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Piktel E, Markiewicz KH, Wilczewska AZ, et al. Int J Nanomedicine. 2020;15:4573–4589. It has been brought to the authors attention that Figure 6E showing nanosystem-mediated DNA fragmentation in DLD-1 colorectal cancer cells is a duplicate of part of Figure 3C that was previously published in the review article: Wnorowska U, Fiedoruk K, Piktel E, et al. J Nanobiotechnology. 2020;18(1):3 (https://doi.org/10.1186/s12951-019-0566-z). In addition, both Figures 5E and 6E were described with incorrect treatment conditions. Data obtained during corresponding experiments were verified; consequently, Figure 5E and 6E were updated using data from the original study. The correct Figure 5 is as follows. Figure 5 Continued. Figure 5 Anticancer activity of ceragenin CSA-131 and ceragenin-containing nanoformulations against lung carcinoma A549 cells. Increase of intracellular levels of reduced thiols in A549 cells treated with CSA-131 (grey bar), MNP@CSA-131 (red bar), CSA-131+MNP (blue bar) and MNP (yellow bar) when compared to untreated control (0 μg/mL; black bar) (A). The percentages of dead cells (black columns), PI-negative cells with low viability (dark grey columns) and healthy cells (light grey columns) in lung carcinoma cells treated with CSA-131, MNP@CSA-131, CSA-131 and MNP or naked MNPs (B). The proliferation of cancer cells treated with CSA-131 (grey squares), MNP@CSA-131 (red circles), CSA-131 + MNP (blue diamonds) and MNP (yellow inverted triangles) when compared to untreated control (black squares) estimated using resazurin-based fluorimetric method (C). Induction of apoptosis in A549 cells by CSA-131 and its magnetic derivatives (D). Percentage of early apoptotic (black columns), late apoptotic/dead cells (dark grey columns) and dead cells (light grey columns). For the purpose of the clarity of the presented data, live cells (Annexin V-negative and 7-AAD-negative) were not presented in the provided figures. Morphological alternations in nuclei of A549 cells upon treatment with CSA-131, MNP@CSA-131 and CSA-131 + MNP when compared to uncreated cells (E). White arrows indicate treatment-induced morphological changes in nuclei of treated cells. All experiments were performed using agents at a concentration of 10 μg/mL for 24 h. (A–D) demonstrate results from 3 to 6 individual experiments ± SD, for panel E results from one representative experiment are shown. * and ^ indicate statistical significance (p-value

Ewelina Piktel,1 Ilona Ościłowska,2 Łukasz Suprewicz,1 Joanna Depciuch,3 Natalia Marcińczyk,4 Ewa Chabielska,4 Przemysław Wolak,5 Tomasz Wollny,6 Marianna Janion,5 Magdalena Parlinska-Wojtan,3 Robert Bucki1 1Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Bialystok, 15-222, Poland; 2Department of Medicinal Chemistry, Medical University of Bialystok, Bialystok, 15-222, Poland; 3Institute of Nuclear Physics Polish Academy of Sciences, Krakow, PL-31342, Poland; 4Department of Biopharmacy, Medical University of Bialystok, Bialystok, 15-222, Poland; 5 Institute of Medical Sciences, Collegium Medicum, Jan Kochanowski University, Kielce, 25-317, Poland; 6Holy Cross Cancer Center in Kielce, Kielce, 25-734, PolandCorrespondence: Robert BuckiDepartment of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Mickiewicza 2C, Bialystok, 15-222, PolandTel + 48 85 748 5793Email buckirobert@gmail.comBackground: Even with considerable improvement in treatment of epithelial ovarian cancer achieved in recent years, an increasing chemotherapy resistance and disease 5-year relapse is recorded for a majority part of patients that encourages the search for better therapeutic options. Gold nanoparticles (Au NPs) due to plethora of unique physiochemical features are thoroughly tested as drug delivery, radiosensitizers, as well as photothermal and photodynamic therapy agents. Importantly, due to highly controlled synthesis, it is possible to obtain nanomaterials with directed size and shape.Methods: In this work, we developed novel elongated-type gold nanoparticles in the shape of nanopeanuts (AuP NPs) and investigated their cytotoxic potential against ovarian cancer cells SKOV-3 using colorimetric and fluorimetric methods, Western blot, flow cytometry, and fluorescence microscopy.Results: Peanut-shaped gold nanoparticles showed high anti-cancer activity in vitro against SKOV-3 cells at doses of 1– 5 ng/mL upon 72 hours treatment. We demonstrate that AuP NPs decrease the viability and proliferation capability of ovarian cancer cells by triggering cell apoptosis and autophagy, as evidenced by flow cytometry and Western blot analyses. The overproduction of reactive oxygen species (ROS) was noted to be a critical mediator of AuP NPs-mediated cell death.Conclusion: These data indicate that gold nanopeanuts might be developed as nanotherapeutics against ovarian cancer.Keywords: gold nanoparticles, ovarian cancer, gold nanopeanuts, anti-cancer therapy, nanotechnology, apoptosis, autophagy

Mateusz Cieśluk,1 Katarzyna Pogoda,2 Piotr Deptuła,1 Paulina Werel,3 Alina Kułakowska,3 Jan Kochanowicz,3 Zenon Mariak,4 Tomasz Łysoń,4 Joanna Reszeć,5 Robert Bucki1 1Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Bialystok PL-15222, Poland; 2Institute of Nuclear Physics, Polish Academy of Sciences, Krakow PL-31342, Poland; 3Department of Neurology, Medical University of Bialystok, Bialystok PL-15276, Poland; 4Department of Neurosurgery, Medical University of Bialystok, Bialystok PL-15276, Poland; 5Department of Pathology, Medical University of Bialystok, Bialystok PL-15269, PolandCorrespondence: Robert BuckiDepartment of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Mickiewicza 2C, Bialystok 15-222, PolandTel +48 85 784 5483Email buckirobert@gmail.comBackground: The tissue-mechanics environment plays a crucial role in human brain physiological development and the pathogenesis of different diseases, especially cancer. Assessment of alterations in brain mechanical properties during cancer progression might provide important information about possible tissue abnormalities with clinical relevance.Methods: With atomic force microscopy (AFM), the stiffness of freshly removed human brain tumor tissue was determined on various regions of the sample and compared to the stiffness of healthy human brain tissue that was removed during neurosurgery to gain access to tumor mass. An advantage of indentation measurement using AFM is the small volume of tissue required and high resolution at the single-cell level.Results: Our results showed great heterogeneity of stiffness within metastatic cancer or primary high-grade gliomas compared to healthy tissue. That effect was not clearly visible in lower-grade tumors like meningioma.Conclusion: Collected data indicate that AFM might serve as a diagnostic tool in the assessment of human brain tissue stiffness in the process of recognizing tumors.Keywords: AFM, brain tumors, human tissue rheology, mechanomarkers, glioblastoma

Ewelina Piktel,1 Karolina H Markiewicz,2 Agnieszka Z Wilczewska,2 Tamara Daniluk,1 Sylwia Chmielewska,1 Katarzyna Niemirowicz-Laskowska,1 Joanna Mystkowska,3 Paulina Paprocka,4 Paul B Savage,5 Robert Bucki1,4 1Department of Medical Microbiology and Nanobiomedical Engineering, Medical University of Bialystok, Bialystok 15-222, Poland; 2Faculty of Chemistry, University of Bialystok, Bialystok 15-245, Poland; 3Department of Materials and Biomedical Engineering, Białystok University of Technology, Białystok 15-351, Poland; 4Department of Microbiology and Immunology, The Faculty of Medicine and Health Sciences, Jan Kochanowski University in Kielce, Kielce 25-317, Poland; 5Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84604, USACorrespondence: Robert Bucki Email buckirobert@gmail.comBackground: Therapeutic efficiency of ceragenins against cancers may be limited by lack of their hemocompatibility when high concentrations of molecules are required to reach a desired result. Synergistic effects observed upon administration of anticancer agents and metal nanoparticles may provide an opportunity to limit toxicity of immobilized ceragenins on the surface of metal nanoparticles and to improve their therapeutic efficiency