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(1) Background: Angiotensin-converting enzyme 2 (ACE2) is a crucial functional receptor of the SARS-CoV-2 virus. Although the scale of infections is no longer at pandemic levels, there are still fatal cases. The potential of the virus to infect the skin raises questions about new preventive measures. In the context of anti-SARS-CoV-2 applications, the interactions of antimicrobial nanomaterials (silver, Ag; diamond, D; graphene oxide, GO and their complexes) were examined to assess their ability to affect whether ACE2 binds with the virus. (2) Methods: ACE2 inhibition competitive tests and in vitro treatments of primary human adult epidermal keratinocytes (HEKa) and primary human adult dermal fibroblasts (HDFa) were performed to assess the blocking capacity of nanomaterials/nanocomplexes and their toxicity to cells. (3) Results: The nanocomplexes exerted a synergistic effect compared to individual nanomaterials. HEKa cells were more sensitive than HDFa cells to Ag treatments and high concentrations of GO. Cytotoxic effects were not observed with D. In the complexes, both carbonic nanomaterials had a soothing effect against Ag. (4) Conclusions: The Ag5D10 and Ag5GO10 nanocomplexes seem to be most effective and safe for skin applications to combat SARS-CoV-2 infection by blocking ACE2-S binding. These nanocomplexes should be evaluated through prolonged in vivo exposure. The expected low specificity enables wider applications.

Agata Lange,1 Marta Kutwin,1 Katarzyna Zawadzka,1 Agnieszka Ostrowska,1 Barbara Strojny-Cieślak,1 Barbara Nasiłowska,2 Aneta Bombalska,3 Sławomir Jaworski1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Center for Biomedical Engineering, Institute of Optoelectronics, Military University of Technology, Warsaw, Poland; 3Department of Optoelectronic Technologies, Institute of Optoelectronics, Military University of Technology, Warsaw, PolandCorrespondence: Agata Lange, Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Ciszewskiego 8, Warsaw, 02-786, Poland, Tel +48 22 593 66 69, Email agata_lange@sggw.edu.plPurpose: Biofilms are one of the main threats related to bacteria. Owing to their complex structure, in which bacteria are embedded in the extracellular matrix, they are extremely challenging to eradicate, especially since they can inhabit both biotic and abiotic surfaces. This study aimed to create an effective antibiofilm nanofilm based on graphene oxide-metal nanoparticles (GOM-NPs).Methods: To create nanofilms, physicochemical analysis was performed, including zeta potential (Zp) (and the nanocomposites stability in time) and size distribution measurements, scanning transmission electron microscopy (STEM), energy dispersive X-ray analysis (EDX), and atomic force microscopy (AFM) of the nanofilm surfaces. During biological analysis, reactive oxygen species (ROS) and antioxidant capacity were measured in planktonic cells treated with the nanocomposites. Thereafter, biofilm formation was checked via crystal violet staining, biofilm thickness was assessed by confocal microscopy using double fluorescent staining, and biofilm structure was analyzed by scanning electron microscopy.Results: The results showed that two of the three nanocomposites were effective in reducing biofilm formation (GOAg and GOZnO), although the nanofilms were c

Marta Kutwin,1 Malwina Sosnowska-Ławnicka,1 Barbara Nasiłowska,2 Agata Lange,1 Mateusz Wierzbicki,1 Sławomir Jaworski1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Institute of Optoelectronics, Military University of Technology, Warsaw, PolandCorrespondence: Marta Kutwin, Email marta_kutwin@sggw.edu.plPurpose: The use of nanotechnology in medicine has gained attention in developing drug delivery systems. GO has the potential to deliver microRNA (miRNA) mimics or antisense structures. MiRNAs regulate gene expression and their dysregulation is implicated in diseases, including cancer. This study aims to observe changes in morphology, viability, mRNA expression of mTOR/PI3K/Akt and PTEN genes in U87, U118, U251, A172 and T98 glioblastoma cells and xenograft models after GO self-assembly with mimic miRNA-7.Methods: Colloidal suspension of graphene oxide (GO) was used for obtaining the GO-mimic miRNA-7 nanosystems by self-assembly method. The ultrastructure, size distribution and ATR-FTIR and UV-Vis spectrum were analyzed. The Zeta potential was measured to verify the stability of obtained nanosystem. The entrapment efficiency, loading capacity and released kinetics of mimic miRNA-7 form GO-mimic miRNA-7 nanosystems were analyzed. The transfection efficiency into the glioblastoma cell lines U87, U118, U251, A172 and T98 of mimic miRNA-7 delivered by GO nanosystems was measure by confocal microscopy and flow cytometry. The changes at mRNA expression level of mTOR, PI3K, AKT1 and PTEN genes was measured by qPCR analysis. The xenograft model of U87 and A172 tumour tissue was performed to analyze the effect at tumor size and volume after GO- mimic miRNA-7 nanosystem administration.Results: The ultrastructure of GO-mimic miRNA-7 nanosystems showed high affinity of mimic miRNA into the GO. The results of transfection efficiency, cell morphology and viability showed that GO -miRNA-7

