10 results on '"Inka Negwer"'
Search Results
2. Overcoming the barrier of CD8+ T cells: Two types of nano-sized carriers for siRNA transport
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Ilja Tabujew, Kaloian Koynov, Nadine Leber, Mark Helm, Christoph Freidel, Inka Negwer, Kalina Peneva, Katharina Landfester, Marleen Willig, Volker Mailänder, and Rudolf Zentel
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Small interfering RNA ,Chemistry ,media_common.quotation_subject ,Genetic enhancement ,0206 medical engineering ,Cell ,Biomedical Engineering ,02 engineering and technology ,General Medicine ,Transfection ,021001 nanoscience & nanotechnology ,020601 biomedical engineering ,Biochemistry ,Biomaterials ,Immune system ,medicine.anatomical_structure ,medicine ,Biophysics ,Gene silencing ,0210 nano-technology ,Internalization ,Cytotoxicity ,Molecular Biology ,Biotechnology ,media_common - Abstract
Bioengineering immune cells via gene therapy offers treatment opportunities for currently fatal viral infections. Also cell therapeutics offer most recently a breakthrough technology to combat cancer. These primary human cells, however, are sensitive to toxic influences, which make the utilization of optimized physical transfection techniques necessary. The otherwise commonly applied delivery agents such as LipofectamineⓇ or strongly cationic polymer structures are not only unsuitable for in vivo experiments, but are also highly toxic to immune cells. This study aimed to improve the design of polymeric carrier systems for small interfering RNA, which would allow efficient internalization into CD8+ T-cells without affecting their viability and thereby removing the current limitations in the field. Here, two new carrier systems for small interfering RNA were tested. One is a cationic diblock copolymer, in which less than 10% of the monomers were modified with triphenylphosphonium cations. This moiety is lipophilic, promotes uptake and it is mostly known for its mitotropic properties. Furthermore, cationic nanohydrogel particles were synthesized in exceedingly small sizes (Rh Statement of significance This study provides insights into the design of polymeric delivery agents as the method of choice for overcoming the limitations of cell manipulation. Until now, CD8+ T-cells, which have become a treatment tool for currently fatal diseases, have not yet been made accessible for gene silencing by polymeric siRNA carrier systems. Choosing appropriate modification approaches for two chemically different polymer structures, we were, in both cases, able to achieve significant uptake in these cells even at low concentrations and without inducing cytotoxicity. These results remove current limitations and pave the way for bioengineering via gene therapy.
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- 2019
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3. The Myb-related protein MYPOP is a novel intrinsic host restriction factor of oncogenic human papillomaviruses
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Fatima Boukhallouk, Luise Florin, Krishnaraj Rajalingam, Frank Stubenrauch, Elena Wüstenhagen, and Inka Negwer
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0301 basic medicine ,Intrinsic Factor ,Keratinocytes ,Cancer Research ,Cellular immunity ,Papillomavirus E7 Proteins ,Antiviral protein ,Uterine Cervical Neoplasms ,Brief Communication ,03 medical and health sciences ,Proto-Oncogene Proteins c-myb ,0302 clinical medicine ,Cell Line, Tumor ,Genetics ,Humans ,MYB ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Human papillomavirus 16 ,Intrinsic factor ,biology ,Human papillomavirus 18 ,Papillomavirus Infections ,Oncogene Proteins, Viral ,Cell biology ,030104 developmental biology ,Profilin ,030220 oncology & carcinogenesis ,DNA, Viral ,Host-Pathogen Interactions ,biology.protein ,Capsid Proteins ,Female ,Transcription Factors - Abstract
The skin represents a physical and chemical barrier against invading pathogens, which is additionally supported by restriction factors that provide intrinsic cellular immunity. These factors detect viruses to block their replication cycle. Here, we uncover the Myb-related transcription factor, partner of profilin (MYPOP) as a novel antiviral protein. It is highly expressed in the epithelium and binds to the minor capsid protein L2 and the DNA of human papillomaviruses (HPV), which are the primary causative agents of cervical cancer and other tumors. The early promoter activity and early gene expression of the oncogenic HPV types 16 and 18 is potently silenced by MYPOP. Cellular MYPOP-depletion relieves the restriction of HPV16 infection, demonstrating that MYPOP acts as a restriction factor. Interestingly, we found that MYPOP protein levels are significantly reduced in diverse HPV-transformed cell lines and in HPV-induced cervical cancer. Decades ago it became clear that the early oncoproteins E6 and E7 cooperate to immortalize keratinocytes by promoting degradation of tumor suppressor proteins. Our findings suggest that E7 stimulates MYPOP degradation. Moreover, overexpression of MYPOP blocks colony formation of HPV and non-virally transformed keratinocytes, suggesting that MYPOP exhibits tumor suppressor properties.
