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- 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.
- ItemGivinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics(Basel : MDPI, 2022) Taiarol, Lorenzo; Bigogno, Chiara; Sesana, Silvia; Kravicz, Marcelo; Viale, Francesca; Pozzi, Eleonora; Monza, Laura; Carozzi, Valentina Alda; Meregalli, Cristina; Valtorta, Silvia; Moresco, Rosa Maria; Koch, Marcus; Barbugian, Federica; Russo, Laura; Dondio, Giulio; Steinkühler, Christian; Re, FrancescaGlioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time-and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from −25% to −75% of protein levels), and reduction in HDAC activity (−25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.
- ItemBacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor(Basel : MDPI, 2021) Drost, Menka; Diamanti, Eleonora; Fuhrmann, Kathrin; Goes, Adriely; Shams, Atanaz; Haupenthal, Jörg; Koch, Marcus; Hirsch, Anna K. H.; Fuhrmann, GregorLiposomes have been studied for decades as nanoparticulate drug delivery systems for cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work, we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound (HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored antimicrobial target. The liposomes were composed of DOPG (18:1 (Δ9-cis) phosphatidylglycerol) and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 °C. Loading of the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm. Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug-resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably hydrophobic, antibiotics.