Seznam abstraktů 2019: SEKCE C - BIONANOTECHNOLOGIE, NANOMATERIÁLY PRO MEDICÍNU

Silicon nanoparticles (Si-NPs), which are biodegradable without specific cytotoxic effects, possess a variety of promising properties for biomedical applications. Si-NPs can act as labels in bioimaging and sensitizer for mild therapy of cancer. These properties allow us to use Si-NPs as promising nano-agents in theranostics (therapy and diagnos-tics) of cancer. Aqueous suspensions of porous Si-NPs are usually fab-ricated by high-energy milling of porous Si (PSi) films. Non-porous crystalline Si-NPs are prepared by different physical methods as laser-ablation, silane decomposition, plasma-assisted synthesis etc. PSi-NPs irradiated by therapeutic ultrasound are found to induce local hyper-thermia and cavitation-induced damages of cancer cells and tumors. Furthermore, PSi NPs with hydrophilic-hydrophobic surface properties are prospective contrast agents for ultrasonic diagnostics. Aqueous sus-pensions of porous and nonporous (laser-ablated) Si-NPs are efficiently heated by therapeutic radiofrequency electromagnetic radiation. Profit-ing from the NP-based hyperthermia, we demonstrate efficient treat-ment of Lewis lung carcinoma in vivo. Moreover, Si-NPs with a high density of electron spin centers have been found to exhibit properties of a contrast agent for MRI.

Tumor cell-specific drug delivery represents the most desirable approach for preventing side effects in non- diseased tissues and increasing drug accumulation in tumor. The aim of this work was the development of magnetic nanoparticles functionalized with folic acid (FA) on their surface for the targeted delivery of anticancer drug Doxorubicin in cancer cells overexpressing the folate receptor. Magnetic nanoparticles were synthesized in the presence of the polysaccharide alginic acid, and functionalized with PEG and folic acid. Both the conjugation of PEG and FA was achieved through EDC chemistry, and characterized with analytical techniques, such as TGA and ATR. The developed nanoparticles were characterized for their physicochemical characteristics (size, z-potential), magnetic properties, colloidal stability, and loading/release of the drug at different pHs (blood pH and pH 6.0). The stimulation of drug release by the concurrent application of alternating magnetic fields was evaluated as well. Optimal nanoparticle compositions were studied for their ability to enter (intracellular uptake) and kill human breast adenocarcinoma cell lines expressing (MDA-MB-231) and not expressing (MCF-7) the FRα receptor. The nanoparticles exhibited higher uptake and toxicity against the MDA-MB-231 cells than against the MCF-7 cells. Their anticancer activity was further enhanced in the presence of a static magnetic field in close proximity with the cells.
ACKNOWLEDGEMENTS: This research has been co-financed by the Operational Program "Human Resources Development, Education and Lifelong Learning" and is co-financed by the European Union (European Social Fund) and Greek national funds. MIS 5005234

The development of micro/nanomotors showing motion in different fluids presents numerous advantages for environmental and biomedical applications [1-2]. The use of biocompatible materials is a prerequisite, and hence the use of mesoporous silica materials imparts a significant convenient support in their fabrication. However, the precise movement manipulations, like the control over the velocity and the direction, are still limited but particularly desirable [3]. Here, we present improved micromotors designed with stimuli-responsive controls. For that we coated mesoporous silica particles with “bottle-brush” polyphosphazene, possessing tailored lower critical solution temperature (LCST) characteristics. Switching the polymer coating between the swollen and collapsed state allows the chemical fuel (hydrogen peroxide) to reversibly access the catalyst immobilized within the silica pores to form oxygen bubbles [4]. The system represents one of the first artificial micromotors with reversible thermoresponsive on-off activation and could be adapted to various future applications, targeting for example to biologically relevant temperatures.
REFERENCES: [1] B. Jurado-Sánchez, J. Wang, Environ. Sci.: Nano, 2018, 5, 1530-1544; [2] S. Wang, X. Liu, Y. Wang, D. Xu, C. Liang, J. Guo, X. Ma, Nanoscale, 2019, DOI: 10.1039/c9nr03393a; [3] S. Balasubramanian, D. Kagan, K. M. Manseh, P. Calvo-Marzal, G. U. Flechsig, J. Wang, Small 2009, 5, 1569-1574; [4] M. Kneidinger, A. Iturmendi, C. Ulbricht, T. Truglas, H. Groiss, I. Teasdale, Y. Salinas, Macromol. Macromol. Rapid. Commun., 2019, submitted.

