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).
- MeSH
- buněčné kultury metody MeSH
- chronická lymfatická leukemie * patologie metabolismus MeSH
- gely chemie MeSH
- kolagen * chemie farmakologie MeSH
- lidé MeSH
- polyethylenglykoly * chemie MeSH
- receptory CXCR4 metabolismus MeSH
- sodná sůl karboxymethylcelulosy * chemie farmakologie MeSH
- techniky 3D buněčné kultury metody MeSH
- tkáňové podpůrné struktury * chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Treating oral diseases remains challenging as API is quickly washed out of the application site by saliva turnover and mouth movements. In situ gels are a class of application forms that present sol-gel transition's ability as a response to stimuli. Their tunable properties are provided using smart polymers responsible for stimuli sensitivity, often providing mucoadhesivity. In this study, antimicrobial in situ gels of thermosensitive and pH-sensitive polymers loaded with silver nanoparticles were prepared and evaluated. The nanoparticles were prepared by green synthesis using Agrimonia eupatoria L. extract. According to the data analysis, the in situ gel with the most promising profile contained 15 % of Pluronic® F-127, 0.25 % of methylcellulose, and 0.1 % of Noveon® AA-1. Pluronic® F-127 and methylcellulose significantly increased the viscosity of in situ gels at 37 °C and shear rates similar to speaking and swallowing. At 20 °C, a behavior close to a Newtonian fluid was observed while being easily injectable (injection force 13.455 ± 1.973 N). The viscosity of the formulation increased with temperature and reached 2962.77 ± 63.37 mPa·s (37 °C). A temperature increase led to increased adhesiveness and rigidity of the formulation. The critical sol-gel transition temperature at physiological pH was 32.65 ± 0.35 °C. 96.77 ± 3.26 % of Ag NPs were released by erosion and dissolution of the gel after 40 min. The determination of MIC showed effect against E. coli and S. aureus (0.0625 mM and 0.5000 mM, respectively). The relative inhibition zone diameter of the in situ gel was 73.32 ± 11.06 % compared to gentamicin sulfate. This work discusses the optimization of the formulation of novel antibacterial in situ gel for oromucosal delivery, analyses the impact of the concentration of excipients on the dependent variables, and suggests appropriate evaluation of the formulation in terms of its indication. This study offers a promising dosage form for local treatment of oral diseases.
Branched copolymer surfactants (BCS) containing thermoresponsive polymer components, hydrophilic components, and hydrophobic termini allow the formation of emulsions which switch from liquid at room temperature to a gel state upon heating. These materials have great potential as in situ gel-forming dosage forms for administration to external and internal body sites, where the emulsion system also allows effective solubilisation of a range of drugs with different chemistries. These systems have been reported previously, however there are many challenges to translation into pharmaceutical excipients. To transition towards this application, this manuscript describes the evaluation of a range of pharmaceutically-relevant oils in the BCS system as well as evaluation of surfactants and polymeric/oligomeric additives to enhance stability. Key endpoints for this study are macroscopic stability of the emulsions and rheological response to temperature. The effect of an optimal additive (methylcellulose) on the nanoscale processes occurring in the BCS-stabilised emulsions is probed by small-angle neutron scattering (SANS) to better comprehend the system. Overall, the study reports an optimal BCS/methylcellulose system exhibiting sol-gel transition at a physiologically-relevant temperature without macroscopic evidence of instability as an in situ gelling dosage form.
