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Die Oberflächenchemie ist mittlerweile in vielen chemischen, biochemischen und anderen Forschungsbereichen präsent und stellt eine interdisziplinäre Forschungsdisziplin dar. Diese Arbeit fokussierte sich auf die Interaktionen von biochemischen Systemen mit Oberflächen, insbesondere auf die Entwicklung von biochemischen Sensoren. Die Optimierung der Sensoroberfläche, insbesondere die Erhöhung der Selektivität durch die Verwendung von Polyglycerol (PG), wurde untersucht. Insgesamt wurden vier Teilprojekte durchgeführt, die sich auf die Unterdrückung unerwünschter Interaktionen, die Einführung von Bioerkennungsstellen, die Stabilisierung von PG-Filmen und die Entwicklung eines leitfähigen Hybridmaterials konzentrierten.
Teilprojekt 1: Stabilisierung von PG-Filmen durch Quervernetzung
Im ersten Abschnitt wurde die Stabilisierung von PG-Filmen durch eine oberflächeninitiierte ringöffnende Polymerisationsreaktion auf SiOx-Substrate realisiert. Dabei wurde die Dicke der Filme (10, 50 und 100 nm) über die Reaktionszeit kontrolliert. Die Quervernetzung erfolgte mit Ethylenglycoldiglycidylether (EGDGE), Divinylsulfon (DVS), Glutaraldehyd (GA), 1,11-Di(mesyl-oxy)-3,6,9-trioxaundecan (TEG-Ms2) und 1,11-Dibrom-3,6,9-trioxaundecan (TEG-Br2). Die Wahl des Quervernetzungsmittels zeigte signifikante Auswirkungen auf die Dicke der Filme und deren Biorepulsivität. Insbesondere die Untersuchungen an PG-EGDGE-Filmen waren vielversprechend, da sie nicht nur Biorepulsivität zeigten, sondern auch eine hohe mechanische Stabilität aufwiesen. Die Erkenntnis, dass dünnere Filme mit einem höheren Elastizitätsmodul gewünscht waren, erforderte eine gezielte Auswahl des Quervernetzungsmittels.
Teilprojekt 2: Einführung einer Bioerkennungsstelle auf einem EGDGE-quervernetzten PG-Film
Im nächsten Abschnitt wurde die Einführung einer Bioerkennungsstelle auf einem EGDGE-quervernetzten PG-Film erforscht. Hierbei kam ein Nitrilotriessigsäure (NTA)-Derivat (NTA4-Cyclen) zum Einsatz, um Histidin (His)-Tag-markierte Proteine selektiv zu binden. Die Modifikation erfolgte durch die Polymerisation von Glycidol auf ein amino-terminiertes Disulfid, gefolgt von einer Quervernetzung mit EGDGE. Die optimierte Synthese des NTA-Derivats ermöglichte nicht nur die erfolgreiche Anbindung von His-markierten Proteinen, sondern auch eine quasi-reversible Anbindung. Diese Erkenntnisse eröffnen neue Möglichkeiten für die Regenerierbarkeit von biochemischen Oberflächen.
