Targeting Intracellular Bacteria with Dual Drug-loaded Lactoferrin Nanoparticles.

Treatment of microbial infections is becoming daunting because of widespread antimicrobial resistance. The treatment challenge is further exacerbated by the fact that certain infectious bacteria invade and localize within host cells, protecting the bacteria from antimicrobial treatments and the host's immune response. To survive in the intracellular niche, such bacteria deploy surface receptors similar to host cell receptors to sequester iron, an essential nutrient for their virulence, from host iron-binding proteins, in particular lactoferrin and transferrin. In this context, we aimed to target lactoferrin receptors expressed by macrophages and bacteria; as such, we prepared and characterized lactoferrin nanoparticles (Lf-NPs) loaded with a dual drug combination of antimicrobial natural alkaloids, berberine or sanguinarine, with vancomycin or imipenem. We observed increased uptake of drug-loaded Lf-NPs by differentiated THP-1 cells with up to 90% proportion of fluorescent cells, which decreased to about 60% in the presence of free lactoferrin, demonstrating the targeting ability of Lf-NPs. The encapsulated antibiotic drug cocktail efficiently cleared intracellular Staphylococcus aureus (Newman strain) compared to the free drug combinations. However, the encapsulated drugs and the free drugs alike exhibited a bacteriostatic effect against the hard-to-treat Mycobacterium abscessus (smooth variant). In conclusion, the results of this study demonstrate the potential of lactoferrin nanoparticles for the targeted delivery of antibiotic drug cocktails for the treatment of intracellular bacteria.

A Monoclonal Human Alveolar Epithelial Cell Line ("Arlo") with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections.

In the development of orally inhaled drug products preclinical animal models regularly fail to predict pharmacological as well as toxicological responses in humans. Models based on human cells and tissues are potential alternatives to animal experimentation allowing for the isolation of essential processes of human biology and making them accessible in vitro. Here, the generation of a novel monoclonal cell line "Arlo," derived from the polyclonal human alveolar epithelium lentivirus immortalized cell line hAELVi via single-cell printing, and its characterization as a model for the human alveolar epithelium as well as a building block for future complex in vitro models is described. "Arlo" is systematically compared in vitro to primary human alveolar epithelial cells (hAEpCs) as well as to the polyclonal hAELVi cell line. "Arlo" cells show enhanced barrier properties with high transepithelial electrical resistance (TEER) of ≈3000 Ω cm2 and a potential difference (PD) of ≈30 mV under air-liquid interface (ALI) conditions, that can be modulated. The cells grow in a polarized monolayer and express genes relevant to barrier integrity as well as homeostasis as is observed in hAEpCs. Successful productive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a proof-of-principle study offers an additional, attractive application of "Arlo" beyond biopharmaceutical experimentation.

Proteoid biodynamers for safe mRNA transfection via pH-responsive nanorods enabling endosomal escape.

The recent success of mRNA vaccines using lipid-based vectors highlights the importance of strategies for nucleotide delivery under the pandemic situation. Although current mRNA delivery is focused on lipid-based vectors, still they need to be optimized for increasing stability, targeting, and efficiency, and for reducing toxicity. In this regard, other vector systems featuring smart strategies such as pH-responsive degradability and endosomal escape ability hold the potential to overcome the current limitations. Here, we report pH-responsive polymeric nanorods made of amino acid-derivatives connected by dynamic covalent bonds called proteoid-biodynamers, as mRNA vectors. They show excellent biocompatibility due to the biodegradation, and outstanding transfection. The biodynamers of Lys, His, and Arg or monomer mixtures thereof were shown to form nanocomplexes with mRNA. They outperformed conventional transfection agents three times regarding transfection efficacy in three human cell lines, with 82-98% transfection in living cells. Also, we confirmed that the biodynamers disrupted the endosomes up to 10-fold more in number than the conventional vectors. We discuss here their outstanding performance with a thorough analysis of their nanorod structure changes in endosomal microenvironments.

Spray-dried pneumococcal membrane vesicles are promising candidates for pulmonary immunization.

Pneumococcal infections represent a global health threat, which requires novel vaccine developments. Extracellular vesicles are secreted from most cells, including prokaryotes, and harbor virulence factors and antigens. Hence, bacterial membrane vesicles (MVs) may induce a protective immune response. For the first time, we formulate spray-dried gram-positive pneumococcal MVs-loaded vaccine microparticles using lactose/leucine as inert carriers to enhance their stability and delivery for pulmonary immunization. The optimized vaccine microparticles showed a mean particle size of 1-2 µm, corrugated surface, and nanocrystalline nature. Their aerodynamic diameter of 2.34 µm, average percentage emitted dose of 88.8%, and fine powder fraction 79.7%, demonstrated optimal flow properties for deep alveolar delivery using a next-generation impactor. Furthermore, confocal microscopy confirmed the successful encapsulation of pneumococcal MVs within the prepared microparticles. Human macrophage-like THP-1 cells displayed excellent viability, negligible cytotoxicity, and a rapid uptake around 60% of fluorescently labeled MVs after incubation with vaccine microparticles. Moreover, vaccine microparticles increased the release of pro-inflammatory cytokines tumor necrosis factor and interleukin-6 from primary human peripheral blood mononuclear cells. Vaccine microparticles exhibited excellent properties as promising vaccine candidates for pulmonary immunization and are optimal for further animal testing, scale-up and clinical translation.