at the same time. The aim of present work is to investigate the anticancer activities and hemocompatibility of nanoformulations consisting of ceragenin CSA-131 united with aminosilane-modified iron oxide-based magnetic nanoparticles (MNP) and prepared by 1) covalent bonding (MNP@CSA-131) or 2) by combining CSA-131 with MNP in 1:1 ratio (CSA-131 + MNP). Possible synergistic interactions between CSA-131 and magnetic nanoparticles were also quantified.Methods: MNP@CSA-131 and CSA-131+MNP were tested in vitro against selected lung and colon cancer cells using colorimetric, fluorimetric and flow cytometry methods.Results: Performed analysis demonstrates that MNP-based nanosystems significantly improve the killing efficiency of tested ceragenin, decreasing the viability of extra 1.37± 4.72% to 76.07± 15.30% cancer cells when compared to free CSA-131. Quantification of synergistic effects indicates the favorable interactions between CSA-131 and magnetic nanoparticles (CI < 1 for all tested doses), revealing at the same time a reduction in effective doses of ceragenin from 1.17 ± 0.61 to 34.57 ± 12.78 times when combined with MNP. We demonstrate that both MNP@CSA-131 and CSA-131+MNP induce significantly apoptosis of cancer cells and prevent the division of colon cancer cells even at relatively low doses of the active compound (10 μg/mL). Importantly, combining CSA-131 with MNP decreases the hemolytic activity of free ceragenin 4.72 to 7.88 times, which indicates a considerable improvement of hemotoxicity profile.Conclusion: Comparative analyses have revealed that both developed CSA-containing nanoformulations due to the utility of synergistic interactions between MNP and CSA-131, which are effective against lung and colon cancer cells. This indicates the new directions in preparation of MNP-based therapeutics, which are relatively easy to synthetize, cost-effective and safe when intravenously administrated.Keywords: ceragenins, anticancer activity, colon cancer, lung cancer, synergistic effects, combinatory therapy

Katarzyna Niemirowicz-Laskowska,1 Katarzyna Głuszek,1 Ewelina Piktel,1 Karlis Pajuste,2 Bonita Durnaś,3 Grzegorz Król,3 Agnieszka Z Wilczewska,4 Paul A Janmey,5 Aiva Plotniece,2 Robert Bucki1 1Department of Microbiological and Nanobiomedical Engineering, Medical University of Bialystok, Bialystok, Poland; 2Laboratory of Membrane Active Compounds and β-Diketones, Latvian Institute of Organic Synthesis, Riga, Latvia; 3Department of Microbiology and Immunology, The Faculty of Health Sciences of the Jan Kochanowski University in Kielce, Kielce, 4Institute of Chemistry, University of Bialystok, Białystok, Poland; 5Department of Physiology, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA Background: 1,4-Dihydropyridine (1,4-DHP) and its derivatives are well-known calcium channel blockers with antiarrhythmic and antihypertensive activities. These compounds exhibit pleiotropic effects including antimicrobial activities that rely on their positive charge and amphipathic nature. Use of magnetic nanoparticles (MNPs) as carriers of 1,4-DHP modulates their properties and enables improved formulations with higher efficacy and less toxicity. Methods: In this study, the antimicrobial and immunomodulatory activities of novel 1,4-DHP derivatives in free form and immobilized on MNPs were determined by evaluating pathogen outgrowth and proinflammatory cytokine release in experimental settings that involve incubation of various 1,4-DHPs with clinical isolates of bacteria or fungi as well as mammalian cell culture models. Results: Conventional immobilization of 1,4-DHP on aminosilane-coated MNPs markedly enhances their antimicrobial activity compared to nonimmobilized molecules, in part because of the higher affinity of these nanosystems for bacterial cell wall components in the presence of human body fluids. Conclusion: Optimized nanosystems are characterized by improved biocompatibility and higher anti-inflammatory properties that provide new opportunities for the therapy of infectious diseases. Keywords: magnetic nanoparticles, 1,4-dihydropyridine, antibacterial, antifungal, immunomodulatory properties

Grażyna Tokajuk,1,2 Katarzyna Niemirowicz,1 Piotr Deptuła,1,3 Ewelina Piktel,1 Mateusz Cieśluk,1 Agnieszka Z Wilczewska,4 Jan R Dąbrowski,3 Robert Bucki1 1Department of Microbiological and Nanobiomedical Engineering, Medical University of Białystok, 2Department of Intergrated Dentistry, Medical University of Białystok, 3Department of Materials and Biomedical Engineering, Białystok University of Technology, 4Institute of Chemistry, University of Białystok, Białystok, Poland Abstract: Nanotechnology offers new tools for developing therapies to prevent and treat oral infections, particularly biofilm-dependent disorders, such as dental plaques and endodontic and periodontal diseases. Chlorhexidine (CHX) is a well-characterized antiseptic agent used in dentistry with broad spectrum activity. However, its application is limited due to inactivation in body fluid and cytotoxicity toward human cells, particularly at high concentrations. To overcome these limitations, we synthesized nanosystems composed of aminosilane-coated magnetic nanoparticles functionalized with chlorhexidine (MNP@CHX). In the presence of human saliva, MNPs@CHX displayed significantly greater bactericidal and fungicidal activity against planktonic and biofilm-forming microorganisms than free CHX. In addition, CHX attached to MNPs has an increased ability to restrict the growth of mixed-species biofilms compared to free CHX. The observed depolarization of mitochondria in fungal cells treated with MNP@CHX suggests that induction of oxidative stress and oxidation of fungal structures may be a part of the mechanism responsible for pathogen killing. Nanoparticles functionalized by CHX did not affect host cell proliferation or their ability to release the proinflammatory cytokine, IL-8. The use of MNPs as a carrier of CHX has great potential for the development of antiseptic nanosystems. Keywords: chlorhexidine, magnetic nanoparticles, antimicrobial properties, anti-biofilm

Katarzyna Niemirowicz,1 Ewelina Piktel,1 Agnieszka Z Wilczewska,2 Karolina H Markiewicz,2 Bonita Durnaś,3 Marzena Wątek,4 Irena Puszkarz,3 Marta Wróblewska,5,6 Wieslawa Niklińska,7 Paul B Savage,8 Robert Bucki1,3 1Department of Microbiological and Nanobiomedical Engineering, Medical University of Bialystok, 2Institute of Chemistry, University of Bialystok, Bialystok, 3Department of Physiology, Pathophysiology and Immunology of Infections, The Faculty of Health Sciences of the Jan Kochanowski University in Kielce, 4Holy Cross Oncology Center of Kielce, Kielce, 5Department of Dental Microbiology, Medical University of Warsaw, 6Department of Microbiology, Central Clinical Hospital in Warsaw, Warsaw, 7Department of Histology and Embryology, Medical University of Bialystok, Bialystok, Poland; 8Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA Abstract: Core–shell magnetic nanoparticles (MNPs) are promising candidates in the development of new treatment methods against infections, including those caused by antibiotic-resistant pathogens. In this study, the bactericidal activity of human antibacterial peptide cathelicidin LL-37, synthetic ceragenins CSA-13 and CSA-131, and classical antibiotics vancomycin and colistin, against methicillin-resistant Staphylococcus aureus Xen 30 and Pseudomonas aeruginosa Xen 5, was assessed alone and in combination with core–shell MNPs. Fractional inhibitory concentration index and fractional bactericidal concentration index were determined by microdilution methods. The potential of combined therapy using nanomaterials and selected antibiotics was confirmed using chemiluminescence measurements. Additionally, the ability of tested agents to prevent bacterial biofilm formation was evaluated using crystal violet staining. In most conditions, synergistic or additive effects were observed when combinations of core–shell MNPs with ceragenins or classical antibiotics were used. Our study revealed that a mixture of membrane-active agents such as LL-37 peptide or ceragenin CSA-13 with MNPs potentialized their antibacterial properties and might be considered as a method of delaying and overcoming bacterial drug resistance. Keywords: synergistic activity, antibiotic-resistant bacteria, LL-37 peptide, ceragenins, magnetic nanoparticles

