Browsing by Author "Loretz, Brigitta"
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- ItemDimethylaminoethyl methacrylate copolymer-siRNA nanoparticles for silencing a therapeutically relevant gene in macrophages(Cambridge : Royal Society of Chemistry, 2015) Jain, Ratnesh; Dandekar, Prajakta; Loretz, Brigitta; Koch, Marcus; Lehr, Claus-MichaelTherapeutic gene silencing using small-interfering RNA (siRNA) for treatment of bacterial infections has been neglected in comparison with cancer and viral infections. The aim of our investigation was to formulate siRNA-loaded nanoparticles, using an established cationic polymethacrylate polymer, to enhance the delivery of siRNA into the cytoplasm of macrophages that host many pathogenic bacterial species, including tuberculosis. Nanoparticles of cationic dimethylaminoethyl methacrylate copolymer (Eudragit[registered sign] E 100) were successfully formulated and were found to efficiently bind the siRNA molecules (Cy3-siRNA, Bfl1/A1 siRNA). The efficiency of nanoparticles in overcoming cellular barriers to intracellular siRNA delivery and the precise pathway of endocytosis of nanoparticles were both confirmed using confocal microscopy. Through efficient siRNA release into the cytoplasm, the siRNA-loaded nanoparticles enabled a five-fold enhancement in the knockdown efficiency of the endogenous host gene Bfl1/A1, when the formulation was compared with free siRNA. Persistence of Bfl1/A1 is useful for phagolysosomal survival of tuberculosis bacteria in macrophages, and the nanoparticles offer a promising concept for exploitation as an anti-tuberculosis therapy.
- ItemKinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles(London : Biomed Central, 2018) Yasar, Hanzey; Biehl, Alexander; De Rossi, Chiara; Koch, Marcus; Murgia, Xabi; Loretz, Brigitta; Lehr, Claus-MichaelBackground: Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA's transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases. Results: A lipid-polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA. Conclusions: Charge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid-polymer NPs.
- 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.
- ItemNano-in-Microparticles for Aerosol Delivery of Antibiotic-Loaded, Fucose-Derivatized, and Macrophage-Targeted Liposomes to Combat Mycobacterial Infections: In Vitro Deposition, Pulmonary Barrier Interactions, and Targeted Delivery(Weinheim : Wiley-VCH, 2022) Huck, Benedikt C.; Thiyagarajan, Durairaj; Bali, Aghiad; Boese, Annette; Besecke, Karen F.W.; Hozsa, Constantin; Gieseler, Robert K.; Furch, Marcus; Carvalho‐Wodarz, Cristiane; Waldow, Franziska; Schwudke, Dominik; Metelkina, Olga; Titz, Alexander; Huwer, Hanno; Schwarzkopf, Konrad; Hoppstädter, Jessica; Kiemer, Alexandra K.; Koch, Marcus; Loretz, Brigitta; Lehr, Claus‐MichaelNontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco-regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline-encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP-1 cells as well as peripheral blood monocyte- and lung-tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry-powder microparticles of spray-dried bedaquiline-loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP-1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.
- ItemA New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms(2021) Schütz, Christian; Ho, Duy-Khiet; Hamed, Mostafa Mohamed; Abdelsamie, Ahmed Saad; Röhrig, Teresa; Herr, Christian; Kany, Andreas Martin; Rox, Katharina; Schmelz, Stefan; Siebenbürger, Lorenz; Wirth, Marius; Börger, Carsten; Yahiaoui, Samir; Bals, Robert; Scrima, Andrea; Blankenfeldt, Wulf; Horstmann, Justus Constantin; Christmann, Rebekka; Murgia, Xabier; Koch, Marcus; Berwanger, Aylin; Loretz, Brigitta; Hirsch, Anna Katharina Herta; Hartmann, Rolf Wolfgang; Lehr, Claus-Michael; Empting, MartinPseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) – a crucial transcriptional regulator serving major functions in PA virulence – can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10−9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development.