Teilprojekt 3: Entwicklung eines leitfähigen und biorepulsiven Hybridmaterials
Das dritte Teilprojekt konzentrierte sich auf die Entwicklung eines leitfähigen und biorepulsiven Hybridmaterials. Ein PG-funktionalisiertes Pyrrol-Derivat (PyPG) wurde elektrochemisch polymerisiert, wodurch Netzwerke aus PG-funktionalisierten Polypyrrol-Strängen (PPG) auf Goldoberflächen entstanden. Die Elektropolymerisation wurde detailliert durch Cyclovoltammetrie (CV) und einer Quarzkristall-Mikrowaage (QCM) charakterisiert. Dabei konnte ein selbstterminierender Mechanismus identifiziert werden, der von der Anwesenheit der verzweigten PG-Gruppen dominiert wird. Mit Hilfe der elektrochemischen Impedanzspektroskopie (EIS) wurde gezeigt, dass dünnere Schichten mit geringerer Schichtdicke hoch leitfähig sind und dass die Veränderungen der Leitfähigkeit tatsächlich nur durch die äußere Oberflächenschicht bestimmt wird. Die Biorepulsivität des Materials wurde durch Proteinadsorptions- und Bakterienadhäsions-Assays nachgewiesen. Der zweite Abschnitt dieses Projekts konzentrierte sich auf die Einführung einer Erkennungsstelle basierend auf Glycosiden, um nicht nur eine Oberflächenbindung, sondern auch eine Quantifizierung von Biomolekülen zu ermöglichen. Die elektrochemische Charakterisierung der Nanoschichtsysteme zeigte eine sehr gute Linearität der Ladungsübertragungsadmittanz (Sct) in Abhängigkeit von der Anbindung der Bakterienstämme. Dies eröffnet Perspektiven für die selektive Quantifizierung von biologischen Analyten mittels elektrochemischer Methoden.
Understanding the functional roles of cells in neuronal circuits and behavior requires the ability to control neuronal activity in an acute and precise way. The field of optogenetics offers a variety of molecular tools to excite or inhibit neurons with light. In the last decade, several strategies have been proposed for reversible silencing of neurotransmission. These tools vary widely in their mechanisms: ranging from opsin-based light-driven ion pumps or anion channels, which are known to hyperpolarize the cell, to alter ionic gradients or cellular biochemistry; over metabotropic receptors, to tools damaging the neurotransmitter release machinery, that allow only long-term silencing as their recovery requires de novo synthesis of targeted proteins. Therefore, the optogenetic toolbox still lacks tools that combine fast activation and fast reversibility with the ability of long-term silencing.
In this study, the optogenetic tool optoSynC (optogenetic synaptic vesicle clustering) was characterized in depth. optoSynC utilizes the light-induced homo-oligomerization of Arabidopsis thaliana cryptochrome 2 (CRY2) for silencing synaptic transmission via clustering of synaptic vesicles (SVs). CRY2 was targeted to the SV membrane by fusion to the SV transmembrane protein synaptogyrin-1 (SNG-1). The silencing kinetics of optoSynC were determined with analyses of swimming and crawling behavior in Caenorhabditis elegans (C. elegans). Pan-neuronally expressed optoSynC reduced swimming locomotion by 80% within 30 s following photo-stimulation (τon ~7.2 s). Locomotion recovered within 15 min in darkness (τoff ~6.5 min). Analysis of crawling behavior indicated even faster activation within 2 s and an almost complete inhibition of the LITE-1-mediated escape response. This escape response occurs at high blue light intensities and results in an increased velocity, which was not detected after optoSynC activation. However, optoSynC can exert its full effect at significantly lower intensities (25 µW/mm²) and is so light-sensitive that it can even be activated at 1.4 µW/mm². optoSynC could be fully recovered and reactivated at least twice without decreased efficiency. With a combination of pharmacological analysis and optogenetics, it has been demonstrated that optoSynC can inhibit neurotransmitter release for several hours. To realize its full potential, optoSynC should be expressed in an sng-1 mutant background. Expression along with endogenous SNG-1 decreases the effectiveness of optoSynC by 50%. Specific expression of optoSynC in cholinergic or GABAergic neurons could robustly inhibit swimming behavior by 55% and 30%, respectively. Moreover, optoSynC can selectively inhibit individual neurons like the nociceptive neuron pair PVD. When PVD is photoactivated by Chrimson, a red-light activatable channelrhodopsin, forward locomotion increases. Activation of optoSynC reduced this behavior by 50%. Besides my experiments in C. elegans, my cooperation partners Dr. Holger Dill and Yilmaz Arda Ateş demonstrated silencing of neurotransmission with optoSynC in zebrafish, and Dr. Shigeki Watanabe and Brady D. Goulden in murine hippocampal neurons.
The clustering of SVs as the mode of action of optoSynC could be confirmed using transmission electron microscopy. Analysis of micrographs of cholinergic synapses stimulated with and without light revealed that distances of neighboring synaptic vesicles in the cytosol were reduced by around 14% after photoactivation. Distances of docked SVs at the plasma membrane remained unaffected. However, near the dense projection, docked SVs accumulated, while other docked SVs were depleted after optoSynC activation. Activation of optoSynC increased the appearances of SVs and dense core vesicles (DCVs) in micrographs. It is unclear whether sizes became physically larger due to lateral pressure by CRY2 aggregates in the SV membrane or if oligomer formation only altered their appearance in micrographs. optoSynC did not accumulate in the plasma membrane to the extent that abnormal structures at the plasma membrane or dense projection were observed. Recycling of SVs remained unaffected by optoSynC as no unusual number of large vesicles was determined. Therefore, optoSynC inhibits neuronal activity mainly by clustering of cytosolic SVs.
To study the precise sequence and timing of events of vesicle mobilization, it is necessary to introduce known stops in the synaptic vesicle cycle which can be achieved by optoSynC. The C. elegans mutation dyn-1(ts-) is a temperature-sensitive dynamin mutation that blocks the recycling of SVs from the plasma membrane and early endosomes at temperatures exceeding 25 °C. By expressing optoSynC in dyn-1(ts-) animals, a novel assay was established, enabling the transfer of SVs between different stages in the SV cycle. Cluster formation of reserve pool SVs blocks the SV cycle before the process of docking and priming begins, while the SV cycle is blocked after the fusion of SVs at high temperatures. It could be demonstrated that behavior returned 15 min after optoSynC activation while animals without optoSynC remained immobile. Unfortunately, the time scale of the recovery from inhibition by optoSynC is too long to effectively study vesicle mobilization.
In spite of the increasing number of biologics license applications, the development of covalent inhibitors is still a growing field within drug discovery. The successful approval of some covalent protein kinase inhibitors, such as ibrutinib (BTK covalent inhibitor) and dacomitinib (EGFR covalent inhibitor), and the very recent discovery of covalent inhibitors for viral proteases, such as boceprevir, narlaprevir, and nirmatrelvir, represent a new milestone in covalent drug development. Generally, the formation of covalent bonds that target proteins can offer drugs diverse advantages in terms of target selectivity, drug resistance, and administration concentration. The most important factor for covalent inhibitors is the electrophile (warhead), which dictates selectivity, reactivity, and the type of protein binding (i.e., reversible or irreversible) and can be modified/optimized through rational designs. Furthermore, covalent inhibitors are becoming more and more common in proteolysis, targeting chimeras (PROTACs) for degrading proteins, including those that are currently considered to be ‘undruggable’. The aim of this review is to highlight the current state of covalent inhibitor development, including a short historical overview and some examples of applications of PROTAC technologies and treatment of the SARS-CoV-2 virus.
Die Charakterisierung reaktiver Intermediate ist experimentell äußerst anspruchsvoll und häufig nicht eindeutig möglich. Die chemische Fachliteratur ist daher durchzogen von Arbeiten, in denen Intermediate anhand chemischer Intuition oder aufgrund spekulativer Interpretationen experimenteller Daten postuliert werden. Auch wenn es die Chemie wie kaum eine andere wissenschaftliche Disziplin geschafft hat, naturgegebene Zusammenhänge intuitiv erfassbar zu machen und es Chemikern somit möglich ist, mit Zettel und Stift Moleküle mit gewünschten Eigenschaften zu designen und komplexe Reaktionen zu planen, so ist doch davon auszugehen, dass zahlreiche in der Literatur postulierte Intermediate nicht korrekt zugeordnet wurden. Durch die massive Steigerung der Leistungsfähigkeit von Computern und Software in den vergangenen Jahrzehnten lassen sich quantenchemisch immer größere Systeme mit hinreichender Genauigkeit behandeln, sodass es in vielen Fällen möglich ist, experimentelle Untersuchungen theoretisch zu evaluieren und auch experimentell nicht nachweisbare Moleküle detailliert zu untersuchen. Insbesondere durch kombinierte experimentelle und quantenchemische Studien können komplexe chemische Zusammenhänge detailliert verstanden werden. Dadurch lassen sich neue Konzepte entwickeln, die eine Weiterentwicklung chemischer Intuition ermöglichen. Anhand dieses Leitbilds wurde in dieser Arbeit die Stoffklasse der Metallopniktinidene sowie daraus abgeleitete Verbindungen quantenchemisch untersucht.
