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Michaela Vojnikova,1,2,* Martina Sukupova,1,3,* Michal Stefanik,1,4 Petra Strakova,3– 5 Jan Haviernik,4 Katerina Kapolkova,1 Eliska Gruberova,1 Klara Raskova,1 Hana Michalkova,1 Pavel Svec,1 Marie Peskova Kudlickova,1 Ivana Huvarova,4 Daniel Ruzek,3– 5 Jiri Salat,3– 5 Vladimir Pekarik,1 Ludek Eyer,3– 5 Zbynek Heger1 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic; 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; 4Department of infectious Diseases and Preventive Medicine, Veterinary Research Institute, Brno, Czech Republic; 5Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic*These authors contributed equally to this workCorrespondence: Zbynek Heger, Research Group for Molecular Biology and Nanomedicine, Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, CZ-613 00, Czech Republic, Tel +420-5-4513-3350, Fax +420-5-4521-2044, Email heger@mendelu.czBackground: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing.Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain.Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH.Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.Keywords: drug delivery, SARS-CoV-2, TBEV, WNV, Zika virus

Hana Stepankova,1,2 Marcin Swiatkowski,3 Rafal Kruszynski,3 Pavel Svec,1 Hana Michalkova,1 Vendula Smolikova,1,2 Andrea Ridoskova,1,4 Zbynek Splichal,1,2 Petr Michalek,1,2 Lukas Richtera,1,2 Pavel Kopel,5 Vojtech Adam,1,2 Zbynek Heger,1,2 Simona Rex1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czechia; 2Central European Institute of Technology, Brno University of Technology, Brno, Czechia; 3Institute of General and Ecological Chemistry, Lodz University of Technology, Lodz, Poland; 4Central European Institute of Technology, Mendel University in Brno, Brno, Czechia; 5Department of Inorganic Chemistry, Faculty of Science, Palacky University, Olomouc, CzechiaCorrespondence: Simona RexDepartment of Chemistry and Biochemistry, Mendel University in Brno, Brno, CzechiaTel +420-5-4513-3350Fax +425-5-4521-2044Email simona.rex@mendelu.czPurpose: The present study deals with the in vitro evaluation of the potential use of coordination compound-based zinc oxide (ZnO) nanoparticles (NPs) for the treatment of triple negative breast cancer cells (TNBrCa). As BrCa is one of the most prevalent cancer types and TNBrCa treatment is difficult due to poor prognosis and a high metastasis rate, finding a more reliable treatment option should be of the utmost interest.Methods: Prepared by reacting zinc carboxylates (formate, acetate, propionate, butyrate, isobutyrate, valerate) and hexamethylenetetramine, 4 distinct coordination compounds were further subjected to two modes of conversion into ZnO NPs – ultrasonication with oleic acid or heating of pure precursors in an air atmosphere. After detailed characterization, the resulting ZnO NPs were subjected to in vitro testing of cytotoxicity toward TNBrCa and normal breast epithelial cells. Further, their biocompatibility was evaluated.Results: The resulting ZnO NPs provide distinct morphological features, size, biocompatibility, and selective cytotoxicity toward TNBrCa cells. They internalize into two types of TNBrCa cells and imbalance their redox homeostasis, influencing their metabolism, morphology, and ultimately leading to their death via apoptosis or necrosis.Conclusion: The crucial properties of ZnO NPs seem to be their morphology, size, and zinc content. The ZnO NPs with the most preferential values of all three properties show great promise for a future potential use in the therapy of TNBrCa.Keywords: carboxylate, hexamethylenetetramine, nanoparticles, redox homeostasis imbalance, triple negative breast cancer, zinc oxide coordination compounds

Zuzana Skubalova,1,2 Simona Rex,1,2 Martina Sukupova,1 Martin Zahalka,1 Petr Skladal,3 Jan Pribyl,3 Hana Michalkova,1,2 Akila Weerasekera,4 Vojtech Adam,1,2 Zbynek Heger1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic; 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 3Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USACorrespondence: Zbynek HegerDepartment of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno CZ-613 00, Czech RepublicTel +420-5-4513-3350Fax +420-5-4521-2044Email heger@mendelu.czIntroduction: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system.Methods: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities.Results: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs’ structural and functional properties.Conclusion: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.Keywords: drug delivery, nanomedicine, lenvatinib, vandetanib

