Development of a high throughput oxidation profiling strategy for monoclonal antibody products.

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

Tailoring lipid nanoparticles for T-cell targeting in allergic asthma: Insights into efficacy and specificity.

Asthma impacts over 300 million patients globally, with significant health implications, especially in cases of its allergic subtype. The disease is characterized by a complex interplay of airway inflammation and immune responses, often mediated by Th2 cell-related cytokines. In this study, we engineered lipid nanoparticles (LNPs) to specifically deliver therapeutic siRNA via the transferrin receptor to T cells. Strain-promoted azide-alkyne cycloaddition (SPAAC) was employed for the conjugation of transferrin ligands to PEGylated lipids in the LNPs, with the goal of enhancing cellular uptake and gene knockdown. The obtained LNPs exhibited characteristics that make them suitable for pulmonary delivery. Using methods such as nanoparticle tracking analysis (NTA) and enzyme-linked immunosorbent assay (ELISA), we determined the average number of transferrin molecules bound to individual LNPs. Additionally, we found that cellular uptake was ligand-dependent, achieving a GATA3 knockdown of more than 50% in relevant in vitro and ex vivo models. Notably, our findings highlight the limitations inherent to modifying the surface of LNPs, particularly with regard to their targeting capabilities. This work paves the way for future research aimed at optimizing targeted LNPs for the treatment of immunologic diseases such as allergic asthma.

Distribution and suitability of pulmonary surfactants as a vehicle for topically applied antibodies in healthy and SARS-CoV-2 infected rodent lungs.

The use of natural pulmonary surfactants (PS) as a drug delivery vehicle for biologics is a more recent therapeutic modality. Herein, we tested different contents of PS regarding their physicochemical properties under stress conditions. The PS content of 12.25 mg/ml (Formulation B) showed desired properties such as an isotonic osmolality ∼300 mOsm/kg and an acceptable viscosity of 8.61 cSt, being lower than in commercially available PS solutions. Formulation B passed the specifications of surface lowering capacities of >80 % total lung capacity and physiologically desired formulation properties were independent of the antibody used in the composition. The identified formulation showed the capability of significantly increasing the oxygen saturation in ex vivo isolated perfused rat lungs, compared to a control and up to 30 min post lavage. In the in vivo setting, we showed that intratracheal administration of a human mAB with and without pulmonary surfactant led to higher amounts of delivered antibody within the alveolar tissue compared to intravenous administration. The antibody with the PS formulation remained longer in the alveolar tissues than the antibody without the PS formulation. Further, SARS-CoV-2 infected Golden Syrian hamsters showed that the intranasally applied antibody reached the site of infection in the alveoli and could be detected in the alveolar region 24 h after the last administration. With this work, we demonstrated that pulmonary surfactants can be used as a pulmonary drug delivery mechanism for antibodies and may subsequently improve the antibody efficacy by increasing the residence time at the desired site of action in the alveolar tissue.

Closing the Gap between Experiment and Simulation─A Holistic Study on the Complexation of Small Interfering RNAs with Polyethylenimine.

Rational design is pivotal in the modern development of nucleic acid nanocarrier systems. With the rising prominence of polymeric materials as alternatives to lipid-based carriers, understanding their structure-function relationships becomes paramount. Here, we introduce a newly developed coarse-grained model of polyethylenimine (PEI) based on the Martini 3 force field. This model facilitates molecular dynamics simulations of true-sized PEI molecules, exemplified by molecules with molecular weights of 1.3, 5, 10, and 25 kDa, with degrees of branching between 50.0 and 61.5%. We employed this model to investigate the thermodynamics of small interfering RNA (siRNA) complexation with PEI. Our simulations underscore the pivotal role of electrostatic interactions in the complexation process. Thermodynamic analyses revealed a stronger binding affinity with increased protonation, notably in acidic (endosomal) pH, compared to neutral conditions. Furthermore, the molecular weight of PEI was found to be a critical determinant of binding dynamics: smaller PEI molecules closely enveloped the siRNA, whereas larger ones extended outward, facilitating the formation of complexes with multiple RNA molecules. Experimental validations, encompassing isothermal titration calorimetry and single-molecule fluorescence spectroscopy, aligned well with our computational predictions. Our findings not only validate the fidelity of our PEI model but also accentuate the importance of in silico data in the rational design of polymeric drug carriers. The synergy between computational predictions and experimental validations, as showcased here, signals a refined and precise approach to drug carrier design.

