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Justyna Śmiałek,1,* Monika Bzowska,2,* Alicja Hinz,2 Renata Mężyk-Kopeć,2 Kamilla Sołtys,2 Paweł Mak1 1Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland*These authors contributed equally to this workCorrespondence: Paweł Mak, Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., Kraków, 30-387, Poland, Tel +48 12 664 6511, Fax +48 12 664 6902, Email pawel.mak@uj.edu.plPurpose: The zoonotic opportunistic pathogen Staphylococcus pseudintermedius 222 produces BacSp222 – an atypical peptide exhibiting the features of a bacteriocin, a virulence factor, and a molecule modulating the host inflammatory reaction. The peptide is secreted in an unmodified form and, additionally, two forms modified posttranslationally by succinylation. This study is a comprehensive report focusing on the proinflammatory properties of such molecules.Methods: The study was performed on mouse monocyte/macrophage-like and endothelial cell lines as well as human neutrophils. The following peptides were studied: BacSp222, its succinylated forms, the form deprived of formylated methionine, and a reference bacteriocin – nisin. The measurements of the nitric oxide (NO) level, induced NO synthase (iNOS) expression, the profile of secreted cytokines, NF-kappa-B activation, reactive oxygen species (ROS) biosynthesis, and the formation of extracellular traps were conducted to evaluate the proinflammatory activity of the studied peptides.Results: BacSp222 and its succinylated forms effectively induced NO production and iNOS expression when combined with IFN-gamma in macrophage-like cells. All natural BacSp222 forms used alone or with IFN-gamma stimulated the production of TNF-alpha, MCP-1, and IL-1-alpha, while the co-stimulation with IFN-gamma increased IL-10 and IL-27. Upregulated TNF-alpha secretion observed after BacSp222 exposition resulted from increased expression but not from membrane TNF-alpha proteolysis. In neutrophils, all forms of bacteriocin upregulated IL-8, but did not induce ROS production or NETs formation. In all experiments, the activities of deformylated bacteriocin were lower or unequivocal in comparison to other forms of the peptide.Conclusion: All naturally secreted forms of BacSp222 exhibit proinflammatory activity against monocyte-macrophage cells and neutrophils, confirming that the biological role of BacSp222 goes beyond bactericidal and cytotoxic effects. The atypical posttranslational modification (succinylation) does not diminish its immunomodulatory activity in contrast to the lower antibacterial potential or cytotoxicity of such modified form established in previous studies.Keywords: cytokines, neutrophils, NF-kappa-B activation, nisin, nitric oxide, posttranslational modifications, Staphylococcus pseudintermedius 222

Alicja Karabasz,1 Monika Bzowska,1 Krzysztof Szczepanowicz2 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Kraków, PolandCorrespondence: Monika BzowskaDepartment of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 7 Gronostajowa Street, Kraków 30-387, PolandTel/Fax +48 12 664 63 88Email monika.bzowska@uj.edu.plKrzysztof SzczepanowiczJerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, 8 Niezapominajek Street, Kraków 30-239, PolandTel/Fax +48 12 639 51 21Email ncszczep@cyf-kr.edu.plAbstract: Polymeric nanomaterials have become a prominent area of research in the field of drug delivery. Their application in nanomedicine can improve bioavailability, pharmacokinetics, and, therefore, the effectiveness of various therapeutics or contrast agents. There are many studies for developing new polymeric nanocarriers; however, their clinical application is somewhat limited. In this review, we present new complex and multifunctional polymeric nanocarriers as promising and innovative diagnostic or therapeutic systems. Their multifunctionality, resulting from the unique chemical and biological properties of the polymers used, ensures better delivery, and a controlled, sequential release of many different therapeutics to the diseased tissue. We present a brief introduction of the classical formulation techniques and describe examples of multifunctional nanocarriers, whose biological assessment has been carried out at least in vitro. Most of them, however, also underwent evaluation in vivo on animal models. Selected polymeric nanocarriers were grouped depending on their medical application: anti-cancer drug nanocarriers, nanomaterials delivering compounds for cancer immunotherapy or regenerative medicine, components of vaccines nanomaterials used for topical application, and lifestyle diseases, ie, diabetes.Keywords: polymeric nanocarriers, biomedical application, anti-cancer, cancer immunotherapy, modern vaccines, regenerative medicine, lifestyle diseases – diabetes

Alicja Karabasz1, Dorota Lachowicz2, Anna Karewicz3, Renata Mezyk-Kopec1, Krystyna Stalińska1, Ewa Werner4,5, Agnieszka Cierniak6, Grzegorz Dyduch7, Joanna Bereta1, Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, Kraków, Poland; 3Faculty of Chemistry, Jagiellonian University, Kraków, Poland; 4Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 5Department of Animal Reproduction and Anatomy, Faculty of Animal Science, University of Agriculture, Krakow, Poland; 6Department of General Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 7Department of Pathomorphology, Jagiellonian University Medical College, Kraków, PolandCorrespondence: Monika BzowskaJagiellonian University in Kraków, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biochemistry, 7 Gronostajowa St., Kraków 30-387, PolandTel +48 12 664 6388Fax +48 12 664 6388Email monika.bzowska@uj.edu.plBackground: Curcumin is a natural polyphenol with anti-inflammatory, chemopreventive and anticancer activity. However, its high hydrophobicity and poor bioavailability limit its medical application. The development of nanocarriers for curcumin delivery is an attractive approach to overcome its low bioavailability and fast metabolism in the liver. We synthesized a blood compatible alginate-curcumin conjugate, AA-Cur, which formed colloidally stable micelles of approximately 200 nm and, as previously shown, exerted strong cytotoxicity against mouse cancer cell lines. Here we analyze in vivo toxicity and antitumor activity of AA-Cur in two different mouse tumor models.Method: Potential toxicity of intravenously injected AA-Cur was evaluated by: i) analyses of blood parameters (morphology and biochemistry), ii) histology, iii) DNA integrity (comet assay), and iv) cytokine profiling (flow cytometry). Antitumor activity of AA-Cur was evaluated by measuring the growth of subcutaneously inoculated colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in control mice vs mice treated with AA-Cur.Results: Injections of four doses of AA-Cur did not reveal any toxicity of the conjugate, thus indicating the safety of its use. AA-Cur elicited moderate anti-tumor activity toward colon MC38-CEA or breast 4T1 carcinomas.Conclusion: The tested conjugate of alginate and curcumin, AA-Cur, is non-toxic and safe, but exhibits limited anticancer activity.Keywords: curcumin, alginate conjugates, in vivo toxicity, antitumor activity