Seznam abstraktů 2024: SEKCE C - BIONANOTECHNOLOGIE, NANOMATERIÁLY PRO MEDICÍNU

Active pharmaceutical ingreadients (APIs) to treat lung diseases, e.g. cancer, acute infections, chronic obstructive pulmunary disease (COPD), are usually administered via typical enteral or parenteral routes, although the lungs can be directly targeted through the airways. Thus, by using inhalable formulations, it is possible to obtain much higher concentration of APIs directly at required site and reduce their amounts distributed systemically [1].
In our group, we developed inhalable stimuli-sensitive drug carriers that are intended to improve the efficacy of lung cancer therapy through guided accumulation directly at the tumour site and controlled API release triggered by an alternating magnetic field resulting in a local increase in temperature [2]. Such carriers are in the form of solid lipid microparticles composed of fatty acids, superparamagnetic iron oxide nanoparticles (SPIONs) and paclitaxel [3].
Recently, we have been working on polymer drug delivery systems of antibiotics and quorum sensing inhibitors for the treatment of bacterial infections in patients with COPD exacerbations [4]. Such systems are based on fast-degrading polyanhydride microparticles loaded with antibiotics (e.g., azithromycin, tobramycin, gentamycin) and quorum-sensing inhibitors (e.g., curcumin, linolenic acid). The microparticles are designed to ensure an appropriate size for inhalation, degrade within a few days, and release drug cargo, which is capable of killing bacteria causing COPD exacerbations and preventing biofilm formation. The system is cytocompatible with lung epithelial cells, as shown by in vitro and ex vivo tests.
ACKNOWLEDGMENTS: This study was supported by the NCN (project No 2019/35/B/ST5/01103) and partly by the Program „Excellence initiative - research university” for the AGH University of Krakow.
REFERENCES: [1] K. Knap, et al, Regenerative Biomaterials, 2023, 10, rbac099; [2] K. Reczyńska, et al, Nanomaterials, 2020, 10(6), art. no. 1076; [3] K. Reczyńska et al, Materials Science and Engineering C, 2020, 111, 110801; [4] K. Kwiecień, et al, Engineering of Biomaterials, 2021, 162, 7-12; [5] K. Knap, et al, Biomaterials Advances, 2023, 153, art. no. 213540.

Various TiO2 nanostructured surfaces are generally recognized as excellent biocompatible materials. Anodic TiO2 nanotube (TNT) layers have emerged as extremely suitable and effective substrates for surface modification of various materials used in biomedical applications, especially TiAlV alloy.
Recently, we compared the biological effect of ultrathin TiO2 coatings, achieved by various numbers of Atomic Layer Deposition (ALD) cycles, on planar and nanotubular surfaces. We demonstrated that ultrathin TiO2 ALD coatings on TNT layers enhanced cell adhesion, growth, and proliferation [1].
Furthermore, we found that 1 cycle of TiO2 ALD coating shaded the impurities of F- and V-based species in tested TiAlV alloys while preserving the original structure and morphology. ALD modification makes tested materials suitable candidates for bone and dental implants [2]. For the first time, single-cell adhesion to modified TNT layers using the ALD method was studied by bio-Atomic Force Microscopy [3].
REFERENCES: [1] Motola, M., Capek, J. et al., ACS Applied Bio Materials (2020); [2] Capek, J., Sepúlveda, M. et al., ACS Applied Materials & Interfaces (2024); [3] Baishya, K., Vrchovecka, K. et al., Applied Surface Science Advances (2023).