Katarzyna Niemirowicz,1 Izabela Prokop,2 Agnieszka Z Wilczewska,3 Urszula Wnorowska,1 Ewelina Piktel,1 Marzena Wątek,4 Paul B Savage,5 Robert Bucki1,6 1Department of Microbiological and Nanobiomedical Engineering, Medical University of Bialystok, 2Department of Medicinal Chemistry, Medical University of Bialystok, 3Institute of Chemistry, University of Bialystok, Bialystok, 4Department of Hematology, Holy Cross Oncology Center of Kielce, Kielce, Poland; 5Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA; 6Department of Physiology, Pathophysiology and Microbiology of Infections, The Faculty of Health Sciences of the Jan Kochanowski University in Kielce, Kielce, Poland Abstract: The pleiotropic activity of human cathelicidin LL-37 peptide includes an ability to suppress development of colon cancer cells. We hypothesized that the anticancer activity of LL-37 would improve when attached to the surface of magnetic nanoparticles (MNPs). Using colon cancer culture (DLD-1 cells and HT-29 cells), we evaluated the effects of MNPs, LL-37 peptide, its synthetic analog ceragenin CSA-13, and two novel nanosystems, ie, MNP@LL-37 and MNP@CSA-13, on cancer cell viability and apoptosis. Treatment of cancer cells with the LL-37 peptide linked to MNPs (MNP@LL-37) caused a greater decrease in cell viability and a higher rate of apoptosis compared with treatment using free LL-37 peptide. Additionally, we observed a strong ability of ceragenin CSA-13 and MNP@CSA-13 to induce apoptosis of DLD-1 cells. We found that both nanosystems were successfully internalized by HT-29 cells, and cathelicidin LL-37 and ceragenin CSA-13 might play a key role as novel homing molecules. These results indicate that the previously described anticancer activity of LL-37 peptide against colon cancer cells might be significantly improved using a theranostic approach. Keywords: anticancer activity, colorectal cancer, ceragenin, cathelicidin LL-37, magnetic nanoparticles

Katarzyna Niemirowicz,1,2 Izabela Swiecicka,3 Agnieszka Z Wilczewska,4 Iwona Misztalewska,4 Beata Kalska-Szostko,4 Kamil Bienias,2 Robert Bucki,1,5,6 Halina Car21Department of Microbiological and Nanobiomedical Engineering, 2Department of Experimental Pharmacology, Medical University of Bialystok, 3Department of Microbiology, 4Institute of Chemistry, University of Bialystok, Bialystok, 5Faculty of Health Sciences, Jan Kochanowski University, Kielce, Poland; 6Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USAAbstract: Superparamagnetic iron oxide nanoparticles (SPIONs) and their derivatives (aminosilane and gold-coated) have been widely investigated in numerous medical applications, including their potential to act as antibacterial drug carriers that may penetrate into bacteria cells and biofilm mass. Pseudomonas aeruginosa is a frequent cause of infection in hospitalized patients, and significant numbers of currently isolated clinical strains are resistant to standard antibiotic therapy. Here we describe the impact of three types of SPIONs on the growth of P. aeruginosa during long-term bacterial culture. Their size, structure, and physicochemical properties were determined using transmission electron microscopy, X-ray diffraction analysis, and Fourier transform infrared spectroscopy. We observed significant inhibition of P. aeruginosa growth in bacterial cultures continued over 96 hours in the presence of gold-functionalized nanoparticles (Fe3O4@Au). At the 48-hour time point, growth of P. aeruginosa, as assessed by the number of colonies grown from treated samples, showed the highest inhibition (decreased by 40%). These data provide strong evidence that Fe3O4@Au can dramatically reduce growth of P. aeruginosa and provide a platform for further study of the antibacterial activity of this nanomaterial.Keywords: antibacterial activity, Pseudomonas aeruginosa, superparamagnetic nanoparticles, iron oxides, gold-coated nanoparticles