Targeted Molecular Therapeutics for Pulmonary Diseases: Addressing the Need for Precise Drug Delivery.

Respiratory diseases are a major concern in public health, impacting a large population worldwide. Despite the availability of therapies that alleviate symptoms, selectively addressing the critical points of pathopathways remains a major challenge. Innovative formulations designed for reaching these targets within the airways, enhanced selectivity, and prolonged therapeutic effects offer promising solutions. To provide insights into the specific medical requirements of chronic respiratory diseases, the initial focus of this chapter is directed on lung physiology, emphasizing the significance of lung barriers. Current treatments involving small molecules and the potential of gene therapy are also discussed. Additionally, we will explore targeting approaches, with a particular emphasis on nanoparticles, comparing targeted and non-targeted formulations for pulmonary administration. Finally, the potential of inhaled sphingolipids in the context of respiratory diseases is briefly discussed, highlighting their promising prospects in the field.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

ApoE-functionalization of nanoparticles for targeted brain delivery-a feasible method for polyplexes?

The blood-brain barrier (BBB) poses a major obstacle in the treatment of all types of central nervous system (CNS) diseases. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach by downregulating disease-related genes via RNA interference. However, the BBB is a formidable barrier for macromolecules such as nucleic acids. In an effort to develop a brain-targeted strategy for siRNA delivery systems formed by electrostatic interactions with cationic polymers (polyplexes (PXs)), we investigated the suitability of the well-known surfactant-based approach for Apolipoprotein E (ApoE)-functionalization of nanoparticles (NPs). The aim of this present work was to investigate if ApoE coating of siRNA PXs formed with cationic branched 25-kDa poly(ethyleneimine) (b-PEI) and nylon-3 polymers without or after precoating with polysorbate 80 (PS 80) would promote successful delivery across the BBB. We utilized highly hydrophobic NM0.2/CP0.8 nylon-3 polymers to evaluate the effects of hydrophobic cyclopentyl (CP) subunits on ApoE binding efficacy and observed successful ApoE binding with and without PS 80 precoating to the nylon-3 but not the PEI polyplexes. Accordingly, ApoE-coated nylon-3 polyplexes showed significantly increased uptake and gene silencing in U87 glioma cells but no benefit in vivo. In conclusion, further optimization of ApoE-functionalized polyplexes and more sophisticated in vitro models are required to achieve more successful in vitro-in vivo translation in future approaches.

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Evaluation of the effects of storage conditions on spray-dried siRNA-LNPs before and after subsequent drying.

In an ideal world, pharmaceutical drugs would have infinite shelf life, no susceptibility to degradation, chemical reactions or loss of efficacy. In reality, these processes occur, however, making it desirable to extend a drugs' shelf life. Nucleic acid-based drugs are most commonly stored as aqueous suspension where they are vulnerable to microbial growth and degradation processes. Drying procedures, such as lyophilization and spray drying, help to reduce the products' residual moisture while increasing the products' shelf life and stability. The present study was designed to evaluate 90 days of storage of spray-dried siRNA-lipid nanoparticles (LNPs) at 4 °C and 25 °C. An updated Onpattro® composition modified with a positively charged helper lipid was used as the LNP carrier system. In an attempt to further reduce the residual moisture of our previously reported formulations, all LNP samples were subjected to a secondary drying step in the spray drying tower for 20 min. The measurement of physicochemical properties of spray-dried and subsequently dried LNPs resulted in sizes of 180 nm, PDI values of 0.1-0.15 and zeta potentials of + 3 mV. Spray drying resulted in residual moisture levels of 3.6-4 % and was reduced by subsequent drying to 2.8-3.1 %. Aerodynamic properties after storage showed discrepancies depending on the storage conditions. MMADs remained at 2.8 µm when stored at 4 °C, whereas an increase to 5 µm at 25 °C was observed. Subsequent drying led to sizes of 3.6-3.8 µm, independent of the storage conditions. Spray-dried LNPs maintained bioactivity resulting in > 95 % protein downregulation and confirming the lack of cytotoxic effects in a lung adenocarcinoma cell line. Furthermore, the spray-dried and subsequently dried LNPs stored for 3 months at 4 °C and 25 °C achieved up to 50 % gene silencing of the house-keeping gene GAPDH after deposition on the mucus layer of Calu-3 cells. This study confirms the stability of spray-dried and subsequently dried LNPs over at least 90 days at 4 °C and 25 °C emphasizing the potential of dry powder inhalation of RNA-loaded LNPs as a therapy option for pulmonary diseases.

