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Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications.

Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

Recent technological advances in the production of in vitro transcribed messenger RNA (IVT‐mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT‐mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT‐mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT‐mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell‐free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification‐dependent steps in the complex delivery process.

Beyond SARS‐CoV2 vaccines, mRNA drugs are being explored to overcome today's greatest healthcare burdens, including cancer and cardiovascular disease. Synthetic mRNA triggers immune responses in transfected cells, which can be reduced by chemically modified nucleotides. However, the side effects of mRNA‐triggered immune activation on cell function and how different nucleotides, such as the N1‐methylpseudouridine (m1Ψ) used in SARS‐CoV2 vaccines, can modulate cellular responses is not fully understood. Here, cellular responses toward a library of uridine‐modified mRNAs are investigated in primary human cells. Targeted proteomics analyses reveal that unmodified mRNA induces a pro‐inflammatory paracrine pattern marked by the secretion of chemokines, which recruit T and B lymphocytes toward transfected cells. Importantly, the magnitude of mRNA‐induced changes in cell function varies quantitatively between unmodified, Ψ‐, m1Ψ‐, and 5moU‐modified mRNA and can be gradually tailored, with implications for deliberately exploiting this effect in mRNA drug design. Indeed, both the immunosuppressive effect of stromal cells on T‐cell proliferation, and the anti‐inflammatory effect of IL‐10 mRNA are enhanced by appropriate uridine modification. The results provide new insights into the effects of mRNA drugs on cell function and cell‐cell communication and open new possibilities to tailor mRNA‐triggered immune activation to the desired pro‐ or anti‐inflammatory application. Synthetic mRNA can serve as a template for protein overexpression but also as an immune stimulant. Unmodified mRNA triggers anti‐viral‐like responses in transfected cells. It induces the secretion of lymphocyte‐attracting chemokines, which recruit T and B lymphocytes. This effect can be gradually tailored by modifying the uridine chemistry, which creates opportunities for deliberately exploiting immune activation in mRNA drugs.

Background A folate-receptor-targeted poly (lactide-co-Glycolide) (PLGA)-Polyethylene glycol (PEG) nanoparticle is developed for encapsulation and delivery of disulfiram into breast cancer cells. After a comprehensive characterization of nanoparticles, cell cytotoxicity, apoptosis induction, cellular uptake and intracellular level of reactive oxygen species are analyzed. In vivo acute and chronic toxicity of nanoparticles and their efficacy on inhibition of breast cancer tumor growth is studied. Results The folate-receptor-targeted nanoparticles are internalized into the cells, induce reactive oxygen species formation, induce apoptosis and inhibit cell proliferation more efficiently compared to the untargeted nanoparticles. The acute and toxicity test show the maximum dose of disulfiram equivalent of nanoparticles for intra-venous injection is 6 mg/kg while show significant decrease in the breast cancer tumor growth rate. Conclusion It is believed that the developed formulation could be used as a potential vehicle for successful delivery of disulfiram, an old and inexpensive drug, into breast cancer cells and other solid tumors. Graphical abstract Disulfiram, an old and inexpensive drug, is encapsulated in folate-targeted PLGA-PEG nanoparticles and delivered into breast cancer cells using passive and active targeting to inhibit tumor growth in mice

Polymeric carriers are gaining increasing attention for the delivery of a range of clinically relevant nucleic acid (NA) payloads, due to their efficacy, safety, and modular chemistry. While the requirements for an \"optimal\" carrier depend strongly on the NA payload, research has largely centered on carrier properties rather than the unique needs of the payload. Therefore, in this review, a novel perspective inspired by protein structural organization is introduced to explore structure-function relationships of polymeric carrier systems, highlighting the diverse requirements of different NA payloads. To gain deeper mechanistic insights, the complicated polymer/NA delivery systems are deconvoluted into four hierarchical levels, including molecular composition, structural features, complexation, and 3D integration. In addition to outlining recent experimental strategies, it is emphasized that the structural diversity and tunability of polymer chemistry provide a foundation for payload-specific carrier design. Critical advances are expected in the molecular design of polymeric carriers to enhance NA delivery efficiency, improve biological safety and incorporate multifunctionality, particularly compared to lipid-based platforms that are currently ahead in clinical applications. A payload-specific design framework may guide development of next-generation polymeric carrier systems for basic research, biotechnology, and therapeutic applications.

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Angiopoietin ligands Ang1 and Ang2 and the Tie2 receptor tyrosine kinases form an endothelial signaling pathway regulating vascular homeostasis and controlling vessel permeability, inflammation and angiogenic responses. Whereas Ang1-mediated Tie2 activation reduces inflammation and endothelial permeability, its antagonist, Ang2 increases it. Increased plasma Ang2 levels are associated with poor outcomes in patients with acute lung injury (ALI), as well as in acute respiratory distress syndrome (ARDS). In the study presented here we tested the effect of a novel synthetic, nucleoside-modified mRNA-76 encoding for a hyperactive Ang1 derived fusion protein (COMP-Ang1) on attenuating post-inflammation vascular leakage. COMP-Ang1 mRNA was formulated into a cationic lipid nanoparticle (cLNP) using an optimized mixture of three different lipids and a microfluidic mixing technology. After intravenous injection, the respective mRNA-loaded LNPs were found to be delivered predominantly to the endothelial cells of the lung, while sparing other vascular beds. Also, the specific multimeric folding of the COMP-Ang1 protein complex appeared to be pivotal for its activity in preventing vascular leakage and in restoring the alveolar-endothelial barrier function in the inflamed and injured pulmonary vasculature. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo by systemic administration in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in an LPS-challenging model.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Introduction and Discussion updated. Names of 2 authors were misspelled.

Purpose: To evaluate the safety and efficacy of viscocanalostomy in the management of medically uncontrollable primary open-angle glaucoma (POAG) in a developing country. Materials and Methods: This is a prospective, non-randomized case series of 14 consecutive eyes with medically uncontrollable POAG, all subjected to viscocanalostomy. The main outcome measure was success rate based on the intraocular pressure (IOP) level achieved post-operatively. The procedure was considered a complete success if IOP was less than 21 mmHg without any anti-glaucoma medication. Qualified success was defined as IOP of less than 21 mmHg with anti-glaucoma medication. All patients had a regular follow-up of at least 12 months. Results: IOP was reduced from a mean baseline value of 27.9 ± 7.3 mmHg (range: 21-40 mmHg) to a mean final value of 16.0 ± 2.7 mmHg (range: 13-22 mmHg), which was statistically highly significant (P < 0.005). The mean number of pre-operative anti-glaucoma medications was 3.0 ± 0.4 (range: 2-4), which was reduced significantly (P < 0.0001) to 0.3 ± 0.6 (range: 0-2) at the last follow-up visit. One year post-operatively, complete success was achieved in 71% and qualified success was observed in 21.4% of patients, summing up to an overall success rate of 92.4%. There were no major complications in any of the patients. Conclusion: Viscocanalostomy could be performed effectively and safely for control of POAG in developing countries.