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Study of the interaction of lung surfactant with selected polymers
Suchá, Klára ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This thesis deals with the study of interactions between pulmonary surfactant (Curosurf) and selected polymers (N,N,N-trimethylchitosan, polyvinylpyrrolidone, sodium hyaluronate). This work also focuses on the inactivation of lung surfactant using bovine serum albumin, which causes an increase in the surface tension of the surfactant, whereby the inactivated surfactant is unable to fulfill its function in the lungs. The addition of polymers to this mixture has been shown to be an effective means of restoring the surface tension of the surfactant. First of all, this work focuses on finding the optimal concentration ratios of polymer and Curosurf, at which their mutual interaction occurs. The method of dynamic light scattering was used to obtain optimal ratios. In the second part, the work is devoted to the determination of surface tension using a du Noüy ring tensiometer. It was found that the addition of all selected polymers led to a reduction of the surface tension of the inactivated surfactant to values close to native Curosurf. This work provided useful information to understand the mechanism of lung surfactant surface tension recovery.
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Rheological characterization of polysaccharide gels with incorporated vesicles
Kalendová, Lucie ; Smilek, Jiří (referee) ; Venerová, Tereza (advisor)
This diploma thesis deals with the rheological characterization of polysaccharide gels with incorporated vesicular systems. Positively charged Ion Pair Amphiphile vesicles (IPA) consisting of cetrimethylammonium bromide, sodium dodecyl sulfate, dioctadecyldimethylammonium chloride and cholesterol were chosen for the incorporation. Other vesicles used for incorporation were oppositely charged liposomes made of phosphatidylcholine (DPPC), phosphatidic acid (PA) and cholesterol. The thesis is divided into two sections. The first one deals with preparation, characterization and stability control (temperature and time stability) of both vesicular systems by using dynamic light scattering (DLS). In this part, the vesicles’ stability was confirmed which made them perfect candidates for the following incorporation. The second part of the thesis describes the preparation and rheological characterization of selected gels. Three types of gels were prepared in total, specifically 1% and 2% gellan gum gels and 4% hyaluronic gel. All of them were studied in water, as well as in solutions of IPA vesicles or DPPC liposomes, both of same concentration (1 mmoll-). The subsequent rheological characterization including amplitude, frequency, and stress relaxation sweeps demonstrates there is no significant difference between reference gels and those containing vesicles. This indicates that vesicles used in this thesis were only freely stored in the gel networks, meaning the structure of the gels didn’t affect their transport properties. This is a significant information for using these systems in drug delivery.
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Micro/macro-scale investigation of the viscoelastic properties of hydrogel materials
Obrusníková, Klára ; Pekař, Miloslav (referee) ; Smilek, Jiří (advisor)
Tato diplomová diplomová práce byla zaměřená na srovnání mezi mikroreologickými metodami a makroreologickým přístupem, zejména při charakterizaci hydrogelů. Dynamický rozptyl světla, fluorescenční korelační spektroskopie a optická pinzeta byly vybrány jako mikroreologické techniky, zatímco oscilační reometrie sloužila jako makroreologický nástroj. Výsledky z dynamického rozptylu světla a fluorescenční korelační spektroskopie pro hydrogely agarózy a gellanu byly porovnány s reometrií a mezi sebou. Hodnoty viskoelastických modulů získané z mikroreologických měření byly výrazně nižší než ty získané pomocí makroreologie. Jak dynamický rozptyl světla, tak fluorescenční korelační spektroskopie nabízejí širší škálu měřitelných frekvencí než reometrie, ale obě metody mají své limitace při charakterizaci hydrogelů. U dynamického rozptylu světla dochází k odrazu světla od gelové sítě, což způsobuje šum a zhoršuje kvalitu získaných výsledků. Fluorescenční korelační spektroskopie, přes svou vyšší specifitu, vykazuje jen velmi malé rozdíly mezi vzorky různých koncentrací. Optická pinzeta je na Fakultě chemické novou metodou, a byla provedena pouze kalibrační měření. Tuhost optické pasti, důležitá hodnota pro kalibraci optické pinzety, byla stanovena pro roztoky glycerolu.