Characterization of critical parameters using an air-liquid interface model with RPMI 2650 cells for permeability studies of small molecules.

The field of nasal drug delivery gained enormously on interest over the past decade. Performing nasal in vivo studies is expensive and time-consuming, but also unfeasible for an initial high-throughput compound and formulation screening. Therefore, the development of fast and high-throughput in vitro models to screen compounds for their permeability through the nasal epithelium and mucosa is constantly expanding. Yet, the protocols used for nasal in vitro permeability studies are varying, which limits the comparability and reproducibility of generated data. This project aimed to elucidate the influence of different culture and assay parameters of RPMI 2650 cells grown under air-liquid interface (ALI) conditions on the transepithelial electrical resistance (TEER) and apparent permeability (Papp) values of five selected reference compounds, covering the range of low to moderate to high permeability. The influence of the passage number, seeding density, and timepoint of airlift was minimal in our approach, while the substrate pore density had a significant influence on the Papp values of carbamazepine, propranolol, and metoprolol, classified as highly permeable compounds, but not on atenolol and aciclovir. Elevation of the experimental concentration of carbamazepine, propranolol, and metoprolol in the donor compartment had an increasing effect on the Papp values, while prolonging the assay time did not have a significant influence. Based on the results reported here, RPMI 2650 cells cultured under ALI conditions offer the possibility of a standardized high-throughput screening model for small molecules and their formulations for in vitro drug permeation studies to predict and select optimal conditions for their nasal delivery.

Preliminary results from the EMoLung clinical study showing early lung cancer detection by the LC score.

BACKGROUND: Lung cancer (LC) causes more deaths worldwide than any other cancer type. Despite advances in therapeutic strategies, the fatality rate of LC cases remains high (95%) since the majority of patients are diagnosed at late stages when patient prognosis is poor. Analysis of the International Association for the Study of Lung Cancer (IASLC) database indicates that early diagnosis is significantly associated with favorable outcome. However, since symptoms of LC at early stages are unspecific and resemble those of benign pathologies, current diagnostic approaches are mostly initiated at advanced LC stages. METHODS: We developed a LC diagnosis test based on the analysis of distinct RNA isoforms expressed from the GATA6 and NKX2-1 gene loci, which are detected in exhaled breath condensates (EBCs). Levels of these transcript isoforms in EBCs were combined to calculate a diagnostic score (the LC score). In the present study, we aimed to confirm the applicability of the LC score for the diagnosis of early stage LC under clinical settings. Thus, we evaluated EBCs from patients with early stage, resectable non-small cell lung cancer (NSCLC), who were prospectively enrolled in the EMoLung study at three sites in Germany. RESULTS: LC score-based classification of EBCs confirmed its performance under clinical conditions, achieving a sensitivity of 95.7%, 91.3% and 84.6% for LC detection at stages I, II and III, respectively. CONCLUSIONS: The LC score is an accurate and non-invasive option for early LC diagnosis and a valuable complement to LC screening procedures based on computed tomography.

Synthesis and application of spermine-based amphiphilic poly(ß-amino ester)s for siRNA delivery.