Controlled magnetic hyperthermia (MHT) is a promising approach for antitumor therapy. There are different opinions about the most effective temperature for MHT - 43°C or above 50°C. Here, we first present a comparative study of the MHT effectiveness for two murine tumors models (4T1 (metastatic breast cancer) and CT26 (non-metastatic colon carcinoma)) depending on the heating temperature (43-60°C) in vitro and in vivo. Cobalt ferrite nanoparticles possessing high specific loss power values (300-500 W·g-1) were synthesized to provide precise heating control in a large temperature window. The effect of MHT on cells in vitro immediately after heating differed depending on the temperature but not on the cell line. However, delayed effect of MHT at 43°C varied also between cultures. Most of 4T1 cells were still alive 24 h after treatment, while CT26 cells were dead. In vivo data showed that MHT at 43°C causes temporary 4T1 tumor volume decrease and does not result in long-term survival. Heating at 48°C and 60°C leads to the complete clearance of the primary tumor and 25% and 40% of long-term survival, respectively. Moreover, MHT inhibits metastases dissemination. In case of CT26 tumor, even 43°C MHT resulted in 100% cured animals. Thus, the temperature must be defined for each tumor type to ensure antitumor effectiveness of MHT.
ACKNOWLEDGEMENT: The study was funded by Ministry of Science and High Education of the Russian Federation in the framework of Increase Competitiveness Program of NUST «MISiS» (№ K2-2019-011), implemented by a governmental decree dated 16^th of March 2013, N 211, and RFBR research Project no. 18-29-09061.

In modern biology and medicine, much attention is paid to the development of methods for non-invasive control of the processes occurring in living organisms, especially at the cellular level. The fluorescence intensity ratio method, exploiting the temperature-dependent luminescence of the thermally coupled energy levels (TCELs), is regarded as a very promising approach for optical temperature sensing due to its reduced sensitivity to measurement conditions and environmental factors.
Aqueous suspensions of NaYF4:Yb3+, Tm3+ upconversion nanoparticles (UCNP), as well as their modifications coated with polyethylene glycol with carboxyl groups were used as the objects of the study. Firstly, we investigated the dependence of the luminescence intensity of the UCNPs in water on the temperature of the suspension in the range of 20-45 oC, which corresponds to the possible use of the UCNP as a temperature sensor in living objects. It was found that the possibility of using these particles as thermal sensors is ensured by the presence of a pair of TCELs of 1G4(a) and 1G4(b). The sensitivity of the described method depends on the temperature and decreases with its growth.
Secondly, possibility of the UCNPs application as temperature sensors in vitro was demonstrated. The temperature of protein incubated with NaYF4:Yb3+, Tm3+ was measured in the range of 33-45 oC. Maximum sensitivity of the method was 0.12o K-1.
ACKNOWLEDGEMENTS: This study was supported by Russian Foundation for Basic Research (Project №18-02-01023) and by Foundation for the Advancement of Theoretical Physics and Mathematics “BASIS” (project No. 18-2-6-198-1).