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Preparation and characterization of triple-IPA for the formation of vesicular systems
Novotná, Ludmila ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This thesis deals with the preparation and characterization of vesicular systems consisting of triple-IPA (ion pair amphiphile with three hydrophobic chains). For the preparation of tripleIPA were used double-chain cationic surfactants dimethyldimyristylammonium bromide (DMSAB), dimethyldipalmitylammonium bromide (DPAB), dimethyldioctadecylammonium bromide (DODAB) and dimethyldioctadecylammonium chloride (DODAC) and the singlechain anionic surfactant sodium dodecyl sulfate (SDS). Among the cationic surfactants, DMSAB and DPAB were selected to be studied more thoroughly. Triple-IPAs were prepared by mixing solutions of oppositely charged surfactants in equimolar ratio, and the resulting triple-IPA precipitate was filtered and dried. From the obtained powder, vesicular systems were prepared by rehydration and sonication. Cholesterol and cationic surfactants, from which the triple-IPAs were prepared, were used to stabilize the cationic vesicles. The properties of the vesicular systems were evaluated by dynamic and electrophoretic light scattering measurements and turbidimetry. The dispersion solutions with the addition of cholesterol were less turbid; therefore, it can be assumed that they supported the formation of cationic vesicles. The optimum cholesterol content is 20 mol. %. At this concentration, the effect of cholesterol on vesicle membrane reorganization becomes apparent and the cationic system remains monodisperse. A positive zeta potential in the stable region was determined for all prepared solutions, which was further increased by the addition of a positive surfactant. The most suitable cationic surfactant for the preparation of triple-IPA is DPAB, which is easy to work with, produces the most monodisperse systems and has the lowest measurement uncertainties.
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Study of interaction of charged catanionic vesicles with oppositely charged polyelectrolytes
Havlíková, Martina ; Lehocký, Marián (referee) ; Táborský, Petr (referee) ; Mravec, Filip (advisor)
The presented dissertation is focused on positively charged catanionic vesicles and their interactions with oppositely charged hyaluronan. The catanionic vesicles were composed of ion pair amphiphile of hexadecyltrimethylammonium-dodecyl sulfate (HTMA-DS), a positively charged double-chain surfactant that electrostatically stabilized the vesicles and cholesterol. Cholesterol may strongly affect the physico-chemical properties of the vesicles, therefore it was necessary to determine its optimal amount in the membrane. Especially, the size and stability of vesicles were studied by dynamic and electrophoretic light scattering, membrane microviscosity by fluorescence anisotropy and hydration of the outer part of the membrane by generalized polarization. It was found that vesicles show ideal properties at a cholesterol content about 40 mol.%. These catanionic vesicles interacted with hyaluronan regardless of its molecular weight. Visible aggregates were observed around their isoelectric point, but they disintegrated with increasing polymer concentration. However, this decay was different for different hyaluronan molecular weights. An increase in ionic strength, or more precisely concentration of sodium chloride above 25 mM had a destabilizing effect on both uncoated and hyaluronan coated vesicles. It was also interesting that increasing the ionic strength affected the microviscosity of the membrane, which became more fluid, but there were no significant changes in the hydration of its outer part. The presented work is also focused on the preparation and characterization of a new type of catanionic vesicles, where the cationic surfactants HTMAB was replaced by Septonex. The same methods were chosen for their characterization as in the case of the HTMA-DS vesicles. This exchange of surfactants had a major effect on the resulting properties of the vesicles, which are stable even at very low cholesterol content. In accordance with long-term stability, a suitable concentration of cholesterol was chosen from 15 mol.% and higher.
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Microrheology with Fluorescence Correlation Spectroscopy
Kábrtová, Petra ; Sedláček, Petr (referee) ; Mondek, Jakub (advisor)
A comparison of three passive microrheological techniques was made with the emphasis on FCS. Fluorescently labelled and unlabelled polystyrene particles were used to probe a microrheological response of glycerol solutions, Mili-Q water and sodium hyaluronate solutions. In addition, for FCS technique an approximated equation for calculation of MSD values was derived and verified. It was found that FCS outmatches current microrheological techniques of DLS and video-based particle tracking by its ability to gain a broader data range including the area in which, until now, it was impossible to describe a microrheological behaviour of samples reliably.
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Hyaluronan micro- and nanoparticles
Mourycová, Jana ; Marián, Lehocký (referee) ; Pekař, Miloslav (advisor)
The aim of this thesis was to prepare hyaluronic acid micro- and nanoparticles based on electrostatic interactions with oppositely charged molecules. Following parameters were monitored: correlation function behavior, the particle size and zeta potential value. At the beginning, it was necessary to study the behavior of hyaluronan in solution by dynamic light scattering measurement. Micro- and nanoparticles were prepared by mixing different volume ratios of negatively charged hyaluronan and positively charged polyarginine or cetyltrimethylammonium bromide. Micro- and nanoparticles were prepared in aqueous solution as well as in 0,15 M sodium chloride solution (physiological solution). In the case of the hyaluronan solution a polydisperse character of hyaluronan was detected. It was found that the dissolution of hyaluronan in the physiological solution gives us the smaller particle size in opposite to particle size obtained from the same concentrations of hyaluronan dissolved in water. Furthermore, it was found that systems composed of hyaluronan and polyarginine create particle size of about 100 nm. Whereas systems consisting of cetyltrimethylaminoum bromide and hyaluronan form larger particles, in units of hundreds of nanometers, the particle size in physiological solution were smaller than the same systems dissolved in aqueous solution.
