Seznam abstraktů 2023: SEKCE C - BIONANOTECHNOLOGIE, NANOMATERIÁLY PRO MEDICÍNU

Monodisperse iron oxide nanoparticles can be tailored via organic shells to have controlled interactions with biomolecules in biofluids. The control over these interactions is a prerequisite for their use in biomedical and biotechnological applications, from contrast agents to drug delivery systems.
We have systematically investigated how to modify superparamagnetic iron oxide nanoparticles with polymer brushes to study both their interactions with proteins and as actuators of thermoresponsive polymer brushes and amphiphilic membrane phase transitions.
My presentation will focus on our work on using grafted polymer brush morphology and topology to create truly stealth superparamagnetic nanoparticles and the use of core-shell nanoparticles to control release from thermoresponsive membrane capsules. Characterizing and testing nanoparticle interactions with proteins and cells is a challenge. I will describe our approach to this topic, including how to capture soft protein corona interactions.
Further, we will demonstrate how the hierarchical self-assembly and magnetic actuation of nanoparticles in membranes can be used to trigger the release of compounds from liposomes or lipopolymersomes with arbitrarily chosen release profiles. In these vesicles, the nanoparticles are used to locally heat the membrane by alternating magnetic fields that leave tissue unaffected. The local heating causes equally local membrane phase transitions that change the membrane permeability. Various designs of such nanoparticle and release systems will be presented and compared.

Nowadays bone is the second most common transplanted tissue, which generates an increasing need for appropriate replacements. A promising approach is the use of three-dimensional scaffolds mimicking the architecture of the natural extracellular matrix, in combination with a mesenchymal stem cell component, which could reduce the risk of immune reaction and decrease the need for reoperation.
In our work, we correlated the use of polylactide-based scaffolds prepared with two different techniques: freeze-dried foams and 3D-printed scaffolds. We tested polymeric PLA/PCL foams with hierarchical structure obtained by addition of water-soluble porogen. 3D-printed PLA polymeric scaffolds were of different inner architectures: honeycomb or linear structure. All scaffold types created a fully interconnected porous network, mimicking the extracellular matrix. As a cellular component, the adipose tissue-derived stem cells (ASCs) were used. The basic criterion for the scaffold type selection was the persisting and dense colonization of the inside area of scaffolds, evaluated on days 6 and 10 by fluorescence staining with phalloidin and DAPI and using the Dragonfly 503 confocal microscope.
All 3D-printed samples have appropriate mechanical properties, cells were spread along the fibers, proliferated well, and colonized the inside of the scaffolds. The most efficient cell penetration occurred in mineralized scaffolds with honeycomb pattern and larger gaps between the fibers. Also, the mineralized foams with hierarchical structure were found to be a promising material, but the penetration of cells deeper inside the material was poorer. ACKNOWLEDGEMENTS: Supported by the Ministry of Health of the Czech Republic (grant No. NU20-08-00208).

The surface of Titanium Grade 2 plates was covered with glancing angle deposited (GLAD) Ti needles. After the deposition, the Ti needles were additionally anodized. As shown by transmission electron microscopy, the morphology and composition of the Ti GLAD needles were affected by the anodization process. According to atomic force microscopy measurement, GLAD needle-covered Ti samples showed decreased roughness after the anodization. Contact angle measurements showed that this had a direct impact on the surface wettability, which changed from hydrophilic to hydrophobic upon anodization. Moreover, the electrochemically active surface area of the Ti GLAD samples, determined coulometrically, was 7 times larger than that of the polished Ti sample used as a reference. Furthermore, the sample surfaces were electrochemically characterized. To determine corrosion resistance properties, the Tafel plots were measured. Overall, compared to the polished Ti sample, the Ti GLAD samples showed better corrosion resistance right after deposition. Comparing the anodized and non-anodized samples, the anodized sample showed further improvement. To learn more about the oxide properties, electrochemical impedance spectroscopy measurements were made. The data were fitted to the suggested electrical circuit models. Finally, the bioassessment with Saos-2 cells revealed osseo-repellent qualities of Ti GLAD samples.