Small interfering RNA (siRNA) can trigger RNA interference (RNAi) to therapeutically silence disease-related genes in human cells. The approval of siRNA therapeutics by the FDA in recent years generated a new hope in novel and efficient siRNA therapeutics. However, their therapeutic application is still limited by the lack of safe and efficient transfection vehicles. In this study, we successfully synthesized a novel amphiphilic poly(ß-amino ester) based on the polyamine spermine, hydrophobic decylamine and 1,4-butanediol diacrylate, which was characterized by 1H NMR spectroscopy and size exclusion chromatography (SEC, Mn = 6000 Da). The polymer encapsulated siRNA quantitatively from N/P 5 on as assessed by fluorescence intercalation while maintaining optimal polyplex sizes and zeta potentials. Biocompatibility and cellular delivery efficacy were also higher than those of the commonly used cationic, hyperbranched polymer polyethylenimine (PEI, 25 kDa). Optimized formulations mediated around 90% gene silencing in enhanced green fluorescence protein expressing H1299 cells (H1299-eGFP) as determined by flow cytometry. These results suggest that spermine-based, amphiphilic poly(ß-amino ester)s are very promising candidates for efficient siRNA delivery.

Spermine-Based Poly(ß-amino ester)s for siRNA Delivery against Mutated KRAS in Lung Cancer.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(ß-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(ß-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(ß-amino ester)s are promising materials for therapeutic siRNA delivery.

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models.

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)-derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

SARS-CoV-2 induced changes in the glycosylation pattern in the respiratory tract of Golden Syrian hamsters.

Even after more than two years of intensive research, not all of the pathophysiological processes of Coronavirus Disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, have been fully elucidated. The initial virus-host interaction at the respiratory epithelium plays a crucial role in the course and progression of the infection, and is highly dependent on the glycosylation pattern of the host cell and of the secreted mucins. Glycans are polysaccharides that can be attached to proteins and thereby add to their stability and functionality. Lectins are glycan-binding proteins that recognize specific glycan motifs, and lectin histochemistry is a suitable tool to visualize and examine glycosylation pattern changes in tissues. In this study we used lectins with different glycan-specificities for the visualization of glycosylation pattern changes in the respiratory tract of SARS-CoV-2 infected Golden Syrian hamsters. While some lectins (LEL, STL) enable the visualization of the damage to alveolar type 1 pneumocytes, other lectins, e.g., GSLI, visualized the loss and subsequent hyperplasia of type 2 pneumocytes. UEAI staining was co-localized with KI67, a proliferation marker. Double staining of lectins LEL, STL and WGA with specific immune cell markers (Iba1, CD68) showed co-localization and the dominant infiltration of monocyte-derived macrophages into infected alveolar tissue. The elucidation of the glycosylation pattern of the respiratory tract cells in uninfected and infected Golden Syrian hamsters revealed physiological and pathological aspects of the disease that may open new possibilities for therapeutic development.

Protein corona investigations of polyplexes with varying hydrophobicity - From method development to in vitro studies.

In the field of non-viral drug delivery, polyplexes (PXs) represent an advanced investigated and highly promising tool for the delivery of nucleic acids. Upon encountering physiological fluids, they adsorb biological molecules to form a protein corona (PC), that influence PXs biodistribution, transfection efficiencies and targeting abilities. In an effort to understand protein - PX interactions and the effect of PX material on corona composition, we utilized cationic branched 10 kDa polyethyleneimine (b-PEI) and a hydrophobically modified nylon-3 polymer (NM0.2/CP0.8) within this study to develop appropriate methods for PC investigations. A centrifugation procedure for isolating hard corona - PX complexes (PCPXs) from soft corona proteins after incubating the PXs in fetal bovine serum (FBS) for PC formation was successfully optimized and the identification of proteins by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method clearly demonstrated that the PC composition is affected by the underlying PXs material. With regard to especially interesting functional proteins, which might be able to induce active targeting effects, several candidates could be detected on b-PEI and NM0.2/CP0.8 PXs. These results are of high interest to better understand how the design of PXs impacts the PC composition and subsequently PCPXs-cell interactions to enable precise adjustment of PXs for targeted drug delivery.