Hana Michalkova,1 Vladislav Strmiska,1 Jiri Kudr,1,2 Zuzana Skubalova,1 Barbora Tesarova,1 Pavel Svec,1 Lukas Richtera,1,2 Ondrej Zitka,1,2 Vojtech Adam,1,2 Zbynek Heger1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno CZ-613 00, Czech Republic; 2Smart Nanodevices Research Group, Central European Institute of Technology, Brno University of Technology, Brno CZ-621 00, Czech RepublicCorrespondence: Zbynek HegerDepartment of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno CZ-613 00, Czech RepublicTel +420 54 513 3350Fax +420 54 521 2044Email heger@mendelu.czBackground: Iron oxide nanoparticles (IONs) have been increasingly utilized in a wide spectrum of biomedical applications. Surface coatings of IONs can bestow a number of exceptional properties, including enhanced stability of IONs, increased loading of drugs or their controlled release.Methods: Using two-step sonochemical protocol, IONs were surface-coated with polyoxyethylene stearate, polyvinylpyrrolidone or chitosan for a loading of two distinct topo II poisons (doxorubicin and ellipticine). The cytotoxic behavior was tested in vitro against breast cancer (MDA-MB-231) and healthy epithelial cells (HEK-293 and HBL-100). In addition, biocompatibility studies (hemotoxicity, protein corona formation, binding of third complement component) were performed.Results: Notably, despite surface-coated IONs exhibited only negligible cytotoxicity, upon tethering with topo II poisons, synergistic or additional enhancement of cytotoxicity was found in MDA-MB-231 cells. Pronounced anti-migratory activity, DNA fragmentation, decrease in expression of procaspase-3 and enhancement of p53 expression were further identified upon exposure to surface-coated IONs with tethered doxorubicin and ellipticine. Moreover, surface-coated IONs nanoformulations of topo II poisons exhibited exceptional stability in human plasma with no protein corona and complement 3 binding, and only a mild induction of hemolysis in human red blood cells.Conclusion: The results imply a high potential of an efficient ultrasound-mediated surface functionalization of IONs as delivery vehicles to improve therapeutic efficiency of topo II poisons.Keywords: doxorubicin, ellipticine, iron oxide, nanoparticles, release kinetics

Pavlina Jelinkova,1 Zbynek Splichal,1,2 Ana Maria Jimenez Jimenez,1,2 Yazan Haddad,1,2 Aninda Mazumdar,1,2 Vishma Pratap Sur,1,2 Vedran Milosavljevic,1,2 Pavel Kopel,1,2 Hana Buchtelova,1 Roman Guran,1,2 Ondrej Zitka,1,2 Lukas Richtera,1,2 Dagmar Hegerova,1,2 Zbynek Heger,1,2 Amitava Moulick,1,2 Vojtech Adam1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska, Brno, Czech Republic; 2Central European Institute of Technology, Brno University of Technology, Purkynova, Brno, Czech Republic Background: Increase in vancomycin (Van)-resistant bacterial strains including vancomycin-resistant Staphylococcus aureus (VRSA) and lack of new effective antibiotics have become a formidable health problem. Materials and methods: We designed a new conjugate composed of Van and a peptide Hecate (Hec; Van/Hec), and its potential antimicrobial activity was evaluated. Results: Results from disk diffusion test, time-kill assay, determination of minimum inhibitory concentration (MIC), microscopy, and comet assay showed strong antimicrobial effects of Van/Hec against wild-type, methicillin-resistant Staphylococcus aureus (MRSA) and VRSA. Microscopy revealed that the exposure to Van/Hec results in disruption of bacterial cell integrity in all tested strains, which was not observed in case of Van or Hec alone. Conclusion: Overall, we showed that the preparation of conjugates from antibiotics and biologically active peptides could help us to overcome the limitation of the use of antibiotic in the treatment of infections caused by multidrug-resistant bacteria. Keywords: vancomycin, antibacterial, Staphylococcus aureus, antibiotic resistance, peptide

Michalkova H, Strmiska V, Kudr J, et al. Int J Nanomedicine. 2019;14:7609–7624The authors have advised due to an error at the time of figure assembly, Figure 1D on page 7614 and Figure 6A on page 7621 is incorrect. Read the original article

