Памяти Александра Евгеньевича Будникова

In photodynamic therapy (PDT), photosensitizers and light are used to cause photochemically induced cell death. The selectivity and the effectiveness of the phototoxicity in cancer can be increased by a specific uptake of the photosensitizer into tumor cells. A promising target for this goal is the folic acid receptor α (FRα), which is overexpressed on the surface of many tumor cells and mediates an endocytotic uptake. Here, we describe a polysaccharide-based nanoparticle system suitable for targeted uptake and its photochemical and photobiological characterization. The photosensitizer 5, 10, 15, 20-tetraphenyl-21H, 23H-porphyrine (TPP) was encapsulated in spermine- and acetal-modified dextran (SpAcDex) nanoparticles and conjugated with folic acid (FA) on the surface [SpAcDex(TPP)-FA]. The particles are successfully taken up by human HeLa-KB cells, and a light-induced cytotoxicity is observable. An excess of free folate as the competitor for the FRα-mediated uptake inhibits the phototoxicity. In conclusion, folate-modified SpAcDex particles are a promising drug delivery system for a tumor cell targeted photodynamic therapy

Dynamic Polysaccharide-based Nanoparticles for Advanced Drug Delivery Applications

Nanoparticles gained great importance in the medical field since their first discovery 50 years ago. Based on the ability to select from a repertoire of materials, shapes, and sizes, nanocarrier can be designed to deliver a wide range of high potent drugs. They can protect and significantly improve the bioavailability of sensitive and pharmaceutically active cargo, that otherwise would not be applicable in biological systems and thereby enabling a broad field of medical applications. This thesis presents the further development of a biocompatible and biodegradable acid-sensitive polysaccharide-based nanocarrier system for the simultaneous delivery of hydrophilic and hydrophobic drugs. L-Asparaginase and etoposide were dual encapsulated in dextran-based nanoparticles by a double emulsion technique. Studies revealed a controlled pH-sensitive release of both drugs and a high synergistic toxicity in K562 cells in vitro. For immunotherapy applications, a biocompatible and targeted transport of active agents is required to enhance the therapeutic effect and limit the undesired side effects. Therefore, the dextran-based nanoparticle system was further advanced in the second project. The introduction of a hydrophilic polyethylene glycol (PEG) layer on the surface of these particles prevents unspecific cellular uptake and prolongs circulation time. Furthermore, specific antibodies as active targeting ligands for dendritic cells were attached on PEG. In vitro and in vivo studies showed that the modified particles were preferred taken up by receptor-mediated endocytosis in dendritic cells, which enables a potential anti-tumor CD8+ cytotoxic T-lymphocytes activation. In addition, based on the pH-responsive modified dextran, an advanced biocompatible horseradish peroxidase-polysaccharide conjugate was developed through formation of a disulfide bridge. The obtained double stimuli-responsive biodegradable hybrid material self assembles in water into spherical nanoparticles. During this process the structure integrity and enzymatic activity of the enzyme conjugate was retained. Nanoparticle degradation behavior was studied in detail with a focus on triggered drug released. The highly potent, indole-3 acetic acid (IAA) prodrug was encapsulated and successfully delivered into the cytosol of HeLa cells. Hereby the released prodrug was oxidized by the horseradish peroxidase particle material, which leads to cellular apoptosis. Both developed dextran-based particle systems represent promising candidates for successful applications in the delivery of therapeutics.Seit ihrer ersten Entdeckung vor 50 Jahren haben Nanopartikel eine große Bedeutung in der Medizin erlangt. Basierend auf der Möglichkeit, aus einem Repertoire an verschiedenen Materialien, Formen und Größen auszuwählen, können Nanocarrier so konzipiert werden, dass sie eine Vielzahl an hochwirksamen Medikamenten transportieren können. Sie können die Bioverfügbarkeit von empfindlichen, pharmazeutisch aktiven Wirkstoffen erhöhen, wodurch ein breites Feld von medizinischen Anwendungen ermöglicht wird. Diese Arbeit stellt die Weiterentwicklung eines biokompatiblen, bioabbaubaren und säureempfindlichen Zucker-basierten Nanopartikels für die gleichzeitige Abgabe von hydrophilen und hydrophoben Medikamenten dar. L-Asparaginase und Etoposid wurden in Dextran-basierten Nanopartikeln mittels einer Doppelemulsionstechnik zusammen verkapselt. Studien zeigten eine kontrollierte pH-sensitive Freisetzung beider Medikamente und eine hohe synergistische Toxizität in K562-Zellen in vitro. Für die Immuntherapie ist ein biokompatibler und gezielter Wirkstofftransport notwendig, um unerwünschte Nebenwirkungen zu reduzieren und die therapeutische Effizienz zu verstärken. Das auf Dextran basierende Nanopartikelsystem wurde daher im zweiten Projekt weiterentwickelt. Die Einführung einer hydrophilen PEG-schicht auf der Oberfläche der Partikel verhindert eine unspezifische Zellaufnahme und verlängert die Zirkulationszeit Darüber hinaus wurden spezifische Antikörper als aktive Zielliganden für dendritische Zellen an PEG gebunden. In vitro- und in vivo-Studien zeigten, dass die modifizierten Partikel bevorzugt durch rezeptorvermittelte Endozytose in dendritische Zellen aufgenommen wurden. Dies ermöglicht eine potenzielle Aktivierung der gegen Tumor wirksamen, zytotoxischen CD8+ T-Lymphozyten. Darüber hinaus wurde auf Basis des pH-responsiven modifizierten Dextrans ein neuartiges, biokompatibles Meerrettich Peroxidase-Polysaccharid-Konjugat entwickelt mittels Bildung einer Disulfidbrücke. Das erhaltene doppelt Stimulus-responsive, biologisch abbaubares Hybridmaterial ordnet sich selbst in Wasser zu kugelförmigen Nanopartikeln zusammen. Hierbei konnten die Strukturintegrität und enzymatische Aktivität des Enzym-Konjugats erhalten bleiben. Nanopartikuläres Abbauverhalten wurde detailliert untersucht, wobei der Schwerpunkt auf einer gesteuerten Medikamentenfreisetzung lag. Das hochwirksame Prodrug Indol-3-Essigsäure wurde verkapselt und erfolgreich in das Zytosol von HeLa-Zellen eingeschleust, wo das freigesetzte Prodrug durch das Meerrettich Peroxidase Partikelmaterial oxidiert wurde und schließlich zur zellulären Apoptose geführt hat. Beide entwickelten Dextran-basierten Partikelsysteme sind vielversprechende Kandidaten für erfolgreiche Anwendungen bei der Verabreichung von Therapeutika

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A

Staphylococcus aureus is one of the most frequent causes of nosocomial and community‐acquired infections, with drug‐resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active‐site cysteine. A broad series of derivatives were synthesized to derive structure‐activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single‐digit micromolar Ki values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase‐mediated adherence of S. aureus cells

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus

Staphylococcus aureus is one of the most frequent causes of nosocomial and community‐acquired infections, with drug‐resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active‐site cysteine. A broad series of derivatives were synthesized to derive structure‐activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single‐digit micromolar Ki values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase‐mediated adherence of S. aureus cells