Agata Lange,1 Arkadiusz Matuszewski,2 Marta Kutwin,1 Agnieszka Ostrowska,1 Sławomir Jaworski1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Animal Environment Biology, Institute of Animal Sciences, Warsaw University of Life Sciences, Warsaw, PolandCorrespondence: Sławomir Jaworski, Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Ciszewskiego 8, Warsaw, 02-786, Poland, Tel +48 225936675, Email slawomir_jaworski@sggw.edu.plPurpose: Biofilms, which are created by most microorganisms, are known for their widely developed drug resistance, even more than planktonic forms of microorganisms. The aim of the study was to assess the effectiveness of agents composed of farnesol and nanoparticles (silver, gold, copper, and zinc oxide) in the degradation of biofilms produced by pathogenic microorganisms.Methods: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were used to create the biofilm structure. Colloidal suspensions of silver, gold, copper, and zinc oxide (Ag, Au, Cu, ZnO) with the addition of farnesol (F) were used as the treatment factor. The size distribution of those composites was analyzed, their zeta potential was measured, and their structure was visualized by transmission electron microscopy. The viability of the microorganism strains was assessed by an XTT assay, the ability to form biofilms was analyzed by confocal microscopy, and the changes in biofilm structure were evaluated by scanning electron microscopy. The general toxicity toward the HFFF2 cell line was determined by a neutral red assay and a human inflammation antibody array.Results: The link between the two components (farnesol and nanoparticles) caused mutual stability of both components. Planktonic forms of the microorganisms were the most sensitive when exposed to AgF and CuF; however, the biofilm str

Malwina Sosnowska,1 Marta Kutwin,1 Piotr Koczoń,2 André Chwalibog,3 Ewa Sawosz1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Department of Chemistry, Institute of Food Sciences, Warsaw University of Life Sciences, Warsaw, Poland; 3Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, DenmarkCorrespondence: André Chwalibog, Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark, Tel +45 40963573, Email ach@sund.ku.dkBackground: The various growth factors change the phenotype of neoplastic cells from sedentary (epithelial) to invasive (mesenchymal), which weaken intercellular connections and promote chemotaxis. It can be assumed that the use of anti-inflammatory polyhydroxyfull nanofilms will restore the sedentary phenotype of neoplastic cells in the primary site of the tumor and, consequently, increase the effectiveness of the therapy.Methods: The studies were carried out on liver cancer cells HepG2, C3A and SNU-449, and non-cancer hepatic cell line THLE-3. Transforming growth factor (TGF), epidermal growth factor and tumor necrosis factor were used to induce the epithelial–mesenchymal transition. C60(OH)40 nanofilm was used to induce the mesenchymal–epithelial transition. Obtaining an invasive phenotype was confirmed on the basis of changes in the morphology using inverted light microscopy. RT-PCR was used to confirm mesenchymal or epithelial phenotype based on e-cadherin, snail, vimentin expression or others. Water colloids at a concentration of 100 mg/L were used to create nanofilms of fullerene, fullerenol, diamond and graphene oxide. The ELISA test for the determination of TGF expression and growth factor antibody array were used to select the most anti-inflammatory carbon nanofilm. Mitochondrial activity and proliferation of cells were measured by XTT and BrdU tests.Results: Cells lost their natural morphology