In this contribution, we examine the diffusion dynamics of methylene blue dye through a layered membrane, constructed with a specific count of nonwoven polypropylene textiles and intervening layers of gelatin hydrogel. The hydrogel ostensibly functions as the diffusion medium, while the nonwoven textile provides structural reinforcement. Due to the large pore structure inherent to polypropylene, the membrane displays minimal dye adsorption. Our research discovered the unusual nature of the dye's diffusion process, revealing a distinct, characteristic concentration profile of methylene blue within the membrane, which is notably inhomogeneous. Using UV-VIS spectroscopy in conjunction with image analysis techniques, we provide an analysis of the temporal progression of dye permeation through the gelatin layers. This examination has led to the identification of a fascinating mechanism governing methylene blue diffusion. These insights substantially augment our understanding of dye diffusion processes within gel matrices and stand to influence the advancement of gel-based application design, including but not limited to sensor technologies and other devices necessitating controlled substance release or absorption mechanisms. Furthermore, the significance of our findings reverberates beyond the immediate study, bearing broad implications for the comprehension of diffusion dynamics within gel substrates on a more comprehensive scale.

The ultra-sonication makes the sound energy to activate to form the nanomaterial and to deposit on various substrates. Acoustic cavitation is the process that includes the formation, growth, and implosive collapse of the gas bubbles that are formed in the liquid. The bubbles are formed due to the decrease in the intermolecular forces by the presence of invisible particles or gas bubbles. The formed bubble grows by diffusion of solute vapor to the volume of the bubble. The localized hotspots forming extreme conditions that reduce the particle consequently stimulating the small-sized nanoparticle particle to place on the substrates by the sonicating system. The ultrasound cavitation forms strong interaction between the substrates and nanomaterial. The ultrasound cavitation in liquid-solid systems creates a shockwaves and micro-jets that are directed towards the solid surface and accelerates the nanoparticle particles to higher velocities, which can lead to strong direct deposition on a substrate. This strong interaction between the polymer chains and nanoparticle led to changes in the backbone chain of the polymer composite when compared to the pristine material. This effective interaction between the substrates and nanocomposite completely eradicates the sensitive, resistant bacteria and bacterial biofilms.

The field of tissue engineering has witnessed significant progress in the development of vascular grafts for critical applications in cardiovascular surgery; however, the current vascular grafts still suffer from certain inadequacies such as thrombosis, intimal hyperplasia, and compliance mismatch. Among the various fabrication techniques, coaxial electrospinning has emerged as a promising method to produce vascular grafts due to its ability to tailor the architecture and composition of fibers for creating core-shell fibers, enabling the incorporation of both miscible and immiscible polymers and functional properties within the same fiber. The importance of material selection in coaxial electrospinning for vascular graft applications lies in determining the graft's mechanical properties, biocompatibility, and degradation characteristics, crucial factors that directly impact its long-term performance and success in cardiovascular tissue engineering. The mechanical properties of vascular grafts are important as they directly influence graft durability, stability, and biomechanical compatibility, critical factors for ensuring successful integration and long-term functionality within the cardiovascular system. This study focuses on the effect of polymer selection and blending for the core and shell parts of the fibers on the tensile strength, burst strength, and compliance of monolayer vascular grafts. The scaffolds having coaxial fibers with PCL, PLA, and their blends either in the core or shell part will be fabricated, and their mechanical properties will be compared.

Today, innovative tools for diagnostics and bioanalytics are needed. They should be able to detect molecules of interest with sufficient sensitivity, preferably label-free and multiplexed, to be usable outside of dedicated laboratories and with less qualified personnel, at minimal costs.
Plasmonic nanostructures promise to provide sensing capabilities with the potential for ultrasensitive and robust assays in a high parallelization and miniaturization, and without the need for markers. When functionalized with certain biomolecules (such as single-stranded DNA or antibodies) which bind the target molecule of interest (e.g., a biomarker like DNA or a protein), they should capture this target molecule with high specificity. Upon binding of target molecules, the localized surface plasmon resonance (LSPR) of these structures is changed, and can be used as sensoric readout. This is possible even on a single nanostructure level, using optical darkfield detection introduced more than 100 years ago, as demonstrated for DNA detection. In contrast to SPR, LSPR senses only in a very thin layer (on the scale of the particle diameter), resulting in an efficient background suppression. Only molecules bound directly at the surface contribute significantly to the signal, but much less molecules farer away.
In order to multiplex this approach, imaging spectrometer setups, e.g. based on a Michelson interferometer or multiple LEDs have been developed, able to readout a whole array of sensors in one step. On the sensor side, microarrays of gold nanoparticle spots were fabricated using spotting of pre-synthesized gold nanoparticles. Such chemically synthesized particles allow for a cost-efficient generation of highly crystalline particles as nanosensors; by using microfluidic approaches, a higher quality and reproducibility can be achieved. Using this microarray approach, a multiplex DNA-targeting detection of fungal pathogens involved in sepsis could be demonstrated. DNA-based signal amplification, e.g. by hybridization chain reaction, improves the sensitivity. Beyond DNA detection, LSPR sensing is also applicable for the detection of protein targets, such as CRP.
In conclusion, the results demonstrate the potential of this LSPR-based array platform for molecular detection of biomolecules of interest, with possible applications in bioanalytics and diagnostics.
ACKNOWLEDGEMENTS: Funding by BMBF (02WIL1521, 01DR20010A and 13N15717) and TAB (2018 VF 0015; 2018 FE 9039).

Nanotherapeutics for brain delivery are currently limited due to the taut nature of the blood-brain barrier (BBB). The modification of the surface of the nanoparticles (NPs) to exploit receptor-mediated traversing of molecules across the BBB has the potential to overcome this hurdle. Due to their facile synthesis and surface functionalization, ferrite NPs have emerged as attractive nanocarriers for gene and drug delivery. This study reports on the synthesis of ferrite NPs targeted for LRP-1 receptor-mediated transcytosis. The as-prepared NPs comprising cobalt ferrite derivatives (viz. CoFe2O4 and Mg0.5Co0.5Fe2O4) were coated with chitosan (CS) and covalently conjugated with Angiopep-2 (ANG), a blood-brain barrier targeting peptide. The physical, chemical, and magnetic properties of the ANG-CS ferrite NPs were characterised by X-ray diffraction (XRD), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FTIR), and nanoparticle tracking analysis (NTA). XRD and VSM results revealed spinel crystalline ferrite core NPs exhibiting superparamagnetic behavior. Particle core sizes were below 13.59 nm. The FTIR spectra showed characteristic peak shifts upon CS-coating and functionalization with Angiopep-2 for all ferrite NPs. Hydrodynamic sizes of 90.4 nm and 152. nm were reported for the ANG-CS-CoFe2O4, and ANG-CS-Mg0.5Co0.5Fe2O4, respectively. ANG-CS-Mg0.5Co0.5Fe2O4 presented the most stability with a zeta potential of +29.0 mV, while ANG-CS-CoFe2O4 has a zeta potential of +19.2 mV. Molecular docking investigations revealed binding energies for the CS-conjugated ANG of -5.67 kcal/mol. These findings suggest that the LRP-1 receptor would not be significantly hindered by the ANG-conjugation to the CS-coated ferrite NPs. These CS-coated cobalt ferrite derivatives thus exhibit potential for targeted CNS delivery and can be further explored for in vitro and in vivo gene and drug delivery studies.

In the past few decades, nanoparticles have been gaining popularity as antimicrobial agents due to their superior properties as opposed to conventional antibiotics (Rutherford et al.; 10.2147/ijn.s300428). Photoactive nanoparticles such as zinc oxide (ZnO) are of special interest as their antimicrobial properties may be influenced using illumination (Jíra et al.; 10.1088/1757-899x/1050/1/012007). To elucidate the effects of nanomaterials and illumination on bacterial cell growth, however, a method to obtain reproducible optical density measurements under the same conditions is needed. In this work, we present such a method for long-term illumination of planktonic cells grown in falcon tubes using reverse spin bioreactors. Custom caps which completely enclosed the bioreactors and falcon tubes were created using 3D modelling and printing. This way, we were able to obtain stable illumination of the bacteria-nanoparticle sample as well as reproducible growth curves, which allowed for the reliable detection of lag phase lengths and maximal growth rates as well as average growth curves. The method was then tested on four cultures of Escherichia coli cells suspended in Mueller Hinton Broth. Half the cultures were exposed to a sub-lethal concentration of commercial ZnO nanoparticles (Sigma Aldrich; 200 nm with micron-sized clusters) and half were illuminated using a visible light source to give all four combinations of these conditions. The experiments were carried out in triplicate for statistical validation and supplemented with colony forming unit (CFU) measurements both before and after exposure, scanning electron microscope (SEM) imaging and minimum inhibitory concentration (MIC) measurements of cells after various exposure conditions. We have established a technique which incorporates real-time monitoring of bacteria growth in the presence of photosensitive nanoparticles and continuous visible light illumination source to study the effects of sub-lethal concentrations of antibacterial photosensitive nanoparticles. Such studies are vitally important for understanding the development of resistance towards novel antimicrobial treatments.

Acne vulgaris is a dermatological severe disease affecting a large portion of the population [1]. ZnO nanoparticles (NPs) represent very efficient agent for the treatment of acne and enhanced skin compatibility, compared to antibiotics. Its antibacterial effect is attributed mainly to the eminent release of biologically active Zn2+ ions from nanoparticles surface and the formation of reactive oxygen species resulting in the death of bacterial cells [2]. On the other hand, the nanoparticles need to be properly dispersed on a suitable carrier, to be applicable on the skin. Fibrous materials have the potential to be great NPs carrier, owing to their unique characteristics, including high porosity, a large specific surface area, and good breathability. Combining ZnO NPs and fibers creates a very promising hybrid material, that is attractive for the treatment of Acne vulgaris [3-5]. In this work, gum arabica, pullulan and ZnO nanoparticles were used as the main material for preparing the therapeutic hybrid material. The polymeric solution with different concentrations (0.30 and 4.5 wt.%) of ZnO NPs were spun by centrifugal spinning into fibers, whose morphological and chemical analyses were carried out (SEM and ED XRF). A relatively homogenous distribution of ZnO within fibers was revealed, which confirms very good prospects of the centrifugal spinning used for fiber synthesis. Most importantly, they were characterized for their antibacterial activity against acne-causing bacteria. The results showed that blended fibers had antibacterial activity for as low ZnO content as 1.5 wt. %. The concept and materials in this work bear clear commercial possibilities and a very promising material for dermatologic application [5]. REFERENCES: [1] R. Balkrishnan et al., Clin. Ther. 77, 2006.; [2] A. Sirelkhatim et al., Nano-Micro Lett. 7, 2015.; [3] M. Rihova et al., J. Appl Polym Sci. 168, 2021.; [4] M. Rihova et al., Carbohydr. Polym. 294, 2022.; [5] M. Rihova et al., Appl. Mater. Today, Ms submitted.

Calcium carbonate (CaCO3) particles are regarded as promising candidates for drug delivery, biomedical, and industrial filler applications due to their good physicochemical properties and biocompatibility. However, a prerequisite for these applications is to obtain particles with desired surface structures and phase compositions, which are intensely determined by synthesis parameters. Herein, we systematically study the synergistic effects of the concentration of reacting salts, reaction time, temperature on the surface morphology, size, and phase composition of CaCO3 particles. It is shown that the crystal growth and phase transition induced the transformation of spherical particles into fusiform, square and needle-like morphologies with increasing temperature, and the increase of concentration increased this transition temperature during this process as well as alleviated the degree of surface undulation of the vaterite agglomerates and the voids between the secondary particles. The size of the spherical particles was determined by the above factors determining the driving forces of the nucleation growth and diffusion of the crystals as well as the time required to reach the kinetic equilibrium. Temperature was the main factor controlling the formation of vaterite, calcite and aragonite, while the concentration is shown to shift the influence of phase transition temperatures to higher values, especially above 50 °C. These results provide reliable experimental supports for studying the crystallographic changes of CaCO3 particles and parameters range for the preparation of designed particles.