Optical biosensors are a modern analytical technology with a potentially broad range of applications in medicine - from unraveling molecular foundations of diseases to their diagnosis and treatment monitoring. Affinity biosensors based on surface plasmons represent the most advanced and mature optical label-free biosensor technology. In this lecture, we present recent advances in the development of plasmonic biosensors for medical diagnostics. Specifically, we report new approaches in plasmonic sensor instrumentation for parallelized detection of multiple analytes, discuss transport of target molecules in microfluidic systems of plasmonic biosensors and present new assays enabling detection of low levels of biomolecules in complex biological media. Finally, we show three examples of clinical applications of plasmonic biosensors, including the monitoring of progression of Myelodysplastic syndromes based on analysis of protein interactions, study of prognostic role of pregnancy associated plasma protein A2 in hemodialysis patients and investigation of molecular interactions related to Alzheimer’s disease.

Statement of the Problem: Early cancer diagnosis is immensely vital to improve treatment outcomes and overall survivals. MicroRNAs as the short non-coding RNA sequences (18-24 nucleotides) are promising biomarkers for early cancer diagnosis due to their altered expression levels during cancer development. However, because of their very low abundance in body fluids, sensitive detection of microRNAs is still formidable. Current reliable miRNA detection methods necessitate amplification while requiring several enzymes to make it costly and time-consuming. Moreover, one of the main limitations in developing any types of biosensors is that the sensitivity enhancement restricts the linear detection range. Methodology & Theoretical Orientation: To resolve the abovementioned issues, here we propose a novel enzyme-free mechanism to detect microRNA-21 (miR-21) in total patient serum in a two-mode electrochemical system to significantly lower the miRNA detection limit while retaining a broad linear detection range. A three-way junction RNA structure is utilized and modified by (i) a methylene blue (MB)-labeled hairpin RNA at its one end, functioning as the capture probe and (ii) the other two ends for being hybridized with barcode gold nanoparticles (MB/barG) as the electrochemical signal boosters. Upon the target miR-21 invasion, the hairpin structure is opened and subsequently hybridized with DNA-assembled gold nanoflower/platinum electrode (GNF@Pt). This brings the MB to the close vicinity of surface resulting in a detectable electrochemical signal (1st mode). To expand the biosensor dynamic detection range and amplify its sensitivity, MB/barG is hybridized to the 1st mode to form a dumbbell-like structure and establish the 2nd mode. Conclusion & Significance: The integrated biosensor could detect 135 aM (324 molecules of miR-21 in only 4 µL sample) and represent a wide linear range from 1 µM to 500 aM in a back-to-back supporting system. The GNF@Pt displayed a remarkable conductivity and addition of MB/barG dramatically increased the MB electrochemical signal by ~230 folds. Further, a novel protocol was introduced to enhance microRNA extraction efficiency from the total serum by ~60%. The developed biosensor showed a high selectivity and specificity towards single base-pair mismatches and could profile different development stages of two cancer serums (breast and liver).

The recent progress in the field of immunoassays has been driven by introduction of various kinds of nanomaterials. In particular, photon-upconversion nanoparticles (UCNPs) proved to be excellent immunoassay labels due to their ability to emit light of shorter wavelengths under near-infrared excitation (anti-Stokes emission), which prevents autofluorescence, minimizes light scattering, and thus reduces the optical background interference. These unique photoluminescent properties allow counting of individual biomolecules labeled with UCNPs by conventional wide-field epiluminescence microscopy and enable the development of single-molecule (digital) immunoassays. We have introduced a novel label based on UCNPs conjugated with streptavidin via poly(ethylene glycol) and applied it in a digital upconversion-linked immunosorbent assay (ULISA) for the detection of a cancer biomarker prostate specific antigen (PSA). The digital readout based on counting of individual immunocomplexes improved the sensitivity 16× compared to conventional analog readout and allowed to reach a limit of detection (LOD) of 23 fg·mL−1 (800 aM). Human serum samples were successfully analyzed achieving an excellent correlation with electrochemiluminescence reference method. The conjugates of UCNPs with streptavidin are also suitable for the detection of pathogenic bacterium Melissococcus plutonius, the causative agent of honeybee disease European foulbrood. The ULISA assay provided an LOD of 340 CFU·mL−1 and successfully analyzed real samples of bees, larvae and bottom hive debris. Due to the high reliability and relatively simple detection scheme, the digital ULISA can pave the way for a new generation of digital immunoassays with a strong potential for commercialization.

Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanocrystals that can be excited by near-infrared light and emit photon-upconversion luminescence of shorter wavelengths. Advantages of UCNPs include near-infrared excitation, multiple and narrow emission bands, negligible autofluorescence and high stability, which make UCNPs ideal luminescence label for use in biological and chemical assays. These assays - e.g. upconversion-linked immunosorbent assay, western blot, lateral flow assay, gel electrophoresis, thin layer chromatography - commonly require the scanning of a planar surface with a high spatial resolution and an excellent sensitivity. The availability of commercial equipment is recently limited because of the novelty of the photon-upconversion phenomenon. Therefore, we report on the construction of photon-upconversion laser scanner. The scanner consists of a laser scanning head, which is attached to a xy-moving stage. The scanning head itself is constructed as an epiluminescence detector with excitation wavelength of 976 nm. A CCD array spectroscope is connected to the laser head and serves as a sensitive detector of photon-upconversion luminescence. The scanner possesses a spatial resolution of 200 µm, the scanning rate is up to 57 points per second and the sensitivity reaches down to single photon-upconversion nanoparticle.

Gold nanoparticles (AuNPs) exhibit localized surface plasmon resonance, a phenomenon which leads to greatly enhanced electromagnetic near field, absorption, and scattering in the VIS-NIR region. This coincidence of nature allows to excite plasmonic AuNPs by commonly used light sources present in optical microscopes. We exploited this feature to asses photothermal properties of AuNPs by designing experiments for laser scanning microscopes, namely confocal and two-photon excitation microscope. These devices equipped with continuous-wave and pulsed lasers allow to study AuNPs arrangements convenient for Surface-Enhanced Raman Scattering (SERS) as well as complex biological systems. First, we developed a routine how to evaluate photothermal stability on a single particle level with possibility to upscale it to extract statistical parameters by image analysis of scanning electron microscopy micrographs. Second, we observed photothermal capabilities of anisometric AuNPs tuned to NIR in different kinds of biological samples in vitro, especially in cancer cell lines. Our goal was to optimize the experimental parameters to achieve either programmed cell death, apoptosis, or necrosis, traumatic cell death. The results reveal some of the aspects of rapidly evolving field of thermoplasmonics. This work was supported by the Czech Science Foundation 18-10897S project and the student project.

In this paper photoluminescence properties of aqueous suspensions of carbon nanoparticles (nanodiamonds, carbon dots and complexes on their basis) with different ratio of surface carbon in sp3- and sp2-hybridizations were studied by methods of Raman, fluorescence and IR absorption spectroscopy. It was found that intensity of photoluminescence correlates with amount of carbon in non-diamond phase on the surface of nanoparticles. Nanoparticles with high non-diamond carbon phase content revealed more intense photoluminescence. The obtained results support the hypothesis about surface nature of photoluminescence of nanodiamonds and carbon dots.