Filmy jsou tenké, flexibilní a transparentní prostředky na rány. Mohou být připraveny jak ze syntetických, tak i přírodních materiálů. V praxi jednoznačně dominuje syntetický polyuretan, avšak výzkum se zaměřuje především na látky přírodního původu. Látkou tělu vlastní, s výbornými filmotvornými vlastnostmi, účastnící se procesu hojení ran je kolagen. Samotné kolagenové filmy však mají slabé mechanické vlastnosti, které lze mimo jiné vylepšit kombinací s dalším materiálem. Takovým materiálem by mohla být karboxymethylcelulosa s prokázaným příznivým účinkem na hojení ran. Filmy tvořené pouze CMC také naráží na slabé mechanické vlastnosti, tudíž by se kombinace obou materiálů mohla jevit jako vhodné řešení daných problémů a navíc by mohlo vzniknout směsné filmové krytí s mnoha výhodnými vlastnostmi pro hojení ran. Cílem našeho experimentu bylo vytvořit směsné filmy pro účely terapie ran tvořené kombinací kolagenu a CMC. Filmy byly připraveny metodou odpaření rozpouštědla a jejich vlastnosti byly porovnávány s vlastnostmi samotných filmů tvořených pouze CMC. V obou případech vznikaly filmy vhodných organoleptických, fyzikálně-chemických i aplikačních vlastností pro uplatnění v terapii ran. Směsné filmy měly v porovnání s těmi tvořenými pouze CMC nižší absorpční kapacitu a lepší mechanickou odolnost. Kombinace kolagenu a CMC pro vytvoření směsných filmů určených k terapii ran tak vedla ke zlepšení vlastností výsledného krytí a má potenciál pro další výzkum.
Films are thin, flexible, and transparent wound dressings. They can be prepared from both synthetic and natural materials. In practice, synthetic polyurethane dominates, but research is mainly focused on substances of natural origin. An endogenous substance with excellent filmforming properties, which is involved in the wound healing process, is collagen. However, collagen films themselves have weak mechanical properties, which can be improved by, among other things, combining collagen with other materials. Such material could be carboxymethylcellulose, which has been shown to affect wound healing positively. Films consisting only of CMC also have weak mechanical properties, so combining both materials seems to be a suitable solution to the given problems, and a wound dressing with many beneficial properties for wound healing could be created. Therefore, our experiment aimed to prepare composite films for wound therapy consisting of a combination of collagen and CMC. The films were prepared by the solvent evaporation method, and their properties were compared with those formed only by CMC. In both cases, films with suitable organoleptic, physicochemical, and application properties for wound therapy were produced. The composite films showed lower absorption capacity and better mechanical resistance compared to those formed only by CMC. The combination of collagen and CMC in composite films intended for wound therapy has thus resulted in improved properties of the resulting dressing and holds potential for further research.
- Klíčová slova
- filmové obvazy, směsné filmy,
- MeSH
- hojení ran MeSH
- klinická studie jako téma MeSH
- kolagen * aplikace a dávkování farmakologie terapeutické užití MeSH
- lidé MeSH
- obvazy * MeSH
- sodná sůl karboxymethylcelulosy aplikace a dávkování farmakologie terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The disconnect between preclinical and clinical results underscores the imperative for establishing good animal models, then gleaning all available data on efficacy, safety, and potential toxicities associated with a device or drug. Mini pigs are a commonly used animal model for testing orthopedic and dental devices because their skeletons are large enough to accommodate human-sized implants. The challenge comes with the analyses of their hard tissues: current methods are time-consuming, destructive, and largely limited to histological observations made from the analysis of very few tissue sections. We developed and employed cryo-based methods that preserved the microarchitecture and the cellular/molecular integrity of mini pig hard tissues, then demonstrated that the results of these histological, histochemical, immunohistochemical, and dynamic histomorphometric analyses e.g., mineral apposition rates were comparable with similar data from preclinical rodent models. Thus, the ability to assess static and dynamic bone states increases the translational value of mini pig and other large animal model studies. In sum, this method represents logical means to minimize the number of animals in a study while simultaneously maximizing the amount of information collected from each specimen.
- MeSH
- fyziologická kalcifikace MeSH
- kryoprezervace metody MeSH
- kryoultramikrotomie metody MeSH
- lebka cytologie MeSH
- miniaturní prasata MeSH
- odběr biologického vzorku metody MeSH
- polyethylenglykoly MeSH
- prasata MeSH
- remodelace kosti MeSH
- sacharosa MeSH
- sodná sůl karboxymethylcelulosy MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulose-derived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.