Nano-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.

Nontuberculous 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.

Transferring Microclusters of P. aeruginosa Biofilms to the Air-Liquid Interface of Bronchial Epithelial Cells for Repeated Deposition of Aerosolized Tobramycin.

As an alternative to technically demanding and ethically debatable animal models, the use of organotypic and disease-relevant human cell culture models may improve the throughput, speed, and success rate for the translation of novel anti-infectives into the clinic. Besides bacterial killing, host cell viability and barrier function appear as relevant but seldomly measured readouts. Moreover, bacterial virulence factors and signaling molecules are typically not addressed in current cell culture models. Here, we describe a reproducible protocol for cultivating barrier-forming human bronchial epithelial cell monolayers on Transwell inserts and infecting them with microclusters of pre-grown mature Pseudomonas aeruginosa PAO1 biofilms under the air-liquid interface conditions. Bacterial growth and quorum sensing molecules were determined upon tobramycin treatment. The host cell response was simultaneously assessed through cell viability, epithelial barrier function, and cytokine release. By repeated deposition of aerosolized tobramycin after 1, 24, and 48 h, bacterial growth was controlled (reduction from 10 to 4 log10 CFU/mL), which leads to epithelial cell survival for up to 72 h. E-cadherin's cell-cell adhesion protein expression was preserved with the consecutive treatment, and quorum sensing molecules were reduced. However, the bacteria could not be eradicated and epithelial barrier function was impaired, similar to the currently observed situation in the clinic in lack of more efficient anti-infective therapies. Such a human-based in vitro approach has the potential for the preclinical development of novel anti-infectives and nanoscale delivery systems for oral inhalation.

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.

A pulmonary mucus surrogate for investigating antibiotic permeation and activity against Pseudomonas aeruginosa biofilms.

BACKGROUND: Pulmonary infections associated with Pseudomonas aeruginosa can be life-threatening for patients suffering from chronic lung diseases such as cystic fibrosis. In this scenario, the formation of biofilms embedded in a mucus layer can limit the permeation and the activity of anti-infectives. OBJECTIVES: Native human pulmonary mucus can be isolated from endotracheal tubes, but this source is limited for large-scale testing. This study, therefore, aimed to evaluate a modified artificial sputum medium (ASMmod) with mucus-like viscoelastic properties as a surrogate for testing anti-infectives against P. aeruginosa biofilms. METHODS: Bacterial growth in conventional broth cultures was compared with that in ASMmod, and PAO1-GFP biofilms were imaged by confocal microscopy. Transport kinetics of three antibiotics, tobramycin, colistin, and ciprofloxacin, through native mucus and ASMmod were studied, and their activity against PAO1 biofilms grown in different media was assessed by determination of metabolic activity and cfu. RESULTS: PAO1(-GFP) cultured in human pulmonary mucus or ASMmod showed similarities in bacterial growth and biofilm morphology. A limited permeation of antibiotics through ASMmod was observed, indicating its strong barrier properties, which are comparable to those of native human mucus. Reduced susceptibility of PAO1 biofilms was observed in ASMmod compared with LB medium for tobramycin and colistin, but less for ciprofloxacin. CONCLUSIONS: These findings underline the importance of mucus as a biological barrier to antibiotics. ASMmod appears to be a valuable surrogate for studying mucus permeation of anti-infectives and their efficacy against PAO1 biofilms.

Identification of a novel sulfonamide non-nucleoside reverse transcriptase inhibitor by a phenotypic HIV-1 full replication assay.

Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

A novel specific edge effect correction method for RNA interference screenings.

MOTIVATION: High-throughput screening (HTS) is an important method in drug discovery in which the activities of a large number of candidate chemicals or genetic materials are rapidly evaluated. Data are usually obtained by measurements on samples in microwell plates and are often subjected to artefacts that can bias the result selection. We report here a novel edge effect correction algorithm suitable for RNA interference (RNAi) screening, because its normalization does not rely on the entire dataset and takes into account the specificities of such a screening process. The proposed method is able to estimate the edge effects for each assay plate individually using the data from a single control column based on diffusion model, and thus targeting a specific but recurrent well-known HTS artefact. This method was first developed and validated using control plates and was then applied to the correction of experimental data generated during a genome-wide siRNA screen aimed at studying HIV-host interactions. The proposed algorithm was able to correct the edge effect biasing the control data and thus improve assay quality and, consequently, the hit-selection step.

Automated genome-wide visual profiling of cellular proteins involved in HIV infection.

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.

Human endogenous retrovirus protein Rec interacts with the testicular zinc-finger protein and androgen receptor.