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- 2018
4. Orthogonal Click Conjugation to the Liposomal Surface Reveals the Stability of the Lipid Anchorage as Crucial for Targeting
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Thomas Fritz, Matthias Worm, Mark Helm, Sophie S. Müller, Holger Frey, Frank Roesch, Tobias L. Ross, Inka Negwer, Matthias Voigt, Kaloian Koynov, and Kathrin Kettenbach
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Glycerol ,Polymers ,Stereochemistry ,Alkyne ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Catalysis ,Folic Acid ,Amphiphile ,Humans ,chemistry.chemical_classification ,Liposome ,Organic Chemistry ,General Chemistry ,Receptor-mediated endocytosis ,021001 nanoscience & nanotechnology ,Lipids ,0104 chemical sciences ,Cholesterol ,Membrane ,Folic acid ,chemistry ,Liposomes ,Drug delivery ,Click chemistry ,Biophysics ,Click Chemistry ,0210 nano-technology - Abstract
Synthetic access to multiple surface decorations are a bottleneck in the development of liposomes for receptor mediated targeting. This opens a complex multiparameter space, exploration of which is severely limited in terms of sample numbers and turnaround times. Here, we unlock this technological barrier by a combination of a milligram-scale liposome formulation using dual centrifugation and orthogonal click chemistry on the liposomal surface. Application of these techniques to conceptually new amphiphilic compounds, which feature norbornene and alkyne groups at the apex of sterically stabilizing, hyperbranched polyglycerol moieties, revealed a particular influence of the membrane anchor of functional amphiphiles. Folic acid residues clicked to cholesterol-based amphiphiles were inefficient in folate-mediated cell targeting, while dialkyl-anchored amphiphiles remained stable in the liposomal membrane and imparted efficient targeting properties. These findings are of specific importance considering the popularity of cholesterol as a lipophilic anchor.
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- 2016
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5. Monitoring drug nanocarriers in human blood by near-infrared fluorescence correlation spectroscopy
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Kaloian Koynov, Leon Capelôa, Manfred Schmidt, Volker Mailänder, Matthias Barz, Meike Schinnerer, Hans-Jürgen Butt, Inka Negwer, Mark Helm, Olga Schäfer, Andreas Best, and Manfred Wagner
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Drug ,Science ,media_common.quotation_subject ,General Physics and Astronomy ,Fluorescence correlation spectroscopy ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Article ,General Biochemistry, Genetics and Molecular Biology ,In vivo ,Humans ,lcsh:Science ,Fluorescent Dyes ,media_common ,Drug Carriers ,Spectroscopy, Near-Infrared ,Multidisciplinary ,Human blood ,Chemistry ,General Chemistry ,021001 nanoscience & nanotechnology ,Fluorescence ,0104 chemical sciences ,Spectrometry, Fluorescence ,Drug delivery ,Biophysics ,Nanoparticles ,lcsh:Q ,Nanocarriers ,0210 nano-technology ,Two-dimensional nuclear magnetic resonance spectroscopy - Abstract
Nanocarrier-based drug delivery is a promising therapeutic approach that offers unique possibilities for the treatment of various diseases. However, inside the blood stream, nanocarriers’ properties may change significantly due to interactions with proteins, aggregation, decomposition or premature loss of cargo. Thus, a method for precise, in situ characterization of drug nanocarriers in blood is needed. Here we show how the fluorescence correlation spectroscopy that is a well-established method for measuring the size, loading efficiency and stability of drug nanocarriers in aqueous solutions can be used to directly characterize drug nanocarriers in flowing blood. As the blood is not transparent for visible light and densely crowded with cells, we label the nanocarriers or their cargo with near-infrared fluorescent dyes and fit the experimental autocorrelation functions with an analytical model accounting for the presence of blood cells. The developed methodology contributes towards quantitative understanding of the in vivo behavior of nanocarrier-based therapeutics., While nanocarrier-based drug delivery is a promising therapeutic approach, in situ characterization of drug nanocarriers in blood remains difficult. Here, the authors demonstrate how the fluorescence correlation spectroscopy can be used to directly characterize drug nanocarriers in flowing blood.