The impact of 2-desaza-annomontine on processes of inflammation and its resolution in leukocytes
(2024)
This present study investigated the effects of the b-carboline derivative C81, also called 2-desaza-annomontine, on the inflammatory response and resolution processes in vivo and in vitro. The study focused on leukocytes and on the elucidation of the underlying pharmacological mode of action. C81 potently reduced the inflammatory response in an imiquimod-induced psoriasis mouse model and additionally resolved the inflammation more quickly. In a CNV model, C81 significantly decreased the microglial infiltration in the inflamed laser lesion in vivo. In vitro experiments revealed that C81 inhibits the migration of macrophages and leukocyte-endothelial cell interaction by reducing the activation of integrins on leukocytes, in particular LFA-1, without affecting the total protein level on the cell surface.
Further experiments revealed that C81 inhibited the expression of EPAC-1, required for Rap1 activation. Consequently, C81 reduced the LPS/PMA-induced Rap1 activity, which is responsible for integrin activation. Moreover, C81 potently reduced the LPS-induced formation of pro-inflammatory mediators, including cytokines and eicosanoids, in macrophages. The C81-derived inhibition of eicosanoid release was surprisingly potent, probably due to the C81-evoked inhibition of cPLA2 expression, resulting in less liberated arachidonic acid, the precursor for eicosanoids. At the same time, C81 only delayed COX-2 expression, but completely diminished LPS-induced mPGES-1 expression. In addition to the potent anti-inflammatory effects in vitro, C81-derived impact was complemented with promising pro-resolving effects. Hence, C81 significantly induced neutrophil apoptosis without affecting the cell viability of other leukocytes, such as macrophages. Accordingly, the caspase 3 activity in neutrophils increased upon C81 treatment. The underlying mechanism altered by C81 was the expression of the anti-apoptotic mediator Mcl-1, which is required for the survival of neutrophils, but not macrophages. Furthermore, neutrophils treated with C81 were significantly better efferocytosed by macrophages. Analyzes of the pharmacological mode of action of C81 revealed DYRK1B as the key target kinase in inflammatory processes in leukocytes. Of note, experiments with pharmacological inhibition of DYRK1B by C81 or the ‘selective’ DYRK1B inhibitor AZ-DYRK1B-33, could not completely exclude the involvement of the CLKs and other DYRKs. Therefore, DYRK1B knockdown and overexpression experiments were conducted to elucidate the impact of DYRK1B alone. Pharmacological inhibition of DYRK1B and DYRK1B knockdown phenocopied the inhibitory effect of C81 on leukocyte adhesion. In parallel, DYRK1B overexpression mitigated the C81-evoked effect, supporting the hypothesis that C81 inhibits DYRK1B to mediate its effects on leukocytes. Furthermore, DYRK1B inhibition and DYRK1B knockdown resulted in depletion of STAT3 phosphorylation. In addition, C81-evoked STAT3 inhibition was again mitigated by DYRK1B overexpression, suggesting a link or even an interaction between DYRK1B and STAT3. Indeed, direct interaction between DYRK1B and STAT3 was confirmed by a NanoBRET assay. Importantly, in vitro experiments demonstrated, that C81 did not affect LPS recognition mechanisms, investigated by TLR-4 and CD14 expression, and other important inflammatory signaling pathways. Although C81 inhibited the regeneration of IkBa, this had no effect on the translocation of the NFkB subunit p65. Furthermore, C81 did not alter the activation of MAPK pathways, including p38, JNK and ERK. As a result, the focus was on the potent inhibition of LPS-nduced STAT3 activation mediated by DYRK1B, which was shown to be IL-6 independent. Indeed, direct STAT3 inhibition by Stattic phenocopied all tested C81-derived effects on leukocytes, including migration, adhesion, pro-inflammatory cytokine expression, eicosanoid formation and cell type specific induction of neutrophil apoptosis. The underlying mechanisms altered by Stattic in terms of migration/adhesion and lipid mediator formation were the same as for C81. STAT3 inhibition led to decreased EPAC1 expression and accordingly to reduced Rap1 activation. In addition, inhibited STAT3 phosphorylation resulted in reduced cPLA2 expression and also in attenuated mPGES-1 expression.