- MeSH
- aptamerová technika SELEX metody MeSH
- aptamery nukleotidové chemie MeSH
- ELISA metody MeSH
- jednovláknová DNA chemie MeSH
- methylcelulosa chemie MeSH
- polymerázová řetězová reakce metody MeSH
- sodná sůl karboxymethylcelulosy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The preparation of an amorphous solid dispersion (ASD) is a promising strategy for improving the poor oral bioavailability of many active pharmaceutical ingredients (APIs). However, poor predictability of ASD long-term physical stability remains a prevalent problem. The purpose of this study was to evaluate and compare the predictive performance of selected models concerning solid-liquid equilibrium (SLE) curve and glass-transition temperature (Tg) line modeling of ibuprofen (IBU) in cellulosic polymers (i.e., hydroxypropyl methylcellulose (HPMC) and hydroxypropyl methylcellulose acetate succinate (HPMCAS)). For SLE curve modeling, an empiricalanalyticalapproach(Kyeremateng et al., 2014)and the Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT) equation of state (EOS) were chosen. Due to the unavailability of PC-SAFT parameters for both polymers, an approximation procedure for parametrization was applied. The Gordon-Taylor equation and Kwei equation were considered for Tg line determination. The impact of various computational set-ups (e.g., model parametrization or extrapolation length) on IBU solubility prediction at storage conditions was thoroughly investigated, assessed and confronted with the results from an 18-month physical stability study. IBU developed stable 20 wt% API content ASDs with both HPMC and HPMCAS.The extrapolation behavior and subsequent ASD thermodynamic stability prediction at storage conditions deduced from the aforementioned models weresignificantly different. Overall, the PC-SAFT EOS predicted higher IBU solubility in both polymers and, thus, a lower recrystallization tendency when compared to the empirical analytical approach. At higherIBU concentrations, liquid-liquid demixing inIBU-polymer systems was predicted by the PC-SAFT EOS, which was in qualitative disagreement with experimental observation.
We have developed a novel simple method for effective preparing gold nanoparticles (AuNPs) intended for utilization in biomedicine. The method is based on gold sputtering into liquid poly(ethylene glycol) (PEG). The PEG was used as a basic biocompatible stabilizer of the AuNP colloid. In addition, two naturally occurring polysaccharides - Chitosan (Ch) and Methylcellulose (MC) - were separately diluted into the PEG base with the aims to enhance the yield of the sputtering without changing the sputtering parameters, and to further improve the stability and the biocompatibility of the colloid. The colloids were sterilized by steam, and their stability was measured before and after the sterilization process by dynamic light scattering and UV-Vis spectrophotometry. The results indicated a higher sputtering yield in the colloids containing the polysaccharides. The colloids were also characterized by atomic absorption spectroscopy (AAS) to reveal the composition of the prepared nanoparticles by transmission electron microscopy (TEM) to visualize the nanoparticles and to evaluate their size and clustering, and by rheometry to estimate the viscosity of the colloids. The zeta-potential of the AuNPs was also determined as an important parameter indicating the stability and the biocompatibility of the colloid. In addition, in vitro tests of antimicrobial activity and cytotoxicity were carried out to estimate the biological activity and the biocompatibility of the colloids. Antimicrobial tests were performed by a drip test on two bacterial strains - Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli. AuNP with chitosan proved to possess the highest antibacterial activity, especially towards the Gram-positive S. epidermidis. In vitro tests on eukaryotic cells, i.e. human osteoblastic cell line SAOS-2 and primary normal human dermal fibroblasts (NHDF), were performed after a 7-day cultivation to determine the effect and the toxic dose of the colloids on human cells. The studied colloid concentrations were in the range from 0.6 μg/ml to 6 μg/ml. Toxicity of the colloids started to reappear at a concentration of 4.5 μg/ml, especially with chitosan in the colloid, where the colloid with a concentration of 6 μg/ml proved to be the most toxic, especially towards the SAOS-2 cell line. However, the PEG and PEG-MC containing colloids proved to be relatively non-toxic, even at the highest concentration, but with a slowly decreasing tendency of the cell metabolic activity.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- buněčné linie MeSH
