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- ItemHuman Primary Monocytes as a Model for in vitro Immunotoxicity Testing: Evaluation of the Regulatory Properties of TiO2 Nanoparticles(DOVE MEDICAL PRESS LTD, 2025-01-30) Švadláková, Tereza; Koláčková, Martina; Kulich, Pavel; Kotouček, Jan; Rosecká, Michaela; Krejsek, Jan; Fiala, Zdeněk; Andrýs, CtiradIntroduction: A critical step preceding the potential biomedical application of nanoparticles is the evaluation of their immunomodulatory effects. Such nanoparticles are expected to enter the bloodstream where they can be recognized and processed by circulating monocytes. Despite the required biocompatibility, this interaction can affect intracellular homeostasis and modulate physiological functions, particularly inflammation. This study focuses on titanium dioxide (TiO2) as an example of relatively low cytotoxic nanoparticles with potential biomedical use and aims to evaluate their possible modulatory effects on the inflammasome-based response in human primary monocytes. Methods: Monocyte viability, phenotypic changes, and cytokine production were determined after exposure to TiO2 (diameter, 25 nm; P25) alone. In the case of the modulatory effects, we focused on NLRP3 activation. The production of IL-1(3 and IL-10 was evaluated after (a) simultaneous activation of monocytes with bacterial stimuli muramyl dipeptide (MDP), or lipopolysaccharide (LPS), and TiO2 (co-exposure model), (b) prior activation with TiO2 alone and subsequent exposure to bacterial stimuli MDP or LPS. The differentiation of TiO2-treated monocytes into macrophages and their polarization were also assessed. Results: The selected TiO2 concentration range (30-120 mu g/mL) did not induce any significant cytotoxic effects. The highest dose of TiO2 promoted monocyte survival and differentiation into macrophages, with the M2 subset being the most prevalent. Nanoparticles alone did not induce substantial production of inflammatory cytokines IL-1(3, IL-6, or TNF-alpha. The immunomodulatory effect on NLRP3 depended on the type of costimulant used. While co-exposure of monocytes to MDP and TiO2 boosted NLRP3 activity, coexposure to LPS and TiO2 inhibited NLRP3 by enhancing IL-10 release. The inhibitory effect of TiO2 on NLRP3 based on the promotion of IL-10 was confirmed in a post-exposure model for both costimulants. Conclusion: This study confirmed a non-negligible modulatory effect on primary monocytes in their inflammasome-based response and differentiation ability.
- ItemElectrospun Ethyl-p-Methoxycinnamate Loaded CS/PEG/PLA Nanofibers(SAGE PUBLICATIONS INC, 2025-01-01) Dinh, Do Thi; Phuong, Ngo Thi; Thuy, Nguyen Thi Thu; Nhung, Pham Thi; Hung, Le Ngoc; Huyen, Do Thi Thanh; An, Nguyen Thai; Žídek, Jan; Le Minh, HaIn this study, the characteristic properties and biological activities of the electrospun nanofibers made of polymers including chitosan (CS), polyethylene glycol (PEG), and polylactic acid (PLA) loaded with 15% of a natural anti-inflammatory and analgesic agent, ethyl p-methoxycinnamate (EPMC), were reported. The morphological and chemical characteristics of the fibers were analyzed using a scanning electron microscope and Fourier transform infrared spectra, respectively. The mechanical properties and hydrophilicity of the nanofibers were also investigated. Research on the release of the bioactive compound EPMC from the nanofibers in phosphate-buffered saline medium showed that about 70% of EPMC was released after 24 h. The significant anti-inflammatory effect of the nanofibers was evaluated via inhibition of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide with an IC50 value of 41.05 +/- 1.75 mu g/mL. Besides, the nanofibers also exhibited anti-bacterial activity and were proved to be safe both in vitro and in vivo. The results suggested that the nanofibers CS/PEG/PLA loaded with ethyl p-methoxycinnamate may be a potential candidate for developing biomedical products related to inflammation.
- ItemOptimization and validation of multiresidual extraction methods for pharmaceuticals in Soil, Lettuce, and Earthworms(Springer-Verlag, 2024-04-27) Mravcová, Ludmila; Amrichová, Anna; Navrkalová, Jitka; Hamplová, Marie; Sedlář, Marian; Zlámalová Gargošová, Helena; Fučík, JanThe presence of human and veterinary pharmaceuticals (PhACs) in the environment poses potential risks. To comprehensively assess these risks, robust multiresidual analytical methods are essential for determining a broad spectrum of PhAC classes in various environmental compartments (soil, plants, and soil organisms). This study optimized extraction methods for analyzing over 40 PhACs from various matrices, including soil, lettuce, and earthworms. A four-step ultrasonic extraction method with varying extraction conditions and subsequent solid phase extraction was developed for soil samples. QuEChERS methods were optimized for extracting PhACs from lettuce and earthworm samples, addressing a literature gap in these less-studied matrices. The quantification of PhACs in soil, lettuce, and earthworm extracts was performed using a single LC-MS/MS method. Following thorough method validation, earthworms and lettuce were exposed to a mixture of 27 pharmaceuticals in a soil environment. The method validation results demonstrated the robustness of these methods for a broad spectrum of PhACs. Specifically, 29 out of 42 PhACs were extracted with an average efficiency >50% and RSD <30% from the soil; 40 out of 42 PhACs exhibited average efficiency >50% and %RSD <30% from the earthworms, while 39 out of 42 PhACs showed average efficiency >50% and RSD <30% from the lettuce. Exposure experiments confirmed the viability of these methods for quantifying a diverse range of PhACs in different environmental compartments. This study presents three thoroughly validated methods for determining more than 40 PhACs in diverse matrices, enabling a comprehensive assessment of PhAC dissemination in the environment.