Finally, the C81-derived depleted Mcl-1 expression was linked to reduced STAT3 inhibition. As C81 abolished STAT3 phosphorylation, less STAT3 was translocated into the nucleus upon LPS stimulation and less STAT3 enrichment at the MCL1 promoter was observed, leading to reduced gene expression and consequently protein levels.
In summary, using the natural product-derived compound C81, the DYRK1B/STAT3 axis was identified as a novel key regulator of inflammatory processes in human leukocytes. This present study revealed that interfering with the DYRK1B-STAT3 signaling can address essential cell functions including leukocyte extravasation, pro-inflammatory mediator release, neutrophil apoptosis and efferocytosis (Figure 1). Furthermore, two different mouse models demonstrated the in vivo relevance of this signaling axis and highlight DYRK1B as an important kinase modulating inflammation and resolution.
K + is the most abundant cytosolic cation in bacteria, and its homeostasis is vital for bacterial survival, playing roles in many essential processes like pH homeostasis, protein synthesis and osmoregulation. When surrounding K + concentrations become very low, bacteria require an active high-affinity uptake system to ensure sufficient cellular K + levels. In many prokaryotes, this system is the K + pump KdpFABC. Peculiarly, KdpFABC forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB), and for a long time, the mechanism of how transport and ATP hydrolysis between these subunits are coordinated remained unclear. By applying a combination of cryo-EM, biochemical assays, and MD simulations, we have been able to shed light on a unique transport mechanism that combines both the channel and P-type ATPase subunits.
At high K + levels, KdpFABC needs to be inhibited to prevent excessive K + accumulation. This is achieved by a phosphorylation of the serine residue in the TGES 162 motif in the A domain of the pump subunit KdpB, which was shown to stall the complex in the E1P intermediate. Using cryo-EM studies under turnover conditions, we illuminated how stalling in this high-energy intermediate is possible.
Furthermore, we identify a previously uncharacterized atypical serine kinase domain in the sensor histidine kinase KdpD as the responsible kinase for KdpB phosphorylation, giving it a dual role in transcriptional and post-translational regulation of the Kdp system.
Amyloid pathology reduces ELP3 expression and tRNA modifications leading to impaired proteostasis
(2023)
Highlights
• Amyloid pathology impacts the tRNA epitranscriptome.
• Expression of the tRNA modifying enzyme ELP3 is reduced in the brains of Alzheimer's disease (AD) patients.
• Expression levels of ELP3 negatively correlate with amyloid plaque burden in AD.
• ELP3 and ELP3-tRNA dependent modifications are relevant for maintaining neuronal proteostasis.
• ELP3 differential expression and tRNA hypomodification are cellular responses to the accumulation of toxic Aβ forms.