Nanoparticles (NPs) were initially considered a promising solution to combat antibiotic-resistant bacteria (10.1039/D4PM00032C). However, recent findings show that bacteria can develop resistance to NPs (10.3389/fmicb.2023.1102615), necessitating a deeper exploration of this phenomenon.
We investigated the potential for resistance development in Escherichia coli (E. coli) when repeatedly exposed to sublethal concentrations of zinc oxide (ZnO) nanoparticles (200 nm, 6% aluminum-doped, Sigma Aldrich) over an extended period. Real-time bacteria growth curves were studied in reverse spin bioreactors, with and without visible light illumination alongside sub-lethal concentrations of ZnO NPs, using an established setup and protocol for highly reproducible experiments (10.37904/nanocon.2023.4813). The experiment involved more than 10 re-exposure cycles, where surviving cells from previous exposures were re-exposed to the same growth conditions. Growth parameters, including lag and log phase lengths, and maximum growth rate, were analyzed. Colony-forming unit (CFU) concentrations and colony morphology were examined after each exposure. Furthermore, bacteria from selected exposure steps underwent minimum inhibitory concentration (MIC) testing and scanning electron microscopy (SEM) analysis.
The results revealed a consistent and specific bacterial response to ZnO NPs and antimicrobial activity enhancement under illumination. Notably, cells exposed to both ZnO NPs and illumination exhibited the longest lag and log phases, the lowest maximum growth rate, and the lowest final optical density (OD) over all exposure steps, with a marked decline in CFUs and increase in log phase length during repeated exposures. In contrast, cells exposed solely to ZnO NPs did not show significant deviations from control groups. The size and shape of bacteria exposed to ZnO NPs and illumination were similar to that of the control and cannot explain the reduction in OD observed during growth. Additionally, there was no reactive oxygen species (ROS, e.g. H2O2) detected from liquid that contained illuminated ZnO NPs, making it highly unlikely that the photoactivation of ZnO is the main factor of the heightened antimicrobial activity of illuminated ZnO. On the other hand, significant differences were observed in cell envelopes after illumination, which could point to the previously described changes in cell membranes, including changes in membrane integrity or ion pump activity (10.3390/antibiotics10030341), under illumination. This may in turn lead to a higher permeability of the cellular membrane towards ZnO NPs, or lower the ability of cells to expel zinc ions, resulting in heightened antimicrobial capacity.

Nanoscale chemical modification and interaction of semiconductor surfaces with organic molecules in gases and solutions is key to many many modern applications from electronic sensors to catalysis and energy conversion. In our work, we investigate at the nanoscale the effect of surface atomic configurations and surface dipole orientation on two polar (Zn-face, O-face) and two non-polar ZnO monocrystalline slabs on several types of application relevant biomolecules (bovine serum albumin - BSA, aminoacids - AA, and thiorphan). We employ atomic force microscopy with molecular resolution to characterize morphology and adhesion of adsorbed molecular assemblies on these ZnO diverse surfaces. The experimental data are correlated with atomistic computations based on DFTB and Force Field MD methods.
The theoretical and experimental results show pronounced differences in each case. BSA adsorbed on ZnO in mono-molecular layers (2-10 nm), arrangement and adhesion of which is controlled by the surface chemistry. Even some chemical bonds are formed spontaneously, via interaction with AA residues, except for O-faced ZnO surface that is rather repelling to BSA. Thiorphan on the other hand tends to form 4 nm or 25 nm nanodots on polar surfaces and rigid 2D-like layers and islands (stacking by 4 nm) on non-polar ZnO surfaces. The simulations prove that it is the effect of electrostatic dipole orientation, not of surface chemistry. Similar effect is observed also for magnetized surfaces with spin up and spin down orientation. These findings may help elucidate many prior studies of ZnO properties and effects in biological environments as well as design new applications.

Surface immobilization of the IgG antibody, which is used as a detection element in immunosensors, requires a special attention. Since this molecule has an Y shape with antigen binding sites located on two Fab arms, its orientation (flat-on, side-on, and vertical: tail-on and head-on) determines the assay efficiency [1,2]. Therefore, it is of utmost interest to examine and control the dominant orientation of surface-immobilized antibodies which depends on the pH of the adsorption solution, the amount Γ of antibody adsorbed, and other factors. However, the IgG orientation is most commonly inferred from indirect methods, prone to high uncertainty. In contrast, antibody orientation can be resolved with TOF-SIMS mass spectrometry, because of the technique sensitivity to the outermost nanometer region of adsorbed proteins and discrimination of the IgG domains (Fc and Fab) with different amino acid composition, enhanced by a multivariate Principal Component Analysis. This method is, however, limited to comparative analysis between samples that hindering an absolute determination of the dominant orientation on the particular surface. In this work, we present the novel approach of surface density dependent studies of antibody orientation with TOF-SIMS and PCA, which allows for direct tracking of orientation changes induced by the increasing molecules surface amount and for an accurate evaluation of the dominant orientation by estimation of share of molecules with head-on and tail-on alignment (fFc fraction) [2,3]. We examined the surface density dependent orientation of antibodies immobilized on silane-modified silicon by physical adsorption (APTES layer) and covalent coupling (APTES layer activated with glutaraldehyde APTES/GA). In particular, differences in dominant vertical orientations are revealed and discussed in terms of relevant molecule-molecule and molecule-surface interactions. Moreover, the impact of the pH of the IgG solution on the dominant vertical orientation of the antibodies immobilized on APTES and APTES/GA is determined and expressed by the fFc fraction. It is shown that under particular pH conditions the dominant antibody orientation can by significantly improved to be close to the ideal tail-on alignment through electrostatic molecules-surface interactions [4].
REFERENCES: [1] Gajos, Katarzyna et al., Applied Surface Science, 594, 153439 (2022); [2] Gajos, Katarzyna et al., Applied Surface Science, 518, 146269 (2020); [3] Gajos, Katarzyna et al., Molecules, 27, 3672 (2022); [4] Gajos, Katarzyna et al., Appl. Surf. Sci., 656, 159644 (2024).

Plasma polymerization of thin film coatings is an effective method for modifying synthetic materials that lack optimal bioapplication properties. Amine plasma polymers (PPs) capable of stable binding with biomolecules and enhanced cell adhesion are suitable for designing biomaterials. Their properties can be tuned concerning the surface charge (from strongly positive to slightly negative) and reactivity for covalent bond formation. The presence of free radicals can explain part of their properties. Therefore, it is essential to investigate their formation for different plasma polymerization conditions, namely radio frequency power absorbed by the discharge in the deposition gas mixture (argon and cyclopropylamine vapors). Electron paramagnetic resonance revealed a significant concentration of free radicals for higher power, attributed to the higher ion energy flux towards the growing PP film. The concentration of free radicals in PPs decreased exponentially over time due to their recombination at the surface or even in the bulk, as they can diffuse through the material. Nevertheless, even after three months, the spin density in PPs remained at 60% of the initial concentration. The amount and kinetics of radicals in the amine PPs and their other chemical properties were correlated to the cell-surface interactions and kinetics of protein immobilization determined by measurements with quartz crystal microbalance sensors.

Structuring conductive diamond surfaces with boron doped diamond as the most popular variant is a challenging task due to the complexities of diamond growth procedure and the inherent properties of the material (hardness, chemical inertness). However, structuring may enhance proclivity to anchor (bio)molecules, increase sensitivity and lower limits of detection in electroanalytical applications or facilitate growth of human neural cells [1-3].
In this contribution, results of our continuous research in the field of engineering BDD surfaces will be presented for:
1) supported and free-standing porous BDD layers for batch and flow-through electrochemical cells,
2) IR laser-processed polycrystalline and chem-mechanically polished BDD layers [3], leading to micrometer-scale spot-structuring of the surface and controlled introduction of sp2 carbon [4],
3) single-crystal BDD layers nanostructured with spherical cavities, achieved via microwave plasma chemical vapor deposition in the presence of silica nanosphere templates.
To address advantages of these novel BDD varieties, their physico-chemical properties and prospective applications will be demonstrated via the electrochemical detection of neurotransmitters in batch and flow-through arrangements or the assessment of DNA adsorption.
ACKNOWLEDGEMENTS: The work is supported by the Czech Science Foundation (project GACR 23-05688S) and by EU (project SenDiSo: CZ.02.01.01/00/22008/0004596).
REFERENCES:
[1] J. Krůšek, I. Dittert, T. Smejkalová, M. Kořínek, K. Gottfriedová, H. Freislebenová, E.
Neuhöferová, L. Klimša, S. Sedláková, A. Taylor, V. Mortet, V. Petrák, V. Benson, V. Petráková, Phys. Status Solidi B 256 (2019) 1800424.
[2] M. Zelenský, J. Fischer, S. Baluchová, L. Klimša, J. Kopeček, M. Vondráček, L. Fekete, J. Eidenschink, F.-M. Matysik, S. Mandal, O.A. Williams, M. Hromadová, V. Mortet, K. Schwarzová-Pecková, A. Taylor, Carbon 203 (2023) 363-376.
[3] J. Hrabovsky, M. Zelensky, J. Sladek, M. Zukerstein, J. Fischer, K. Schwarzova-Peckova, A. Taylor, M. Veis, S. Mandal, O.A. Williams, N.M. Bulgakova, Applied Surface Science 639 (2023) 158268J.