More than 2000 human endogenous retrovirus (HERV) sequences are present in the human genome, yet only a few are intact and able to produce proteins. The normal functions of these, if any, are unknown, but some HERV proteins have been implicated in cancers, in particular germ-cell cancers. For instance, it has been documented that (i) patients with germ-cell tumours frequently produce antibodies against HERV proteins; (ii) transgenic mice expressing HERV-K (HML-2) rec are prone to testicular carcinoma in situ; and (iii) Rec can bind and suppress a guardian of germline stem-cell pluripotency, the promyelocytic leukaemia zinc-finger protein (PLZF). This study identified the PLZF-related testicular zinc-finger protein (TZFP) as a binding partner of HERV-K (HML-2) Rec. Interactions occurred via the N- and C-terminal domains of Rec and the C-terminal DNA-binding zinc-finger domain of TZFP (aa 375-450). Not much is known about the function of TZFP. The protein is expressed predominantly in the testis, where it functions as a transcriptional repressor that is active during specific stages of spermatogenesis. The most intensely studied function of TZFP is that of a co-repressor of the activated androgen receptor (AR). Here, it was shown that Rec can form a trimeric complex with TZFP and AR, and can relieve the TZFP-mediated repression of AR-induced transactivation. In addition, Rec was able to overcome the direct transcriptional repression by TZFP of the c-myc gene promoter in reporter assays. Thus, HERV-K (HML-2) Rec may function as an oncoprotein by de-repressing oncogenic transcription factors such as AR.

Integrase interactor 1 (Ini1/hSNF5) is a repressor of basal human immunodeficiency virus type 1 promoter activity.

Integrase interactor 1 (Ini1/hSNF5/BAF47/SMARCB1), the core subunit of the ATP-dependent chromatin-remodelling complex SWI/SNF, is a cellular interaction partner of the human immunodeficiency virus type 1 (HIV-1) integrase. Ini1/hSNF5 is recruited to HIV-1 pre-integration complexes before nuclear migration, suggesting a function in the integration process itself or a contribution to the preferential selection of transcriptionally active genes as integration sites of HIV-1. More recent evidence indicates, however, that, whilst Ini1/hSNF5 is dispensable for HIV-1 transduction per se, it may have an inhibitory effect on the early steps of HIV-1 replication but facilitates proviral transcription by enhancing Tat function. These partially contradictory observations prompted an investigation of the immediate and long-term effects of Ini1/hSNF5 depletion on the basal transcriptional potential of the virus promoter. Using small interfering RNAs, it was shown that Ini1/hSNF5-containing SWI/SNF complexes mediate transcriptional repression of the basal activity of the integrated HIV-1 long terminal repeat. Transient depletion of Ini1/hSNF5 during integration was accompanied by an early boost of HIV-1 replication. After the reappearance of Ini1/hSNF5, expression levels decreased and this was associated with increased levels of histone methylation at the virus promoter in the long term, indicative of epigenetic gene silencing. These results demonstrate the opposing effects of Ini1/hSNF5-containing SWI/SNF complexes on basal and Tat-dependent transcriptional activity of the HIV-1 promoter. It is proposed that Ini1/hSNF5 may be recruited to the HIV-1 pre-integration complex to initiate, immediately after integration, one of two mutually exclusive transcription programmes, namely post-integration latency or high-level, Tat-dependent gene expression.

Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

RNAi using siRNA is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Although their use in epithelial cells is relatively easy and straightforward, transfection in other cell types is still challenging. The authors report the optimization of transfection conditions for Raw 267.4 macrophage cells. The herein described procedure makes use of automated confocal microscopy, enhanced green fluorescent protein (EGFP)-expressing macrophages, and fluorescently labeled siRNAs to simultaneously quantify both siRNA uptake and silencing efficiency. A comparison of 10 commercial transfectants was performed, leading to the selection of the transfectant giving the highest reproducible knock-down effect without inducing cell toxicity or cell activation. Several buffers used for siRNA/lipid complex assembly were tested, and such a study revealed the crucial importance of this parameter. In addition, a kinetics study led to the determination of the optimal siRNA concentration and the best time window for the assay. In an original approach aimed at simultaneously optimizing both the high-throughput screening process and biological factors, optimal reagent volumes and a process flowchart were defined to ensure robust silencing efficiencies during screening. Such an account should pave the way for future genome-wide RNAi research in macrophages and present an optimization procedure for other "hard-totransfect" cell lines.

Ini1/hSNF5 is dispensable for retrovirus-induced cytoplasmic accumulation of PML and does not interfere with integration.

Retroviral infection triggers the cytoplasmic translocation of two Crm1-dependent shuttle factors, namely the Ini1 (integrase interactor 1, hSNF5) and the promyelocytic leukemia (PML) protein. Blocking nuclear export of shuttle factors by leptomycin B increases the efficiency of retroviral integration, suggesting that some may mediate antiviral activity. While PML was shown to counteract proviral establishment, it remained unclear whether Ini1, a protein implicated in various processes during human immunodeficiency virus replication, has the same potential. Employing RNA interference-mediated knock-down of Ini1, we show here that the simultaneous accumulation of both proteins in the cytoplasm likely reflects two non-interdependent phenomena. Furthermore, Ini1 does not interfere with retroviral integration, as cells lacking Ini1 show no increased infection susceptibility.