Abstract
Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by accumulation of β-amyloid aggregates and loss of proteostasis. Transfer RNA (tRNA) modifications play a crucial role in maintaining proteostasis, but their impact in AD remains unclear. Here, we report that expression of the tRNA modifying enzyme ELP3 is reduced in the brain of AD patients and amyloid mouse models and negatively correlates with amyloid plaque mean density. We further show that SH-SY5Y neuronal cells carrying the amyloidogenic Swedish familial AD mutation (SH-SWE) display reduced ELP3 levels, tRNA hypomodifications and proteostasis impairments when compared to cells not carrying the mutation (SH-WT). Additionally, exposing SH-WT cells to the secretome of SH-SWE cells led to reduced ELP3 expression, wobble uridine tRNA hypomodification, and increased protein aggregation. Importantly, correcting tRNA deficits due to ELP3 reduction reverted proteostasis impairments. These findings suggest that amyloid pathology dysregulates proteostasis by reducing ELP3 expression and tRNA modification levels, and that targeting tRNA modifications may be a potential therapeutic avenue to restore neuronal proteostasis in AD and preserve neuronal function.
Ataxia Telangiectasia (A-T) is a rare monogenetic, autosomal recessive disorder with an incidence of 1 in 40,000-100,000 live births caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The encoded serine/threonine protein kinase (ATM) plays a major role in DNA damage response as well as apoptosis, cell cycle regulation, cell survival, oxidative stress response and genomic stability. Biallelic mutations result in partial or complete loss of ATM expression and/or ATM protein activity. A-T is a disease characterized by progressive cerebellar degeneration, telangiectasia, immunodeficiency (impaired B- and T-cell development), recurrent sinopulmonary infections, radiation sensitivity, premature aging, and a predisposition to cancer. Life expectancy of these patients is highly compromised, with only around 50% expected to reach 20 years of age. Malignancies and pulmonary diseases are the two main causes of death. There is currently no therapy available for A-T patients. There are symptomatic treatments available (e.g. immunoglobulin replacement therapy, therapy with antioxidants, and the administration of growth hormone or glucocorticoids as anti- inflammatory hormones) and in some patients, allogeneic hematopoietic stem cell transplantations from matched donors were performed with improved disease outcome. Unfortunately, suitable donors are not available for most patients. An autologous hematopoietic stem cell (HSC)-directed gene therapy approach is a promising alternative, since no matching donor is needed. The patient’s own cells are used, modified ex-vivo (e.g. delivering a healthy copy of the gene with viral vectors or directly correcting the mutation with gene editing). Afterwards, modified HSCs are given back to the patient thereby repopulating the bone marrow and re-establishing the whole blood system. The aim of this project was to develop a gene transfer tool for Atm.
In the first part of this project, retroviral vectors containing the full-length murine Atm cDNA were generated. Gene transfer of Atm with retroviral vectors is challenging, as the Atm cDNA is 9.1 kb in size reaching the packaging capacity of retroviral vectors. Although the foamy viral vector is described to have superior abilities to transfer large sequences, produced titers of the foamy viral Atm vectors were low and transductions of Atm-deficient fibroblasts were inefficient. In contrast, gene transfer of Atm with gammaretroviral and lentiviral vectors was possible, and because lentiviral vectors harboring the full-length Atm coding sequence were produced with the highest viral titers, this vector was used to transduce Atm-deficient fibroblasts. Following transduction, ATM protein levels were restored (40 - 50% of wild-type level). In addition, transduced cells showed increased levels of phosphorylated ATM downstream substrates (γH2AX, pKap1 and p-p53) after irradiation, demonstrating functional reconstitution. However, efficient transduction of murine lineage marker negative cells, the target cells for an Atm gene therapy approach, was not possible and viability of these cells was highly compromised after transduction.
Therefore, a dual vector system was developed in the second part of the project to circumvent the packaging limit of retroviral vectors. Protein halves were fused with split inteins which catalyze their self-excision followed by the formation of a full-length protein in a process called protein trans-splicing. The split Atm cDNA was delivered with lentiviral vectors and sufficient viral titers were achieved for efficient double transduction of Atm-deficient fibroblasts. Whereas the reconstitution of full-length ATM protein was low in cells transduced with vectors containing Npu split inteins, the use of Rma split inteins showed superior reconstitution. When comparing reconstitution levels with two different split sites within the ATM protein, no major differences were observed. Because a proof of ATM functionality could not be shown with these vector pairs, the F2A site used to co-deliver a marker gene was replaced by an IRES element. After transduction with split intein Atm vectors containing IRES elements, the level of ATM protein reached only 10% of the wild-type level. Nevertheless, an increased amount of pKap1 and p-p53 was detected demonstrating a functional kinase activity of reconstituted ATM protein. Furthermore, a partial repair of cell cycle defects in Atm-deficient fibroblasts was demonstrated.