An In Vitro Approach to Model EMT in Breast Cancer.

During the progression from ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC), cells must overcome the physically restraining basement membrane (BM), which compartmentalizes the epithelium from the stroma. Since the extracellular matrix (ECM) of the epithelial and stromal compartments are biochemically and physically distinct from one another, the progression demands a certain degree of cellular plasticity for a primary tumor to become invasive. The epithelial-to-mesenchymal transition (EMT) depicts such a cell program, equipping cancer cells with features allowing for dissemination from the epithelial entity and stromal invasion at the single-cell level. Here, the reciprocal interference between an altering tumor microenvironment and the EMT phenotype was investigated in vitro. BM-typical collagen IV and stroma-typical collagen I coatings were applied as provisional 2D matrices. Pro-inflammatory growth factors were introduced to improve tissue mimicry. Whereas the growth on coated surfaces only slightly affected the EMT phenotype, the combinatorial action of collagen with growth factor TGF-ß1 induced prominent phenotypic changes. However, EMT induction was independent of collagen type, and cellular accessibility for EMT-like changes was strongly cell-line dependent. Summarizing the entire body of data, an EMT-phenotyping model was used to determine cellular EMT status and estimate EMT-like changes. The miR200c-mediated reversion of mesenchymal MDA-MB-231 cells is reflected by our EMT-phenotype model, thus emphasizing its potential to predict the therapeutic efficacy of EMT-targeting drugs in the future.

Microfluidic mixing as platform technology for production of chitosan nanoparticles loaded with different macromolecules.

In recent decades, biotechnological drugs have emerged as relevant therapeutic tools. However, therapeutic molecules can exert their activity only if properly formulated and delivered into the body. In this regard, nano-sized drug delivery systems have been shown to provide protection, stability, and controlled release of payloads, increasing their therapeutic efficacy. In this work, a microfluidic mixing technique for the preparation of chitosan-based nanoparticles was established with the capability of easily exchanging macromolecular biological cargos such as the model protein ß-Galactosidase, mRNA, and siRNA. The nanoparticles obtained showed hydrodynamic diameters ranging from 75 nm to 105 nm, low polydispersity of 0.15 to 0.22 and positive zeta potentials of 6 mV to 17 mV. All payloads were efficiently encapsulated (>80 %) and the well-known cytocompatibility of chitosan-based nanoparticles was confirmed. Cell culture studies demonstrated increased cellular internalization of loaded nano-formulations compared to free molecules as well as successful gene silencing with nano-formulated siRNA, suggesting the ability of these nanoparticles to escape the endosome.

Shaping the future from the small scale: dry powder inhalation of CRISPR-Cas9 lipid nanoparticles for the treatment of lung diseases.

INTRODUCTION: Most lung diseases are serious conditions resulting from genetic and environmental causes associated with high mortality and severe symptoms. Currently, treatments available have a palliative effect and many targets are still considered undruggable. Gene therapy stands as an attractive approach to offering innovative therapeutic solutions. CRISPRCas9 has established a remarkable potential for genome editing with high selectivity to targeted mutations. To ensure high efficacy with minimum systemic exposure, the delivery and administration route are key components that must be investigated. AREAS COVERED: This review is focused on the delivery of CRISPRCas9 to the lungs, taking advantage of lipid nanoparticles (LNPs), the most clinically advanced nucleic acid carriers. We also aim to highlight the benefits of pulmonary administration as a local delivery route and the use of spray drying to prepare stable nucleic-acid-based dry powder formulations that can overcome multiple lung barriers. EXPERT OPINION: Exploring the pulmonary administration to deliver CRISPRCas9 loaded in LNPs as a dry powder increases the chances to achieve high efficacy and reduced adverse effects. CRISPRCas9 loaded in LNP-embedded microparticles has not yet been reported in the literature but has the potential to reach and accumulate in target cells in the lung, thus, enhancing overall efficacy and safety.

Targeted GATA3 knockdown in activated T cells via pulmonary siRNA delivery as novel therapy for allergic asthma.

GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma.