SESSION C - BIONANOTECHNOLOGY, NANOMATERIALS IN MEDICINE

The mechanical and biomechanical properties of the cell's extracellular matrix is a factor in their later development. In this regard, elastic biocompatible materials as a surface for cultivating cells are in recent years, becoming an exciting field of study. Hydrogels are a hydrophilic 3D network of synthetic polymers or biopolymers. They can have tunable mechanical properties, controllable degradability, or they can be designed for long-time release of treatments. Here, hydrogels from Hyaluronic acid (HA), Bovine serum albumin (BSA) and gelatin was prepared, via carbodiimide chemistry. Morphology and mechanical properties of these hydrogels was characterized via Atomic Force Microscopy (AFM). Young's modulus of HA/BSA and HA/BSA/gel were 124.7 ± 15.9 and 20.3 ± 0.5 kPa, respectively. Mouse embryonic fibroblasts were then cultivated on hydrogels and on the glass surface as a reference. Mechanical and morphological properties of fibroblasts were examined by AFM and via phalloidin-TRITC staying. Relationship between the structure of cells and the type of surface was seen. Cells grown on glass were more flattened with an evenly linear actin structure. On hydrogels, fibroblasts were more star-shaped with non-linear actin structures and many filopodia. Furthermore, height of cells was higher was lower compared to cells on both types of hydrogels (0.36 µm vs. 0.56 µm vs. 1.03 µm). Young's modulus of cells on fibroblast was highest, which relates to the whole range of structural changes linked with surface stiffness. In conclusion, this type of hydrogel might be utilized in the future as a better surface material for cell cultivation.
Acknowledgements: CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2018127, is gratefully acknowledged for the financial support of the measurements at the CF Nanobiotechnology. The work was also supported by the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868)

Atomic force microscopy (AFM) combined with stem cell derived human cardiomyocytes (CMs) enables dynamic follow-up of cardiac contractions (e.g. beating rate, contraction and relaxation time), simultaneously with other CMs biomechanical properties. Today majority of drugs entering clinical usage, needs to be tested on arrhythmic adverse effects, nevertheless effect on cardiomyocyte contraction is not routinely employed, only related to cardiac pathologies. AFM-based biosensor allows in-vitro disease modelling but also enables to monitor the effect of CMs-contraction affecting drugs. Until today only few selected drugs modulating contractility and spontaneous pacing were described in animal models. This work for the first time demonstrates that basic biomechanical parameters, such as average value of contraction force and the beat rate represent valuable pharmacological indicators of different phenotypic effects on cells without genetic burden. Presented method is robust and has low computational requirements, while keeping optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement). The cardiac stimulating activities of basic beta-adrenergic stimulators such as isoproterenol and adrenalin, but also drugs utilized in pneumology as ipratropium, or salbutamol were tested. Stimulating drugs, e.g. methylxanthines and caffeine, presented aberrant cardiomyocyte response, confirming arrhythmogenic potential, and force related fluctuations. In experiment those could be dimished by beta-blockers as metoprolol. Spontaneous contraction irregularities quantification and related contractility changes allows precise scaling of potential negative effects adding new safety level to clinically relevant drug testing. AFM combined with cardiomyocytes can serve as robust screening platform for direct drug effects, especially contractility, which is hard to describe by other screening methods. Acknowledgements: Supported by Ministry of Health of the Czech Republic, grant AZV NU20-06-00156, we acknowledge CF Nanobiotechnology supported by MEYS CR (LM2018127). References: Pesl M, Pribyl J, Acimovic I, Vilotic A, Jelinkova S, Salykin A, Lacampagne A, Dvorak P, Meli AC, Skladal P, Rotrekl V. Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing. Biosens Bioelectron. 2016 Nov 15;85:751-757. doi: 10.1016/j.bios.2016.05.073. Epub 2016 May 28. PMID: 27266660.

Atomic force microscopy (AFM) is a highly sensitive non-invasive surface method able to provide insight into cells' mechanical parameters. Membrane and sub-membrane development, as well as internal cellular properties, can be monitored.
The stiffness of cells is a fundamental phenomenon that reflects changes in cell physiology. More importantly, changes in cell mechanical properties are also often found to be closely associated with various disease conditions. Cell mechanics are mainly dependent on cytoskeletal architecture.
The development of cryopreserved cells' mechanical properties (stiffness) after thawing was studied using AFM. Cell stiffness was mapped and thus monitored in time and space under nearly physiological conditions (i.e., in culture medium and at elevated temperature). In AFM force spectroscopy mode, cells are indented at many sites, and their complete elastic responses are recorded, enabling them to reconstruct a stiffness map. We measured the frozen cell surface stiffness immediately after thawing; they, when the dynamics of development of the cell stiffness were monitored in time up to 24 hours. Moreover, the AFM spectroscopy was combined with fluorescence-based staining of the cytoskeleton, thus enabling to directly correlate cytoskeleton development with stiffness mapping.
Acknowledgements: We acknowledge CF Nanobiotechnology supported by MEYS CR (LM2018127).

References
1. Golan, M. et al. AFM Monitoring the Influence of Selected Cryoprotectants on Regeneration of Cryopreserved Cells Mechanical Properties. Front. Physiol. 9, (2018).1.
2. Golan, M. et al. Cryopreserved Cells Regeneration Monitored by Atomic Force Microscopy and Correlated With State of Cytoskeleton and Nuclear Membrane. IEEE Transactions on NanoBioscience 17, 485-497 (2018).

Interactions between trycyclic antidepressants and zinc oxide nanoparticles have been studied. Amitriptyline and nortryptyline, which were immobilized from buffer medium at 37 °C, were selected as models. The key factor influencing the adsorption of these substances on zinc oxide is pH. During the adsorption, an acid-base equilibrium was established between the buffer, zinc oxide and the antidepressant. These pH changes were also reflected in the surface charge of the adsorbent and the values of the zeta potential, which was also monitored. Both substances have a similar structure, yet they differ in their acid-base properties and also in their ability to form micelles. These different properties caused different values of the amount adsorbed on the surface of the zinc oxide. Adsorption isotherms show an sigmoidal course. The highest adsorbed amount of amitriptyline on ZnO is 6.38 mmol g-1, in the case of nortryptyline it is 3.68 mmol g-1. The adsorption is directed in accordance with the pseudo-second-order kinetic equation. The values from the kinetic equations, as well as the changes in the zeta potential, indicate different mechanisms of immobilization of amitriptyline and nortryptyline on the surface of zinc oxide nanoparticles. In the case of both substances, due to the interaction with zinc oxide, they were significantly removed from the liquid medium; in the case of amitryptyline, the residual concentration was less than 10% at an initial concentration of 5 mmol L-1.