- ItemSpray-dried lactose-leucine microparticles for pulmonary delivery of antimycobacterial nanopharmaceuticals(New York, NY [u.a.] : Springer, 2021) Thiyagarajan, Durairaj; Huck, Benedikt; Nothdurft, Birgit; Koch, Marcus; Rudolph, David; Rutschmann, Mark; Feldmann, Claus; Hozsa, Constantin; Furch, Marcus; Besecke, Karen F. W.; Gieseler, Robert K.; Loretz, Brigitta; Lehr, Claus-MichaelPulmonary delivery of nanocarriers for novel antimycobacterial compounds is challenging because the aerodynamic properties of nanomaterials are sub-optimal for such purposes. Here, we report the development of dry powder formulations for nanocarriers containing benzothiazinone 043 (BTZ) or levofloxacin (LVX), respectively. The intricacy is to generate dry powder aerosols with adequate aerodynamic properties while maintaining both nanostructural integrity and compound activity until reaching the deeper lung compartments. Microparticles (MPs) were prepared using vibrating mesh spray drying with lactose and leucine as approved excipients for oral inhalation drug products. MP morphologies and sizes were measured using various biophysical techniques including determination of geometric and aerodynamic mean sizes, X-ray diffraction, and confocal and focused ion beam scanning electron microscopy. Differences in the nanocarriers’ characteristics influenced the MPs’ sizes and shapes, their aerodynamic properties, and, hence, also the fraction available for lung deposition. Spay-dried powders of a BTZ nanosuspension, BTZ-loaded silica nanoparticles (NPs), and LVX-loaded liposomes showed promising respirable fractions, in contrast to zirconyl hydrogen phosphate nanocontainers. While the colloidal stability of silica NPs was improved after spray drying, MPs encapsulating either BTZ nanosuspensions or LVX-loaded liposomes showed the highest respirable fractions and active pharmaceutical ingredient loads. Importantly, for the BTZ nanosuspension, biocompatibility and in vitro uptake by a macrophage model cell line were improved even further after spray drying.
- ItemSynthesis and Biopharmaceutical Characterization of Amphiphilic Squalenyl Derivative Based Versatile Drug Delivery Platform(Lausanne : Frontiers Media, 2020) Ho, Duy-Khiet; Christmann, Rebekka; Murgia, Xabier; De Rossi, Chiara; Frisch, Sarah; Koch, Marcus; Schaefer, Ulrich F.; Loretz, Brigitta; Desmaele, Didier; Couvreur, Patrick; Lehr, Claus-MichaelLimited drug loading capacity (LC), mostly below 5% w/w, is a significant drawback of nanoparticulate drug delivery systems (DDS). Squalenoylation technology, which employs bioconjugation of squalenyl moiety and drug, allows self-assemble of nanoparticles (NPs) in aqueous media with significantly high LC (>30% w/w). The synthesis and particle preparation of squalenoylated prodrugs are, however, not facile for molecules with multiple reactive groups. Taking a different approach, we describe the synthesis of amphiphilic squalenyl derivatives (SqDs) as well as the physicochemical and biopharmaceutical characterizations of their self-assembled NPs as DDSs. The SqDs included in this study are (i) cationic squalenyl diethanolamine (ii) PEGylated SqD (PEG 750 Da), (iii) PEGylated SqD (PEG 3,000 Da), and (iv) anionic squalenyl hydrogen sulfate. All four SqDs self-assemble into NPs in a size range from 100 to 200 nm in an aqueous solution. Furthermore, all NP derivatives demonstrate appropriate biocompatibility and adequate colloidal stability in physiological relevant pH environments. The mucoprotein binding of PEGylated NPs is reduced compared to the charged NPs. Most importantly, this technology allows excellent LC (at maximum of 45% w/w) of a wide range of multifunctional compounds, varying in physicochemical properties and molecular weight. Interestingly, the drug release profile can be tuned by different loading methods. In summary, the SqD-based NPs appear as versatile drug delivery platforms.
- 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.