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- 2018
6. Histidine-rich glycoprotein-induced vascular normalization improves EPR-mediated drug targeting to and into tumors
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Maike Baues, Willi Jahnen-Dechent, Michal Pechar, Fabian Kiessling, Robert Pola, Matthias Barz, Gert Storm, Twan Lammers, Inka Negwer, Benjamin Weber, Kaloian Koynov, Benjamin Theek, Felix Gremse, Afd Pharmaceutics, Pharmaceutics, and Biomaterials Science and Technology
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Histidine-rich glycoprotein ,UT-Hybrid-D ,Pharmaceutical Science ,Vascular normalization ,02 engineering and technology ,Permeability ,Article ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Drug Delivery Systems ,0302 clinical medicine ,Polymethacrylic Acids ,Cell Line, Tumor ,Neoplasms ,medicine ,Animals ,Methacrylamide ,Tissue Distribution ,pHPMA ,Fibrosarcoma ,Mice, Knockout ,chemistry.chemical_classification ,Drug Carriers ,Proteins ,021001 nanoscience & nanotechnology ,medicine.disease ,Pathophysiology ,Up-Regulation ,Mice, Inbred C57BL ,HRG ,Nanomedicine ,Tumor targeting ,chemistry ,Targeted drug delivery ,Permeability (electromagnetism) ,030220 oncology & carcinogenesis ,Drug delivery ,Cancer research ,EPR ,0210 nano-technology ,Glycoprotein - Abstract
Tumors are characterized by leaky blood vessels, and by an abnormal and heterogeneous vascular network. These pathophysiological characteristics contribute to the enhanced permeability and retention (EPR) effect, which is one of the key rationales for developing tumor-targeted drug delivery systems. Vessel abnormality and heterogeneity, however, which typically result from excessive pro-angiogenic signaling, can also hinder efficient drug delivery to and into tumors. Using histidine-rich glycoprotein (HRG) knockout and wild type mice, and HRG-overexpressing and normal t241 fibrosarcoma cells, we evaluated the effect of genetically induced and macrophage-mediated vascular normalization on the tumor accumulation and penetration of ~10 nm-sized polymeric drug carriers based on poly(N-(2-hydroxypropyl) methacrylamide). Multimodal and multiscale optical imaging was employed to show that normalizing the tumor vasculature improves the accumulation of fluorophore-labeled polymers in tumors, and promotes their penetration out of the blood vessels deep into the tumor interstitium.
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- 2018
7. A modular approach for multifunctional polymersomes with controlled adhesive properties
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Kaloian Koynov, Julien Petit, Jennifer Schultze, Marcin Makowski, Oliver Bäumchen, Stephan Herminghaus, Katharina Landfester, Frederik R. Wurm, Laura Thomi, and Inka Negwer
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Chemistry ,Nanotechnology ,Context (language use) ,02 engineering and technology ,General Chemistry ,Adhesion ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,01 natural sciences ,0104 chemical sciences ,Synthetic biology ,Membrane ,Covalent bond ,Polymersome ,Molecule ,Surface modification ,0210 nano-technology - Abstract
The bottom-up approach in synthetic biology involves the engineering of synthetic cells by designing biological and chemical building blocks, which can be combined in order to mimic cellular functions. The first step for mimicking a living cell is the design of an appropriate compartment featuring a multifunctional membrane. This is of particular interest since it allows for the selective attachment of different groups or molecules to the membrane. In this context, we report on a modular approach for polymeric vesicles, so-called polymersomes, with a multifunctional surface, namely hydroxyl, alkyne and acrylate groups. We demonstrate that the surface of the polymersome can be functionalized to facilitate imaging, via fluorescent dyes, or to improve the specific adhesion to surfaces by using a biotin functionalization. This generally applicable multifunctionality allows for the covalent integration of various molecules in the membrane of a synthetic cell.