- ItemThe pharmaceutical quality of freeze-dried tablets containing therapeutic bacteriophages against Pseudomonas aeruginosa and Staphylococcus aureus(Elsevier, 2025-01-07) Komárková, Marie; Benešík, Martin; Černá, Eva; Sedláčková, Lucie; Moša, Marek; Vojtová, Lucy; Franc, Aleš; Pantůček, RomanThe preparation of a solid dosage form containing bacteriophages, which meets pharmaceutical requirements and ensures long-term stability of the phage effect, is significant for implementing phage therapy in practice. A commonly used method for processing phages into a solid form is freeze-drying into a so-called freeze-dried cake; however, to date there have been no studies examining the pharmacopeial parameters of freeze-dried tablets with bacteriophages. In this study, we describe the preparation and properties of freeze-dried tablets containing a cocktail of purified pseudomonal bacteriophage DSM 33593 from the genus Pbunavirus and staphylococcal bacteriophage DSM 33473 from the genus Kayvirus (108 PFU/tablet) as the active ingredient. Maltodextrin was used as a tablet filler, and D-mannitol was used as a cryoprotectant. The tablet preparation process resulted in a decrease in phage titer by no more than 1 log PFU/mL. For Pbunavirus, the titer values in tablet and liquid form were comparable. Kayvirus was more stable in tablet form than in liquid form after six months of storage at 25 degrees C (a decrease of 1.9 +/- 0.8 log PFU/mL and 3.8 +/- 0.7 log PFU/mL, respectively). The uniformity of mass of singledose preparations, uniformity of content of single-dose preparations, and their disintegration complied with pharmacopeial requirements. The uniformity of dosage units of the tablets was maintained over three months. A microscopic examination of the internal part of the tablet revealed a heterogeneous structure, which does not affect the required pharmacopeial properties of the tablets. This study highlights the potential of freeze-dried tablets for long-term preservation of the phage effect at room temperature.
- ItemIn vitro culture of leukemic cells in collagen scaffolds and carboxymethyl cellulose-polyethylene glycol gel(PeerJ, 2024-12-06) Svozilova, Hana; Vojtová, Lucy; Matulová, Jana; Bruknerova, Jana; Poláková, Veronika; Radová, Lenka; Doubek, Michael; Plevová, Karla; Pospíšilová, ŠárkaBackground: Chronic lymphocytic leukemia (CLL) is a common adult leukemia characterized by the accumulation of neoplastic mature B cells in blood, bone marrow, lymph nodes, and spleen. The disease biology remains unresolved in many aspects, including the processes underlying the disease progression and relapses. However, studying CLL in vitro poses a considerable challenge due to its complexity and dependency on the microenvironment. Several approaches are utilized to overcome this issue, such as co-culture of CLL cells with other cell types, supplementing culture media with growth factors, or setting up a three-dimensional (3D) culture. Previous studies have shown that 3D cultures, compared to conventional ones, can lead to enhanced cell survival and altered gene expression. 3D cultures can also give valuable information while testing treatment response in vitro since they mimic the cell spatial organization more accurately than conventional culture. Methods: In our study, we investigated the behavior of CLL cells in two types of material: (i) solid porous collagen scaffolds and (ii) gel composed of carboxymethyl cellulose and polyethylene glycol (CMC-PEG). We studied CLL cells' distribution, morphology, and viability in these materials by a transmitted-light and confocal microscopy. We also measured the metabolic activity of cultured cells. Additionally, the expression levels of MYC, VCAM1, MCL1, CXCR4, and CCL4 genes in CLL cells were studied by qPCR to observe whether our novel culture approaches lead to increased adhesion, lower apoptotic rates, or activation of cell signaling in relation to the enhanced contact with co-cultured cells. Results: Both materials were biocompatible, translucent, and permeable, as assessed by metabolic assays, cell staining, and microscopy. While collagen scaffolds featured easy manipulation, washability, transferability, and biodegradability, CMC-PEG was advantageous for its easy preparation process and low variability in the number of accommodated cells. Both materials promoted cell-to-cell and cell-to-matrix interactions due to the scaffold structure and generation of cell aggregates. The metabolic activity of CLL cells cultured in CMC-PEG gel was similar to or higher than in conventional culture. Compared to the conventional culture, there was (i) a lower expression of VCAM1 in both materials, (ii) a higher expression of CCL4 in collagen scaffolds, and (iii) a lower expression of CXCR4 and MCL1 (transcript variant 2) in collagen scaffolds, while it was higher in a CMC-PEG gel. Hence, culture in the material can suppress the expression of a pro-apoptotic gene ( MCL1 in collagen scaffolds) or replicate certain gene expression patterns attributed to CLL cells in lymphoid organs (low CXCR4, high CCL4 in collagen scaffolds) or blood (high CXCR4 in CMC-PEG).