Itaconic Acid Increases the Efficacy of Tobramycin against Pseudomonas aeruginosa Biofilms

The search for novel therapeutics against pulmonary infections, in particular Pseudomonas aeruginosa (PA) biofilm infections, has been intense to deal with the emergent rise of antimicrobial resistance. Despite the numerous achievements in drug discovery and delivery strategies, only a limited number of therapeutics reach the clinic. To allow a timely preclinical development, a formulation should be highly effective, safe, and most importantly facile to produce. Thus, a simple combination of known actives that enhances the therapeutic efficacy would be a preferential choice compared to advanced drug delivery systems. In this study, we propose a novel combination of an anti-inflammatory agent—itaconic acid (itaconate, IA)—and an approved antibiotic—tobramycin (Tob) or ciprofloxacin (Cipro). The combination of Tob and IA at a molar ratio of 1:5 increased the biofilm eradicating efficacy in the strain PA14 wild type (wt) by ~4-fold compared to Tob alone. In contrast, such effect was not observed for the combination of IA with Cipro. Subsequent studies on the influence of IA on bacterial growth, pyocyanin production, and Tob biofilm penetration indicated that complexation with IA enhanced the transport of Tob through the biofilm. We recommend the simple and effective combination of Tob:IA for further testing in advanced preclinical models of PA biofilm infections

Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems

Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin–pDNA coacervate in its center. Thermal stabilization enhances the core’s colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications

Preferential uptake of chitosan-coated PLGA nanoparticles by primary human antigen presenting cells

Biodegradable polymeric nanoparticles (NP) made from poly (lactid-co-glycolide) acid (PLGA) and chitosan (CS) hold promise as innovative formulations for targeted delivery. Since interactions of such NP with primary human immune cells have not been characterized, yet, here we assessed the effect of PLGA or CS-PLGA NP treatment on human peripheral blood mononuclear cells (PBMC), as well as on monocyte-derived DC (moDC). Amongst PBMC, antigen presenting cells (APC) showed higher uptake of both NP preparations than lymphocytes. Furthermore, moDC internalized CS-PLGA NP more efficiently than PLGA NP, presumably because of receptor-mediated endocytosis. Consequently, CS-PLGA NP were delivered mostly to endosomal compartments, whereas PLGA NP primarily ended up in lysosomes. Thus, CS-PLGA NP confer enhanced delivery to endosomal compartments of APC, offering new therapeutic options to either induce or modulate APC function and to inhibit pathogens that preferentially infect APC

Lipid–Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media

To 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

Lipid-Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells : Impact of Lipid Composition on Performance in Different Media

To 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

Disrupting Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Latent Replication with a Small Molecule Inhibitor

Kaposi's sarcoma-associated herpesvirus (KSHV) can establish latent lifelong infections in infected individuals. During viral latency, the latency-associated nuclear antigen (LANA) mediates the replication of the latent viral genome in dividing cells and tethers them to mitotic chromosomes, thus ensuring their partitioning into daughter cells during mitosis. This study aims to inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) latent replication by targeting the LANA-DNA interaction using small molecular entities. Drawing from first-generation inhibitors and using growth vectors identified through STD-NMR, we expanded these compounds using Suzuki-Miyaura cross-coupling. This led to a deeper understanding of SAR achieved by microscale thermophoresis (MST) measurements and cell-free tests via electrophoretic mobility shift assays (EMSA). Our most potent compounds successfully inhibit LANA-mediated replication in cell-based assays and demonstrate favorable in vitro ADMET-profiles, including suitable metabolic stability, Caco-2 permeability, and cytotoxicity. These compounds could serve as qualified leads for the future refinement of small molecule inhibitors of KSHV latent replication

Tofacitinib Loaded Squalenyl Nanoparticles for Targeted Follicular Delivery in Inflammatory Skin Diseases

Tofacitinib (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

Intracellular Dynamics of Extracellular Vesicles by Segmented Trajectory Analysis

The analysis of nanoparticle (NP) dynamics in live cell studies by video tracking provides detailed information on their interactions and trafficking in the cells. Although the video analysis is not yet routinely used in NP studies, the equipment suitable for the experiments is already available in most laboratories. Here, we compare trajectory patterns, diffusion coefficients, and particle velocities of NPs in A549 cells with a rather simple experimental setup consisting of a fluorescence microscope and openly available trajectory analysis software. The studied NPs include commercial fluorescent polymeric particles and two subpopulations of PC-3 cell-derived extracellular vesicles (EVs). As bioderived natural nanoparticles, the fluorescence intensities of the EVs limited the recording speed. Therefore, we studied the effect of the recording frame rate and analysis parameters to the trajectory results with bright fluorescent commercial NPs. We show that the trajectory classification and the apparent particle velocities are affected by the recording frame rate, while the diffusion constants stay comparable. The NP trajectory patterns were similar for all NP types and resembled intracellular vesicular transport. Interestingly, the EV movements were faster than the commercial NPs, which contrasts with their physical sizes and may indicate a greater role of the motor proteins in their intracellular transports.Peer reviewe

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs’ technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro–in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics