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Alicja Hinz,1 Sylwia Stankiewicz,1,2 Jacek Jakub Litewka,2,3 Paweł E Ferdek,3 Maja Sochalska,4 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Doctoral School of Exact and Natural Sciences Jagiellonian University, Kraków, Poland; 3Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 4Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, PolandCorrespondence: Monika Bzowska, Email monika.bzowska@uj.edu.plPurpose: Given the increased use of polyethylene glycol (PEG) in refining the therapeutic activity of medicines, our research focuses on explaining the potential mechanism of immune reactions associated with this polymer. We aim to investigate the interaction of different types of PEG with mouse and human immune cells, thereby contributing to understanding PEG interactions with the immune system and verifying the proinflammatory activity of the tested polymers.Patients and Methods: Mouse macrophage and neutrophil cell lines, human peripheral blood mononuclear cells, and polymorphonuclear cells isolated from healthy donors were exposed to various PEGs. ROS, NO, and cytokine production were analyzed using fluorescence intensity, absorbance, or cytometric measurements. Toll-like receptor (TLR) signaling was verified using HEK-blue-reporter cell lines. Finally, neutrophil trap formation was studied using immunofluorescence labeling, and calcium imaging was performed using a Ca2+-sensitive indicator and fluorescence microscope.Results: Our findings show that specific PEG and mPEG are not toxic to tested mouse and human cells. However, they exert proinflammatory activity against human immune cells, as evidenced by the increased secretion of proinflammatory cytokines, such as IFN-a2, IFN-γ, TNF-α, MCP-1, IL-8, IL-17A, and IL-23. This phenomenon is independent of PEG signaling via TLR. Additionally, mPEG10 induced the formation of neutrophil extracellular traps and intracellular calcium signaling.Conclusion: Our finding suggests that some PEG types have proinflammatory activity against human immune cells, manifesting in the upregulated production of cytokines and neutrophils trap releasing.Keywords: drug delivery, immunomodulation, in vitro nanotoxicity, nanoparticle functionalization, PEGylated therapeutics

Purpose: Given the increased use of polyethylene glycol (PEG) in refining the therapeutic activity of medicines, our research focuses on explaining the potential mechanism of immune reactions associated with this polymer. We aim to investigate the interaction of different types of PEG with mouse and human immune cells, thereby contributing to understanding PEG interactions with the immune system and verifying the proinflammatory activity of the tested polymers., Patients and Methods: Mouse macrophage and neutrophil cell lines, human peripheral blood mononuclear cells, and polymorphonuclear cells isolated from healthy donors were exposed to various PEGs. ROS, NO, and cytokine production were analyzed using fluorescence intensity, absorbance, or cytometric measurements. Toll-like receptor (TLR) signaling was verified using HEK-blue-reporter cell lines. Finally, neutrophil trap formation was studied using immunofluorescence labeling, and calcium imaging was performed using a Ca 2+ -sensitive indicator and fluorescence microscope., Results: Our findings show that specific PEG and mPEG are not toxic to tested mouse and human cells. However, they exert proinflammatory activity against human immune cells, as evidenced by the increased secretion of proinflammatory cytokines, such as IFN-a2, IFN-γ, TNF-α, MCP-1, IL-8, IL-17A, and IL-23. This phenomenon is independent of PEG signaling via TLR. Additionally, mPEG10 induced the formation of neutrophil extracellular traps and intracellular calcium signaling., Conclusion: Our finding suggests that some PEG types have proinflammatory activity against human immune cells, manifesting in the upregulated production of cytokines and neutrophils trap releasing., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Hinz et al.)

Background: Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations., Methods: EVs were isolated from three hiPSC lines cultured under normoxia (21% O 2 ; EV-N) or reduced oxygen concentration (hypoxia): 3% O 2 (EV-H3) or 5% O 2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis., Results: We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFβ/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue., Conclusions: In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration., (© 2023. The Author(s).)