Even though immunoassays are considered the gold standard among bioanalytical methods, conventional immunoassay labels often possess disadvantages, e.g., low stability and sensitivity in the case of enzymes or photobleaching in the case of fluorophores. To address these drawbacks, new nanomaterial-based labels are being studied (1). Photon-upconversion nanoparticles (UCNPs) are lanthanide-doped nanocrystals exhibiting anti-Stokes emission, enabling low assay background due to the reduction of autofluorescence and light scattering (2).
Our work focused on the development of a dot-blot immunoassay with UCNPs as detection labels. First, UCNPs were synthesized, and their surface was modified with a PEG-based linker to achieve hydrophilicity and low levels of non-specific interactions. This was followed by the conjugation with streptavidin, preparing a sensitive biorecognition element. Human serum albumin was chosen as a model biomarker for immunoassay optimizations. Each assay step was optimized to achieve the highest signal-to-background ratios and, consequently, the lowest detection limits (LOD). After that, the assay was successfully adjusted to detect other clinically important biomarkers, prostate-specific antigen as a prostate cancer biomarker and cardiac troponin as a biomarker of myocardial infarction. Achieved LODs were 0.24 ng/mL for HSA, 0.62 ng/mL for troponin, and 9.4 pg/mL for PSA.
The obtained data showed that dot-blots with UCNP labels can become an alternative to conventional microtiter plate assays (MTP). Conventionally, dot-blots lack the sensitivity of the MTP assays and are mainly used for the semi-quantitative detection of analytes. Due to the outstanding performance of UCNPs as labels, it is possible to transform dot blots into a reliable quantitative detection platform for the detection of low-abundance biomarkers.
(1) Farka Z, Juřík T, Kovář D, Trnková L, Skládal P. Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem Rev. 2017;117(15):9973-10042.
(2) Hlaváček A, Farka Z, Mickert MJ, Kostiv U, Brandmeier JC, Horák D, et al. Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging. Nat Protoc. 2022;17(4):1028-72.

Although cadmium-based quantum dots (QDs) exhibit superior optical properties, their potential applications are significantly limited due to the high risk of toxic cadmium ion release. Therefore, there is a strong demand for the development of non-toxic alternatives. In this study, we focused on two classes of cadmium-free nanomaterials: carbon nanodots (CNDs) and molybdenum disulfide (MoS₂) QDs. These materials not only offer desirable optical properties but also exhibit low toxicity, good biocompatibility, and excellent water dispersibility, making them attractive candidates for a wide range of applications, including heavy metal ion sensing. We explored the capability of both label-free CNDs and MoS₂ QDs to function as fluorescence quenching-based sensors for Fe³⁺ ions, with a focus on the less extensively studied nonlinear optical properties in an expanded spectral range of wavelengths. Utilizing tunable femtosecond laser pulses for the two-photon induced luminescence technique, we optimized the excitation wavelength in the near-infrared region to achieve maximum of two-photon brightness. Additionally, we performed temperature-dependent measurements to elucidate the potential mechanisms underlying the observed quenching effects under one- and two-photon excitation conditions. The results confirm that both CNDs and MoS₂ QDs exhibit significant fluorescence quenching in the presence of Fe³⁺ ions, demonstrating their potential as sensitive and selective two-photon active sensors suitable for applications in e.g. environmental systems.
ACKNOWLEDGEMENTS:We acknowledge support from the National Science Centre, Poland under Grant no.
UMO-2018/30/E/ST5/00718.

Due to the ever-increasing threat of Antimicrobial resistance (AMR) to human health globally, there is a constant need for novel strategies for rapid detection and identification of live bacteria. Amongst these technologies, quantum dots with their unique tuneable optical properties are small enough to traverse the bacteria membrane and become activated upon interaction with light of suitable energy even after internalisation without, crucially, causing bacteria death.
We observed pronounced differences in the growth profiles of bacteria (E. coli) when cultured in the presence of carbon quantum dots (CQD), nitrogen-doped CQD (N-CQD) or iron/nitrogen co-doped CQD (Fe/N-CQD). Bacteria growth was not inhibited even during exposure to relatively high concentrations (1.5 mg/mL). Bacteria exposed to CQD and Fe/N-CQD had comparable growth profiles to the untreated bacteria. Interestingly, we observed enhanced maximum growth rate, shortened lag phase, and higher final optical density for bacteria exposed to the highest concentration of N-CQD. Pre-illumination of the colloid using solar simulated light prior to the addition of bacteria resulted in further enhancement of the effect. All these effects decreased towards lower N-CQD concentrations. Moreover, photoluminescence of N-CQD with bacteria was more pronounced than that of CQD and Fe/N-CQD, possibly because of localisation around the bacteria surface as observed using scanning electron microscopy. Absorbance measurements of colloidal CQD, N-CQD and Fe/N-CQD confirmed long-term stability (5 days). Such a material has potential use for incorporation into rapid diagnostic systems for bacteria detection in liquid with minimal cytotoxic complications for the environment.