- chitosan chemie MeSH
- dynamický rozptyl světla MeSH
- Escherichia coli účinky léků MeSH
- koloidy chemie MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- methylcelulosa chemie MeSH
- polysacharidy chemie MeSH
- stabilita léku MeSH
- Staphylococcus epidermidis účinky léků MeSH
- sterilizace MeSH
- velikost částic MeSH
- zlato chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: To assess the efficacy of the novel clinical formulation of fenretinide (LAU-7b) for the treatment of allergic asthma. To study the association between LAU-7b treatment in allergic asthma and the modulation of very long chain ceramides (VLCC). METHODS: We used two allergens (OVA and HDM) to induce asthma in mouse models and we established a treatment protocol with LAU-7b. The severity of allergic asthma reaction was quantified by measuring the airway resistance, quantifying lung inflammatory cell infiltration (Haematoxylin and eosin stain) and mucus production (Periodic acid Schiff satin). IgE levels were measured by ELISA. Immunophenotyping of T cells was done using Fluorescence-activated cell sorting (FACS) analysis. The analysis of the specific species of lipids and markers of oxidation was performed using mass spectrometry. RESULTS: Our data demonstrate that 10 mg/kg of LAU-7b was able to protect OVA- and HDM-challenged mice against increase in airway hyperresponsiveness, influx of inflammatory cells into the airways, and mucus production without affecting IgE levels. Treatment with LAU-7b significantly increased percentage of regulatory T cells and CD4+ IL-10-producing T cells and significantly decreased percentage of CD4+ IL-4-producing T cells. Our data also demonstrate a strong association between the improvement in the lung physiology and histology parameters and the drug-induced normalization of the aberrant distribution of ceramides in allergic mice. CONCLUSION: 9 days of 10 mg/kg of LAU-7b daily treatment protects the mice against allergen-induced asthma and restores VLCC levels in the lungs and plasma.
- MeSH
- alergeny imunologie MeSH
- bronchiální astma farmakoterapie imunologie metabolismus MeSH
- ceramidy metabolismus MeSH
- fenretinid terapeutické užití MeSH
- klinické protokoly MeSH
- methylcelulosa chemie MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- ovalbumin imunologie MeSH
- příprava léků MeSH
- Pyroglyphidae imunologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Endo-glucanase (cellulase) and xylanase have high industrial demand due to their vast application in industrial processes. This study reports statistical based experimental optimization for co-production of endo-glucanase and xylanase from Bacillus sonorensis BD92. Response surface methodology (RSM) involving central composite design (CCD) with full factorial experiments (23) was applied to elucidate the components that significantly affect co-production of endo-glucanase and xylanase. The optimum co-production conditions for endo-glucanase and xylanase were as follows: carboxymethyl cellulose (CMC) 20 g/L, yeast extract 15 g/L, and time 72 h. The maximum endo-glucanase and xylanase production obtained was 1.46 and 5.69 U/mL, respectively, while the minimum endo-glucanase and xylanase production obtained was 0.66 and 0.25 U/mL, respectively. This statistical model was efficient because only 20 experimental runs were necessary to assess the highest production conditions, and the model accuracy was very satisfactory as coefficient of determination (R2) was 0.95 and 0.89 for endo-glucanase and xylanase, respectively. Further, potential application of these enzymes for saccharification of lignocellulosic biomass (wheat bran, wheat straw, rice straw, and cotton stalk) was also investigated. The results revealed that the biomass was susceptible to enzymatic saccharification and the amount of reducing sugars (glucose and xylose) increased with increase in incubation time. In conclusion, Bacillus sonorensis BD92 reveals a promise as a source of potential endo-glucanase and xylanase producer that could be useful for degrading plant biomass into value-added products of economic importance using precise statistically optimized conditions.
- MeSH
- Bacillus růst a vývoj metabolismus MeSH
- biomasa * MeSH
- celulasa biosyntéza MeSH
- endo-1,4-beta-xylanasy biosyntéza MeSH
- fermentace MeSH
- hydrolýza MeSH
- průmyslová mikrobiologie metody MeSH
- rýže (rod) metabolismus MeSH
- sodná sůl karboxymethylcelulosy MeSH
- statistické modely MeSH
- Publikační typ
- časopisecké články MeSH