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- 2018
8. Overcoming the barrier of CD8
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Ilja, Tabujew, Marleen, Willig, Nadine, Leber, Christoph, Freidel, Inka, Negwer, Kaloian, Koynov, Mark, Helm, Katharina, Landfester, Rudolf, Zentel, Kalina, Peneva, and Volker, Mailänder
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Cytotoxicity, Immunologic ,Organophosphorus Compounds ,Polymers ,Cations ,Humans ,Nanoparticles ,Hydrogels ,CD8-Positive T-Lymphocytes ,Particle Size ,RNA, Small Interfering ,Endocytosis ,RNA Transport - Abstract
Bioengineering immune cells via gene therapy offers treatment opportunities for currently fatal viral infections. Also cell therapeutics offer most recently a breakthrough technology to combat cancer. These primary human cells, however, are sensitive to toxic influences, which make the utilization of optimized physical transfection techniques necessary. The otherwise commonly applied delivery agents such as Lipofectamine
- Published
- 2017
9. Modulation of mitochondriotropic properties of cyanine dyes by in organello copper-free click reaction
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Hans-Jürgen Butt, Kaloian Koynov, Markus Hirsch, Tom Brown, Inka Negwer, Kalina Peneva, Stefka Kaloyanova, and Mark Helm
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0301 basic medicine ,chemistry.chemical_element ,Biochemistry ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Confocal laser scanning microscopy ,Fluorescence Resonance Energy Transfer ,Organic chemistry ,Animals ,Cyanine ,Molecular Biology ,Fluorescent Dyes ,Microscopy, Confocal ,Organic Chemistry ,fungi ,Carbocyanines ,Copper ,Mitochondria ,Rats ,030104 developmental biology ,Förster resonance energy transfer ,chemistry ,Mitochondrial targeting ,Click chemistry ,Biophysics ,Molecular Medicine ,Click Chemistry ,Conjugate - Abstract
Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper-free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Forster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3-Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.
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- 2017
10. The Cytoskeletal Adaptor Obscurin-Like 1 Interacts with the Human Papillomavirus 16 (HPV16) Capsid Protein L2 and Is Required for HPV16 Endocytosis
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Elena Wüstenhagen, W. M. Kast, Inka Negwer, Gilles A. Spoden, Fatima Boukhallouk, Marc A. Schneider, Kaloian Koynov, Luise Florin, and L. Hampe
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0301 basic medicine ,Keratinocytes ,viruses ,Immunology ,Endocytic cycle ,Endocytosis ,Microbiology ,Clathrin ,Cell Line ,03 medical and health sciences ,Transduction (genetics) ,Tetraspanin ,Viral entry ,Virology ,Two-Hybrid System Techniques ,Caveolin ,Humans ,Human papillomavirus 16 ,biology ,Papillomavirus Infections ,Signal transducing adaptor protein ,Oncogene Proteins, Viral ,Virus Internalization ,Cell biology ,Virus-Cell Interactions ,Cytoskeletal Proteins ,030104 developmental biology ,Insect Science ,Gene Knockdown Techniques ,Host-Pathogen Interactions ,biology.protein ,Capsid Proteins ,HeLa Cells - Abstract
The human papillomavirus (HPV) capsid protein L2 is essential for viral entry. To gain a deeper understanding of the role of L2, we searched for novel cellular L2-interacting proteins. A yeast two-hybrid analysis uncovered the actin-depolymerizing factor gelsolin, the membrane glycoprotein dysadherin, the centrosomal protein 68 (Cep68), and the cytoskeletal adaptor protein obscurin-like 1 protein (OBSL1) as putative L2 binding molecules. Pseudovirus (PsV) infection assays identified OBSL1 as a host factor required for gene transduction by three oncogenic human papillomavirus types, HPV16, HPV18, and HPV31. In addition, we detected OBSL1 expression in cervical tissue sections and noted the involvement of OBSL1 during gene transduction of primary keratinocytes by HPV16 PsV. Complex formation of HPV16 L2 with OBSL1 was demonstrated in coimmunofluorescence and coimmunoprecipitation studies after overexpression of L2 or after PsV exposure. We observed a strong colocalization of OBSL1 with HPV16 PsV and tetraspanin CD151 at the plasma membrane, suggesting a role for OBSL1 in viral endocytosis. Indeed, viral entry assays exhibited a reduction of viral endocytosis in OBSL1-depleted cells. Our results suggest OBSL1 as a novel L2-interacting protein and endocytosis factor in HPV infection. IMPORTANCE Human papillomaviruses infect mucosal and cutaneous epithelia, and the high-risk HPV types account for 5% of cancer cases worldwide. As recently discovered, HPV entry occurs by a clathrin-, caveolin-, and dynamin-independent endocytosis via tetraspanin-enriched microdomains. At present, the cellular proteins involved in the underlying mechanism of this type of endocytosis are under investigation. In this study, the cytoskeletal adaptor OBSL1 was discovered as a previously unrecognized interaction partner of the minor capsid protein L2 and was identified as a proviral host factor required for HPV16 endocytosis into target cells. The findings of this study advance the understanding of a so far less well-characterized endocytic pathway that is used by oncogenic HPV subtypes.
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- 2016
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