Nanodiamonds (NDs) and metal nanoparticles (NPs) are widely investigated for their diverse applications, from biomedicine to industry, due to their unique properties at the nanoscale. In this work, the effect of surface plasmon resonance for different nanostructures (i.e. clusters and dimers of gold, silver and diamond nanoparticles) were studied theoretically by using the Finite Element Method (FEM) in COMSOL Multiphysics with the RF module. We analyzed the extinction cross section spectra and electric field enhancement maps for varying parameters (interparticle distance, size and surrounding medium) of nanoparticle clusters including dimers and larger aggregates with diamond at their core. This study aims to provide a comprehensive understanding of the plasmonic behavior in mixed nanoparticle systems. Our findings shows that electric field enhancement and the resonance wavelength is affected by interparticle distance, nanoparticle size, the surrounding medium and the presence of diamond nanoparticles at the center of the cluster. For instance, the resonance wavelength changes from 532 nm to 544 nm as the interparticle distance decreases from 5 nm to 1 nm for a nanodimer with a 20 nm gold NP and 50 nm diamond NP in water. The introduction of diamond NP at the center of the Au and Ag nanoparticle clusters and varying the number of NPs in the system significantly affect the electric field enhancement, its localization, and resonance wavelengths from original 520 nm (Au NP) or 380 nm (Ag NP). The results underscore the critical role of nanoparticle geometry and composition in tailoring plasmonic properties, highlighting their potential applications in nanophotonic devices, thermal therapy, and sensing technologies.

Emissions from mobile sources, released directly in streets at breathing level, are one of the major sources of air pollution in many urban areas. Air pollution, in turn, is one of the gravest environmental hazards, causing premature death of millions of people worldwide. Particles from high temperature processes, primarily carbonaceous particles from engine exhaust and metallic particles from friction brakes, contain semi-volatile organic compounds and metals , and are on the order of units to tens nm diameter. Such small particles have been reported to deposit efficiently in lung alveoli and to penetrate into bloodstream, and to travel along the olfactory nerve to the brain, causing or contributing to a range of acute and chronic illnesses. The toxicity attributed to exhaust particles can be reduced by oxidation catalysts and particle filters, yet despite their massive introduction, our air is far from clean. Brake wear nanoparticles can be reduced by improved friction materials, electrodynamic braking and sensible driving style. As in nearly all categories of emissions sources, small percentage of not properly operating devices contributes a large part of total emissions, highlighting the importance of appropriate design, manufacture, calibation, maintenance and usage. High variability of emissions over time and among sources, particle dispersion and physical and chemical processes cause nanoparticle distribution in the air to vary with both location and time. As of yet, there is no reliable, cheap, simple nanoparticle sensor for general public use. Despite considerable improvements mainly in the technology, nanoparticles in our air remain a considerable challenge.

The availability of water for firefighters is a key factor in ensuring fire safety in the area. That is why natural and artificial water sources can be used without limitations, according to the Water Act. In recent years, the idea of using alternative water sources, in particular treated wastewater and rainwater, has also been discussed. The rainfall over the last three decades has been more localised but much more intense, which led to a crisis situation where long-term water scarcity is alternated with an immediate surplus. Rescue services then deal with cases where, although there is sufficient water in a natural stream, it is not available for pumping equipment due to unsuitable terrain. One option is to use treated wastewater from sewage treatment plants, where there is a constant supply of sufficient water for pumping and access is provided by paved roads. For this reason, water samples were collected from open fire ponds, closed fire ponds, WWTP outfall, open watercourse and closed watercourse. The aim of this paper is thus to perform ecotoxicological tests on samples mentioned in accordance with the European standard ISO 11348-3:2007 Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio Fischeri (Luminiscent bacteria test) and Czech legislation (Law No. 541/2020 Coll., Waste Act and Decree No. 8/2021 Coll., Waste Catalogue), to determine whether this water is ecotoxicologically safe to use in firefighting.

The variety of nanostructures surrounding us today is enormous. The use of nanomaterials is very extensive because of the unique properties of nanoparticles (NPs) arising from their nanometre dimensions. When NPs enter a cell or a living organism, biomolecules adhere to their surface and form a so-called protein corona. This affects not only the entry of NPs into the cell but also the overall toxicity of NPs and their fate in the cell. The secondary toxicity of NPs is caused by the interaction of molecules on their surface, which greatly limits their use in medicine and pharmacy. These interactions between NPs and the biological environment have so far been studied by outdated indirect ensemble methods. Our approach plans to examine individual NPs and is focused on the rate of protein corona formation and its stability by studying individual anchored nanoparticles under liquid using modified atomic force microscopy.