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Preparation and characterization of complex liposomal for drug delivery systems
Szabová, Jana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the preparation and characterization of stealth liposomes and their combination with trimethylchitosan (TMC). This complex could find application in the field of inhalation administration. Stealth liposomes were prepared from neutral phophatidylcholine, negatively charged fosfatidic acid and polyethyleneglycol bounded to phosphatidylethanolamine. We have managed to prepare stealth liposomes with suitable properties that should guarantee passive targeting without evocation an immune response, despite the content of the negative component. We also found a suitable method of preparation for stealth liposome–TMC complex, where the change of size and zeta potential confirmed the non–covalent bound between two components despite the content of the polyethyleneglycol.
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Use of some encapsulation techniques to controlled release of active substances in food and cosmetics products.
Skoumalová, Petra ; Rittich, Bohuslav (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The presented doctoral thesis is focused on preparation, characterization and application of organic micro- and nanoparticles as transport systems for active components and some their complex natural sources. Active component were packed into liposomes and polysaccharide particles. As active components were used caffeine, some drugs – clotrimazole and ibuprofen, further antioxidants and vitamins. Antimicrobial herbs and spices extract, antimicrobial peptides lysozyme, nisin and other antimicrobial ingredients were encapsulated too. Encapsulation of selected hydrolytic enzymes was tested, too. Particles were also used for encapsulation of probiotic strains Bifidobacterium breve and Lactobacillus acidophilus and prebiotic components. These prebiotics were co-encapsulated into capsules with probiotic cells. Natural extracts were encapsulated e.g. extracts of guarana, ginseng, goji, green barley, propolis, black, green and white tea, coffee, fruit and vegetable extracts. The efficiency of encapsulation was determined by HPLC/PDA and by spectrophotometry. Long-term stability of particles and amount of released component in model/real foods, in model cosmetic conditions and in a model physiological environment were monitored too. Size of prepared liposomes and polysaccharide particles was determined by dynamic light scattering and by light microscopy and electron microscopy, respectively. Stability of the particles was measured using a zeta potential. Also, analytical centrifugation was used to measurement of sedimentation velocity and stability of the prepared particles. The antimicrobial activity were tested using two Gram-positive (Bacillus subtilis, Micrococcus luteus), two Gram-negative (Escherichia coli, Serratia marcescens) bacteria and one fungal strains (Candida glabrata). For determining the antimicrobial properties of active component and prepared particles two the most widely used methods were used - agar diffusion method and broth dilution method. The viability of probiotic strains were performed using flow cytometry and fluorescence microscopy. Encapsulation of active component was successful in all types of particles. Liposome showed a very good long-term stability mainly in water conditions with neutral pH and polysaccharide particles were stable in acidic conditions. Prepared particles showed a very good stability in model stomach environment, while in model intestines environments particles were disintegrated and active component were released. Prepared particles with encapsulated caffeine as well as other tested antioxidants and vitamins could be used to modern types of energy drinks, food supplements and also for some cosmetics applications. Encapsulated antimicrobial components could be used for food application as well as for cosmetics and pharmaceutical application like antimicrobial wound formulation. Encapsulated enzymes can be used for controlled release of proteases in wound healing, as delivery systems in digestive tract and as a part of pharmaceutical preparative and food supplements for enzyme therapy. The study revealed that encapsulation of probiotics and also co-encapsulation of probiotics with prebiotics exhibited longer stability of particles and survival bacterial cells. So, prepared particles are suitable for use to food product with beneficial effects on the human body.
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Stability of vesicular complexes against changes in their concentration
Velichová, Veronika ; Marková, Kateřina (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on the stability of vesicular systems that consist of ion pair amphiphile. These catanionic vesicles were prepared from single-chained surfactants CTAB cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate), which were stabilized by adding double-chained surfactant DODAC (dimethyldioctadecylammonium chloride) with 43 mol.% cholesterol. The aim of the thesis was to verify the stability of vesicular systems against changes in their concentration. For the measurement, a concentration series was prepared by diluting vesicles with deionized water. The stability was evaluated by dynamic (DLS) and electrophoretic (ELS) light scattering measurements at weekly intervals for four weeks. By DLS method were determined the average values of the size of vesicles and their changes during the measurement, the method ELS provided the information about changes of charge with different concentration in time. Samples with a higher concentration, i.e. no dilution till tenfold dilution of the vesicle solution, were marked as stable, higher values of average sizes and lower values of zeta potential were measured at solution with lower concentrations, according to which such diluted vesicles can be described as unstable. There were also visual observations made, but no visible aggregation in the solutions was observed. Furthermore, the aggregation behavior of the vesicles was observed by fluorescence spectroscopy using pyrene as fluorescent probe.
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