Simona Dostalova,1,2 Katerina Vasickova,1 David Hynek,1,2 Sona Krizkova,1,2 Lukas Richtera,1,2 Marketa Vaculovicova,1,2 Tomas Eckschlager,3 Marie Stiborova,4 Zbynek Heger,1,2 Vojtech Adam1,2 1Department of Chemistry and Biochemistry, Mendel University in Brno, 2Central European Institute of Technology, Brno University of Technology, Brno, 3Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, University Hospital Motol, Charles University, 4Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Abstract: Due to many adverse effects of conventional chemotherapy, novel methods of targeting drugs to cancer cells are being investigated. Nanosize carriers are a suitable platform for this specific delivery. Herein, we evaluated the long-term stability of the naturally found protein nanocarrier apoferritin (Apo) with encapsulated doxorubicin (Dox). The encapsulation was performed using Apo’s ability to disassemble reversibly into its subunits at low pH (2.7) and reassemble in neutral pH (7.2), physically entrapping drug molecules in its cavity (creating ApoDox). In this study, ApoDox was prepared in water and phosphate-buffered saline and stored for 12 weeks in various conditions (-20°C, 4°C, 20°C, and 37°C in dark, and 4°C and 20°C under ambient light). During storage, a very low amount of prematurely released drug molecules were detected (maximum of 7.5% for ApoDox prepared in PBS and 4.4% for ApoDox prepared in water). Fourier-transform infrared spectra revealed no significant differences in any of the samples after storage. Most of the ApoDox prepared in phosphate-buffered saline and ApoDox prepared in water and stored at -20°C formed very large aggregates (up to 487% of original size). Only ApoDox prepared in water and stored at 4°C showed no significant increase in size or shape. Although this storage caused slower internalization to LNCaP prostate cancer cells, ApoDox (2.5 µM of Dox) still retained its ability to inhibit completely the growth of 1.5×104 LNCaP cells after 72 hours. ApoDox stored at 20°C and 37°C in water was not able to deliver Dox inside the nucleus, and thus did not inhibit the growth of the LNCaP cells. Overall, our study demonstrates that ApoDox has very good stability over the course of 12 weeks when stored properly (at 4°C), and is thus suitable for use as a nanocarrier in the specific delivery of anticancer drugs to patients. Keywords: anticancer therapy, doxorubicin-loaded apoferritin, encapsulation, long-term stability, protein nanocarriers

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Since 1980, prostate specific antigen (PSA) has been using for screening of prostate carcinoma, followed by other diagnostic approaches. The relative reliability of these examinations is redeemed by painful, invasive damage of tissue. Hence, nowadays arises the requirement for molecules that could be utilized for diagnostic and/or prognostic purposes. In this study we present a pilot experiment that deals with the potential of the amino acid - proline. Previously we showed that proline may serve as an auxiliary diagnostic marker. Using comparison of two groups of patients (n = 30) we showed that proline may also provide very useful information about the treatment outcome after surgery for prostate carcinoma. Urinary samples obtained from patients after radical prostatectomy, in whom incomplete removal of prostate cancer tissue was suspected, contained higher levels of proline, when compared to the group of patients with remission. Although the prognostic potential of proline is promising, comprehensive study of large cohort of patients with long term follow-up is required to confirm our hypothesis. [ABSTRACT FROM AUTHOR]

Objective: The aim of our study was to prepare a protocol for pre-treatment of clinical urinary samples, subsequently employed for acquirement of amino acid profiles via ion exchange chromatography with detection in visible spectrum (IEC/ Vis). Further for clinically interesting biomolecules as sarcosine and taurine the optimization was carried out to shorten the analysis time. Design: Methodological Material and methods: Urine specimens from patients diagnosed with prostate cancer (n = 500) were collected within 1 year and stored in -80°C. Samples were processed for analysis of amino acid profiles through acidic hydrolysis in a microwave reactor. Using optimized conditions (80 W, 120°C, 25 bar, 105 min), 500 μL sample, mixed with 500 μL of 35% hydrochloric acid hydrolyzed. That resulted in product, which was subsequently diluted in buffer of sodium cycle (0.2 mol/L NaCl, 60 C6H8O7 mmol/L, 1.5 mmol/L and 0.4% N3Na S(CH2CH2OH)2). After centrifugation (25000 g at 4°C, 20 min), the samples were neutralized (0.6 mol/l NaOH) and analyzed using ion-exchange liquid chromatography with post-column derivatization with ninhydrin, using the detection wavelength λ = 440 and 570 nm (IEC/Vis). For analysis of sarcosine and taurine, the samples were prepared by evaporation of 250 μL of sample employing a nitrogen evaporator (40 min, temperature 60°C. Nitrogen pressure of 1 bar) and resuspended with buffer of sodium cycle (250 μL). Results and conclusion: The analyses of amino acid profiles offer interesting clinical information not only in non-invasively collected urinary samples, but also in other organic matrices. Employment of nitrogen evaporator for sample pre-treatment leads to reduction of manipulation with sample and its combination with shortened analyses times of sarcosine and taurine may serve as a sensitive and low-cost method for analysis of clinical specimens. [ABSTRACT FROM AUTHOR]