Pokročilé biomateriály

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Optimization 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, Jan
The 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.
The 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, Roman
The 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.
In 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, Šárka
Background: 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).
Porcine model of a complicated skin and soft tissue infection caused by Pseudomonas aeruginosa
(CZECH ACADEMY AGRICULTURAL SCIENCES, 2024-09-10) Lipový, Břetislav; Vacek, Lukáš; Polaštík Kleknerová, Dominika; Jeklová, Edita; Lišková, Lenka; Holoubek, Jakub; Matýsková, Dominika; Růžička, Filip
Pseudomonas aeruginosa poses a significant threat to both immunocompetent and immunocompromised individuals, often resulting in life -threatening infections. With increasing antimicrobial resistance, novel therapeutic strategies are urgently needed. Although animal models are crucial for preclinical studies, limited data are available for porcine models, more specifically for P. aeruginosa complicated skin and soft tissue infections (cSSTIs). This study presents a novel porcine model inducing and sustaining cSSTI for 14 days. Six pigs (120 wounds) were used for the development of infections, and within this group, two pigs (40 wounds) were used to evaluate the progression of the cSSTI infection. The model demonstrated bacterial loads of more than 10 7 CFU/ gram of tissue or higher. The cSSTI fully developed within three days and remained well above these levels until day 14 post -infection. Due to the immunocompetence of this model, all the immunological processes associated with the response to the presence of infection and the wound healing process are preserved.
Pharmaceutical Metabolite Identification in Lettuce (Lactuca sativa) and Earthworms (Eisenia fetida) Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry and In Silico Spectral Library
(Springer-Verlag, 2024-09-10) Fučík, Jan; Fučík, Stanislav; Rexroth, Sascha; Sedlář, Marian; Zlámalová Gargošová, Helena; Mravcová, Ludmila
Pharmaceuticals released into the aquatic and soil environments can be absorbed by plants and soil organisms, potentially leading to the formation of unknown metabolites that may negatively affect these organisms or contaminate the food chain. The aim of this study was to identify pharmaceutical metabolites through a triplet approach for metabolite structure prediction (software-based predictions, literature review, and known common metabolic pathways), followed by generating in silico mass spectral libraries and applying various mass spectrometry modes for untargeted LC-qTOF analysis. Therefore, Eisenia fetida and Lactuca sativa were exposed to a pharmaceutical mixture (atenolol, enrofloxacin, erythromycin, ketoprofen, sulfametoxazole, tetracycline) under hydroponic and soil conditions at environmentally relevant concentrations. Samples collected at different time points were extracted using QuEChERS and analyzed with LC-qTOF in data-dependent (DDA) and data-independent (DIA) acquisition modes, applying both positive and negative electrospray ionization. The triplet approach for metabolite structure prediction yielded in a total of 3,762 pharmaceutical metabolites, and in silico mass spectral library was created based on these predicted metabolites. This approach resulted in the identification of 26 statistically significant metabolites (p<0.05), with DDA+ and DDA- outperforming DIA modes by successfully detecting 56/67 sample type:metabolite combinations. Lettuce roots had the highest metabolite count (26), followed by leaves (6) and earthworms (2). Despite the lower metabolite count, earthworms showed the highest peak intensities, closely followed by roots, with leaves displaying the lowest intensities. Common metabolic reactions observed included hydroxylation, decarboxylation, acetylation, and glucosidation, with ketoprofen-related metabolites being the most prevalent, totaling 12 distinct metabolites. In conclusion, we developed a high-throughput workflow combining open-source software with LC-HRMS for identifying unknown metabolites across various sample types.

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