Over the last few years, several studies have shown that monolayer-protected gold nanoparticles (MPGNs) can be functionalized to produce nanodevices with unique properties and applications, e.g. in nanomedicine and diagnostics, chemosensing, and even catalysis (nanozymes).[1-9] The molecules forming the outer coating monolayer are the main contributor to the nanoparticle’s functionality. The self-organized nature of the monolayer offers a straightforward path to realizing large multifunctional chemical systems with a level of complexity resembling that of proteins.[10] Remarkably, recent experiments have shown ribonuclease-like activity of selected MPGNs.[11] These have been demonstrated to cleave efficiently small organic substrates resembling nucleic acids. In this case, nanocatalysis was facilitated by catalytic Zn2+ ion(s) on the surface of MPGNs.[12] This is reminiscent of metal-dependent nucleases that cleave nucleic acids.[13] Here, we apply computational simulations to understand the structure, organization, and dynamics of the nanozyme/substrate interface at the atomic level revealing unique self-organization of the coating monolayer through specific non-covalent interactions.[10,13-15] The integration of computational modeling and experiments (e.g. kinetic and NMR measurements) allows us to clarify the role of the dynamic rearrangement of coating groups and metal ions for phosphodiester bond cleavage, thus achieving a better understanding of MPGN-mediated catalysis.
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(3) Su, S. et al. Nanoscale 2013, 5, 2589.
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(6) Gao, N. et al. Nano Res 2016, 9, 1079.
(7) Qu et al. Chem Rev 2019, 119, 4357.
(8) Wei, H. et al. Chem Soc Rev 2019, 48, 1004.
(9) Yan X. et al. Acc Chem Res 2019 - doi: 10.1021/acs.accounts.9b00140
(10) Riccardi, L. et al. Chem (Cell Press) 2017, 3, 92.
(11) Diez-Castellnou, M. et al. J Am Chem Soc 2014, 136, 1158.
(12) Diez-Catellnou, M. et al. Front Chem 2019, 7, 469.
(13) Riccardi L. et al. Nat Rev Chem 2018, 7, 100.
(14) Franco-Ulloa, S. et al. J Chem Theory Comput 2019, 15, 2022.
(15) Riccardi, L. et al. ACS Nano 2019, 6, 7127.

Acknowledgement:
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 843117 (CompNanozymes).

Atomic Force Microscope (AFM) can be effectively used as a nano sensor transducer; as was demonstrated in cell based biosensing of drug effects. AFM based nano-indentation gives a way to measure the stiffness of bio samples and localize this mechanical parameter with nanometre resolution. AFM operates under nearly physiological conditions- i.e. in a liquid medium under either laboratory or elevated temperature. In the nanoindentation mode (often called force mapping or force volume), the sample is indented at many sites and its complete elastic response is recorded which enables to reconstruct its stiffness map. Combination of this mode with high sampling rate of tapping mode has led in development of hybrid mode, when short force-distance curves are recorded with kilohertz sampling rate, thus leading to a detailed topography images combined with mechanical characterization of the surface. Complex characterization of macromolecules and their complexes is therefore possible.
Detailed study of structure of selected glycosaminoglycans will be presented. Moreover, change of structure and mechanical properties of hyaluronan due to interaction with myeloperoxidase was studied by AFM spectroscopy. Similarly, a complex of chromatin and BAF protein (barrier-to-autointegration factor) was studied in order to characterize detailed structure changes, as well as to map local mechanical properties and their changes as a result of the specific interaction. ACKNOWLEDGEMENTS: CIISB research infrastructure project LM2015043 supported the AFM measurements at the Core Facility Nanobiotechnology. REFERENCES: M. Pesl, J. Pribyl et al., “Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing”. Biosensors and Bioelectronics 85:751, 2016.

Zinc oxide nanoparticles (ZnO NP) have shown great potential as a novel antibacterial material at a time when resistance towards conventional antibiotics is becoming more prevalent. We report bacteria inactivation by ZnO NP with novel hedgehog-like morphology using model gram-negative (E. coli) and gram-positive (S. aureus) bacteria. E. coli exposed to the novel ZnO hedgehog NP during growth resulted in 4 orders of magnitude reduction in viable cell concentration after 24 h, which is more than 2 orders higher reduction compared to commercially available ZnO NPs with nominal sizes from 50 nm to 20 um. There was a positive correlation between hedgehog NP concentration and bacteria cell concentration reduction within the range tested 0.1 - 1.0 mg/mL. S. aureus was less sensitive to ZnO NP exposure and inactivation effect of various ZnO NP, was comparable. The effect can be thus attributed to direct mechanical damage of the bacterial mebrane that is the most effective for the novel hedgehog ZnO NP. This conclusion was corroborated also by disk diffusion assays.