Marta Kutwin,1 Malwina Sosnowska,1 Agnieszka Ostrowska,1 Maciej Trzaskowski,2 Agata Lange,1 Mateusz Wierzbicki,1 Sławomir Jaworski1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, 02-786, Poland; 2Centre for Advanced Materials and Technologies CEZAMAT, Warsaw University of Technology, Warsaw, 02-822, PolandCorrespondence: Marta Kutwin, Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, 8 Ciszewskiego street, Warsaw, 02-786, Poland, Tel +48-225936671, Email marta_kutwin@sggw.edu.plIntroduction: Graphene oxide (GO) is a single layer of carbon atoms with unique properties, which are beneficial due to its surface functionalisation by miRNA. miRNAs are a non-coding small form of RNA that suppress the expression of protein-coding genes by translational repression or degradation of messenger RNA. Antisense miRNA-21 is very promising for future investigation in cancer therapy. This study aimed to detect cytokine expression levels after the administration of GO-antisense miRNA-21 into U87, U118, U251 and T98 glioma cell lines.Methods: U87, U118, U251 and T98 glioma cell line were investigated in term of viability, human cytokine expression level at protein and genes after treatment with GO, GO-antisense miRNA-21 and antisense miRNA-21. The delivery of antisense miRNA-21 into the glioma cell at in vitro investigation were conducted by GO based transfection and electroporation.Results: The results of the protein microarray and gene expression profile showed that complexes of GO-antisense miRNA-21 modified the metallopeptidase inhibitor 2 (TIMP-2), interleukin-6 (IL-6), interleukin 8 (IL-8), intercellular adhesion molecule 1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) expression level compared to transfection by electroporation of antisense miRNA-21 at investigated glioblastoma cell lines. The TIMP-2 protein and gene expression level was upregulated after antisens

The resistance of microorganisms to antibiotics is a crucial problem for which the application of nanomaterials is among a growing number of solutions. The aim of the study was to create a nanocomposite (composed of graphene oxide and silver nanoparticles) with a precise mode of antibacterial action: what enables textiles to be coated in order to exhibit antibacterial properties. A characterization of nanomaterials (silver nanoparticles and graphene oxide) by size distribution, zeta potential measurements, TEM visualization and FT-IR was performed. The biological studies of the nanocomposite and its components included the toxicity effect toward two pathogenic bacteria species, namely Pseudomonas aeruginosa and Staphylococcus aureus, interaction of nanomaterials with the outer layer of microorganisms, and the generation of reactive oxygen species and lipid peroxidation. Afterwards, antibacterial studies of the nanocomposite’s coated textiles (cotton, interlining fabric, polypropylene and silk) as well as studies of the general toxicity towards a chicken embryo chorioallantoic membrane model were conducted. The toxicity of the nanocomposite used was higher than its components applied separately (zones of growth inhibition for P. aeruginosa for the final selected concentrations were as follows: silver nanoparticles 21 ± 0.7 mm, graphene oxide 14 ± 1.9 mm and nanocomposite 23 ± 1.6 mm; and for S. aureus were: silver nanoparticles 27 ± 3.8 mm, graphene oxide 14 ± 2.1 mm, and nanocomposite 28 ± 0.4 mm. The viability of P. aeruginosa and S. aureus after treatment with selected GO-Ag decreased to 27% and 31%, respectively, compared to AgNPs, when the viability of both species was 31% and 34%, accordingly). The coated textiles showed encouraging antibacterial features without general toxicity towards the chicken embryo chorioallantoic membrane model. We demonstrated that graphene oxide might constitute a functional platform for silver nanoparticles, improving the antibacter

Glioblastoma (GBM) is the most common primary and aggressive tumour in brain cancer. Novel therapies, despite achievements in chemotherapy, radiation and surgical techniques, are needed to improve the treatment of GBM tumours and extend patients’ survival. Gene delivery therapy mostly uses the viral vector, which causes serious adverse events in gene therapy. Graphene-based complexes can reduce the potential side effect of viral carries, with high efficiency of microRNA (miRNA) or antisense miRNA delivery to GBM cells. The objective of this study was to use graphene-based complexes to induce deregulation of miRNA level in GBM cancer cells and to regulate the selected gene expression involved in apoptosis. The complexes were characterised by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy and zeta potential. The efficiency of miRNA delivery to the cancer cells was analysed by flow cytometry. The effect of the anticancer activity of graphene-based complexes functionalised by the miRNA sequence was analysed using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) assays at the gene expression level. The results partly explain the mechanisms of miRNA deregulation stress, which is affected by graphene-based complexes together with the forced transport of mimic miR-124, miR-137 and antisense miR-21, -221 and -222 as an anticancer supportive therapy

