poly(ethyleneimine) (PEI), poly(L-lysine) (PLL), or (N,N-diethylamino)ethyldextran (DEAE) in combination with the polyanions dextran sulfate (DS) or cellulose sulfate (CS) were prepared. PECNPs' physicochemical properties (size, net charge) were characterized by dynamic light scattering and particle charge detector measurements. Biocompatibility was investigated using human mesenchymal stromal cells (hMSCs) cultured on immobilized PECNP films (5-50 nmol·cm-2) by analysis for metabolic activity of hMSCs in dependence of PECNP surface concentration by MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay, as well as cell morphology (phase contrast microscopy). Results: PECNPs ranging between ~50 nm and 150 nm were prepared. By varying the ratio of polycations and polyanions, PECNPs with a slightly positive (PEC+NP) or negative (PEC-NP) net charge were obtained. The PECNP composition significantly affected cell morphology and metabolic activity, whereas the net charge had a negligible influence. Therefore, we classified PECNPs into "variant systems" featuring a significant dose dependency of metabolic activity (DEAE/CS, PEI/DS) and "invariant systems" lacking such a dependency (DEAE/DS, PEI/CS). Immunofluorescence imaging of fluorescein isothiocyanate isomer I (FITC)-labeled PECNPs suggested internalization into hMSCs remaining stable for 8 days. Conclusion: Our study demonstrated that PECNP composition affects hMSC behavior. In particular, the PEI/CS system showed biocompatibility in a wide concentration range, representing a suitable system for local drug delivery from PECNP-functionalized bone substitute materials.
- ItemApplication of new lysine-based peptide dendrimers D3K2 and D3G2 for gene delivery: Specific cytotoxicity to cancer cells and transfection in vitro(San Diego, Calif. : Elsevier, 2020) Gorzkiewicz, Michal; Konopka, Malgorzata; Janaszewska, Anna; Tarasenko, Irina I.; Sheveleva, Nadezhda N.; Gajek, Arkadiusz; Neelov, Igor M.; Klajnert-Maculewicz, BarbaraIn order to enhance intracellular uptake and accumulation of therapeutic nucleic acids for improved gene therapy methods, numerous delivery vectors have been elaborated. Based on their origin, gene carriers are generally classified as viral or non-viral vectors. Due to their significantly reduced immunogenicity and highly optimized methods of synthesis, nanoparticles (especially those imitating natural biomolecules) constitute a promising alternative for virus-based delivery devices. Thus, we set out to develop innovative peptide dendrimers for clinical application as transfection agents and gene carriers. In the present work we describe the synthesis of two novel lysine-based dendritic macromolecules (D3K2 and D3G2) and their initial characterization for cytotoxicity/genotoxicity and transfection potential in two human cell line models: cervix adenocarcinoma (HeLa) and microvascular endothelial (HMEC-1). This approach allowed us to identify more cationic D3K2 as potent delivery agent, being able to increase intracellular accumulation of large nucleic acid molecules such as plasmids. Moreover, the dendrimers exhibited specific cytotoxicity towards cancer cell line without showing significant toxic effects on normal cells. These observations are promising prognosis for future clinical application of this type of nanoparticles. © 2019 The Authors