In parallel to the development of a gene transfer tool for Atm, preliminary experiments were performed in Atm-deficient mice to create optimal transplantation conditions for gene-corrected HSCs that could be performed in the future. Because Atm-deficient mice are highly sensitive to irradiation, conventional conditioning regimes (e.g. total body irradiation or myeloablative conditioning with chemotherapeutics) cannot be used prior to HSC transplantation. Therefore, Atm-deficient mice were pretreated with different conditioning regimens and subsequently received a bone marrow transplantation. Mice that did not receive preconditioning prior to transplantation showed no chimerism in peripheral blood, bone marrow or spleen samples, indicating that preconditioning of mice is required for donor cell engraftment. A non-myeloablative conditioning regimen with cyclophosphamide and immunosuppressive CD4 and CD8 antibodies and the application of a mobilizing agent (Plerixafor) one hour before transplantation showed the highest chimerism in recipient mice. None of the mice developed a thymic tumor, and lymphoid-biased differentiation of the donor cells was observed, as chimerism was highest in T cells in the blood, bone marrow and spleen. In addition, chimerism was higher in lymphoid progenitor cells than in myeloid progenitors. Blood counts (white blood cell and lymphocyte counts) were normal 20 weeks after transplantation (comparable to wild-type mice), making this preconditioning regime suitable for Atm-deficient mice.
Taken together, this data paves the way for using split intein-based lentiviral vectors for Atm delivery in preclinical models and opens new possibilities for developing gene therapy for A-T patients.
SEDDS-loaded mucoadhesive fiber patches for advanced oromucosal delivery of poorly soluble drugs
(2022)
To date, buccal administration of lipophilic drugs is still a major challenge due to their poor solubility in saliva and limited penetration into mucosal tissues. To overcome these limitations, we developed electrospun patches combining the benefits of mucoadhesive fibers and self-emulsifying drug delivery systems (SEDDS).
The fiber system comprises a combination of mucoadhesive thiolated polyacrylic acid fibers and SEDDS-loaded fibers fabricated by parallel electrospinning. The resulting mucoadhesive electrospun SEDDS patches were systemically investigated for fiber characteristics, self-emulsification, mucoadhesion, drug penetration into porcine buccal tissue and biocompatibility.
The patches showed high encapsulation efficiency for SEDDS without causing fiber defects or leakage. SEDDS incorporation enhanced the spinning process and reduced the fiber diameter and fiber size distribution. Hydration-dependent self-emulsification provided a controlled release of curcumin being encapsulated in nano-scaled o/w emulsion for over 3 h. Due to the thiolated polyacrylic acid fibers, the buccal residence time of patches was 200-fold prolonged. Further, they promoted a significantly increased drug penetration into buccal tissue compared to fiber patches without SEDDS. Finally, biocompatibility and improved therapeutic effects of curcumin-loaded patches on human keratinocytes and fibroblasts were confirmed.
Mucoadhesive electrospun SEDDS patches represent a promising approach to overcome current challenges in the oromucosal delivery of lipophilic drugs to unlock their full therapeutic potential.
Highlights
• Self-emulsifying drug delivery systems (SEDDS) were electrospun into polymer fibers patches for oromucosal drug delivery.
• High loading capacity of the fibers with SEDDS was shown (up to 50% of polymer weight).
• 200-fold prolonged buccal patch residence time was achieved by parallel electrospinning of polyacrylic acid thiomer fibers.
• SEDDS-loaded fibers ensured prolonged release of curcumin and led to 1200-fold increase in aqueous solubility.
• In contrast to conventional fiber patches, SEDDS-loaded mucoadhesive fibers enabled deep mucosal penetration of curcumin.