Purpose: Surgical resection of hepatocellular carcinoma can be associated with recurrence resulting from the degeneration of residual volume of the liver. The objective was to assess the possibility of using a biocompatible nanofilm, made of a colloid of diamond nanoparticles (nfND), to fill the side after tumour resection and optimize its contact with proliferating liver cells, minimizing their cancerous transformation. Methods: HepG2 and C3A liver cancer cells and HS-5 non-cancer cells were used. An aqueous colloid of diamond nanoparticles, which covered the cell culture plate, was used to create the nanofilm. The roughness of the resulting nanofilm was measured by atomic force microscopy. Mitochondrial activity and cell proliferation were measured by XTT and BrdU assays. Cell morphology and a scratch test were used to evaluate the invasiveness of cells. Flow cytometry determined the number of cells within the cell cycle. Protein expression in was measured by mass spectrometry. Results: The nfND created a surface with increased roughness and exposed oxygen groups compared with a standard plate. All cell lines were prone to settling on the nanofilm, but cancer cells formed more relaxed clusters. The surface compatibility was dependent on the cell type and decreased in the order C3A >HepG2 >HS-5. The invasion was reduced in cancer lines with the greatest effect on the C3A line, reducing proliferation and increasing the G2/M cell population. Among the proteins with altered expression, membrane and nuclear proteins dominated. Conclusion: In vitro studies demonstrated the antiproliferative properties of nfND against C3A liver cancer cells. At the same time, the need to personalize potential therapy was indicated due to the differential protein synthetic responses in C3A vs HepG2 cells. We documented that nfND is a source of signals capable of normalizing the expression of many intracellular proteins involved in the transformation to non-cancerous cells.

There are numerous applications of graphene in biomedicine and they can be classified into several main areas: Delivery systems, sensors, tissue engineering and biological agents. The growing biomedical field of applications of graphene and its derivates raises questions regarding their toxicity. We will demonstrate an analysis of the toxicity of two forms of graphene using four various biological models: Zebrafish (Danio rerio) embryo, duckweed (Lemna minor), human HS-5 cells and bacteria (Staphylococcus aureus). The toxicity of pristine graphene (PG) and graphene oxide (GO) was tested at concentrations of 5, 10, 20, 50 and 100 µg/mL. Higher toxicity was noted after administration of high doses of PG and GO in all tested biological models. Hydrophilic GO shows greater toxicity to biological models living in the entire volume of the culture medium (zebrafish, duckweed, S. aureus). PG showed the highest toxicity to adherent cells growing on the bottom of the culture plates-human HS-5 cells. The differences in toxicity between the tested graphene materials result from their physicochemical properties and the model used. Dose-dependent toxicity has been demonstrated with both forms of graphene.

Malwina Sosnowska,1 Marta Kutwin,1 Barbara Strojny,1 Mateusz Wierzbicki,1 Dominik Cysewski,2 Jarosław Szczepaniak,1 Mateusz Ficek,3 Piotr Koczoń,4 Sławomir Jaworski,1 André Chwalibog,5 Ewa Sawosz1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland; 2Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland; 3Department of Metrology and Optoelectronics, Gdansk University of Technology, Gdansk, Poland; 4Department of Chemistry, Institute of Food Sciences, Warsaw University of Life Sciences, Warsaw, Poland; 5Department of Veterinary and Animal, Sciences, University of Copenhagen, Frederiksberg, DenmarkCorrespondence: André ChwalibogDepartment of Veterinary and Animal Sciences, University of Copenhagen, Groennegaardsvej 3, Frederiksberg, 1870, DenmarkTel +45 40963573Email ach@sund.ku.dkMalwina SosnowskaDepartment of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, PolandTel +48 225936667Email malwina_sosnowska@sggw.edu.plPurpose: Surgical resection of hepatocellular carcinoma can be associated with recurrence resulting from the degeneration of residual volume of the liver. The objective was to assess the possibility of using a biocompatible nanofilm, made of a colloid of diamond nanoparticles (nfND), to fill the side after tumour resection and optimize its contact with proliferating liver cells, minimizing their cancerous transformation.Methods: HepG2 and C3A liver cancer cells and HS-5 non-cancer cells were used. An aqueous colloid of diamond nanoparticles, which covered the cell culture plate, was used to create the nanofilm. The roughness of the resulting nanofilm was measured by atomic force microscopy. Mitochondrial activity and cell proliferation were measured by XTT and BrdU assays. Cell morphology and a scratch test were used to evaluate the invasiveness of cells. Flow cytometry determin