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Analysis of coronavirus by QRT-PCR and possible therapy by nanoliposomal carriers of recombinant antigens
Krchová, Lenka ; doc. RNDr. Milan Bartoš. Ph.D. (referee) ; Turánek, Jaroslav (advisor)
This thesis deals with the study of coronaviruses, their diagnostics and subsequently the possible construction of a recombinant vaccine based on liposomal carriers of recombinant antigens. The experimental part of the thesis touches upon two levels. First, the diagnosis of SARS-CoV-2 virus was measured and investigated by real-time PCR. Biological material of a large number of patients was worked with. Together with the basic determination of the presence of the virus, the stability of the viral particle over time, its resistance to temperature changes, the effect of dilution of the carrier medium on the Ct value and other sub-experiments were investigated. From all the metadata, the most useful variant for pathogen diagnosis is finally evaluated. The experimental part is then followed by a possible therapy with recombinant vaccines. This issue is very topical and moves the whole professional community. Liposomes have been prepared which serve as carriers of bioactive substances in recombinant vaccines. The liposomes were homogenized by extrusion to the desired size and variously surface modified. Antigen binding, stability over time and possible degradation were investigated. Liposomes were characterized by DLS, nanoflow cytometry and electron microscopy.
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Solid form of liposomal systems for inhalation
Fardová, Lucie ; Kalina, Michal (referee) ; Mravec, Filip (advisor)
This thesis deals with the preparation of solid liposomal systems for inhalation delivery. Liposomal systems were prepared by rehydration of a phospholipid film from dipalmitoylphosphatidylcholine. A sucrose solution used for film rehydration served as a protectant during spray drying. Additional liposome samples contained cholesterol, phosphatidic acid, or polyethylene glycol for better stabilization. The prepared liposome solutions were examined using scanning transmission electron microscopy, dynamic light scattering, and electrophoretic light scattering methods. Dried powders were measured using infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, and determination of particle aerodynamic size. The results demonstrate the successful preparation of all liposomal systems, their drying into powder form, and subsequent reconstruction after rehydration with water or buffer. Based on the observed properties, liposomes with polyethylene glycol appeared to be the most suitable.
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Analysis of coronavirus by QRT-PCR and possible therapy by nanoliposomal carriers of recombinant antigens
Krchová, Lenka ; doc. RNDr. Milan Bartoš. Ph.D. (referee) ; Turánek, Jaroslav (advisor)
This thesis deals with the study of coronaviruses, their diagnostics and subsequently the possible construction of a recombinant vaccine based on liposomal carriers of recombinant antigens. The experimental part of the thesis touches upon two levels. First, the diagnosis of SARS-CoV-2 virus was measured and investigated by real-time PCR. Biological material of a large number of patients was worked with. Together with the basic determination of the presence of the virus, the stability of the viral particle over time, its resistance to temperature changes, the effect of dilution of the carrier medium on the Ct value and other sub-experiments were investigated. From all the metadata, the most useful variant for pathogen diagnosis is finally evaluated. The experimental part is then followed by a possible therapy with recombinant vaccines. This issue is very topical and moves the whole professional community. Liposomes have been prepared which serve as carriers of bioactive substances in recombinant vaccines. The liposomes were homogenized by extrusion to the desired size and variously surface modified. Antigen binding, stability over time and possible degradation were investigated. Liposomes were characterized by DLS, nanoflow cytometry and electron microscopy.
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The Preparation and the Characterization of the Cationic Liposomes Carrying New Immunoadjuvant.
Houšť, Jiří ; Nesměrák, Karel (advisor) ; Kozlík, Petr (referee)
The aim of this diploma thesis was preparation, characterization and determination of encapsulation efficiency of the cationic liposomes composed of dimethyldioctadecylammonium bromide (DDAB) and cholesterol carrying new drug MT05 with an immunoadjuvant effect. The influence of the temperature of sonication bath and the influence of the volume of liposomal suspension on the average size of liposomes and their polydispersity index was monitored. The most effective liposome preparation by sonication bath was at temperature of 60 řC. The volume of liposomes undergoing sonication did not influence the resulting values of the average size of liposomes and their polydispersity index. The time of sonication time was 6 hours and could be shortened by using sonication bath with higher output. The determination of encapsulation efficiency was carried out in three separated experiments by HPLC-MS/MS. The encapsulation efficiency of the cationic liposomes was 30.1 ± 8.5 % in the first experiment, 43 ± 25 % in the second, and 32 ± 25 % in the third. The amount of DDAB was determined only in the liposomes prepared in the third experiment. The amount of DDAB in the purified liposomes was 78.9 ± 3.7 % in the first replicate, 65.4 ± 1.8 % in the second and 53.8 ± 1.4 % in the third. The actual molar ratio of MT05...
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Artificial phospholipid membranes - method of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
Heterogenita biologických membrán vedla ke vzniku širokého spektra zjednodušených modelových systémů, jejichž uspořádání, velikost a tvar se dají přizpůsobovat různým aktuálním požadavkům. Existují dva rozdílné přístupy k vytváření umělých fosfolipidových membrán. První z nich je založen na vzniku membrán ve vodném prostředí. Do této skupiny patří černé lipidické membrány, dvojvrstvy na pevném podkladu, dvojvrstvy vzniklé z monovrstev na rozhraní vody a vzduchu a liposomy. Ve druhém případě vznikají dvojvrstevné membrány v množství organické fáze metodou dvojvrstev na kapičkovém rozhraní. Každý typ umělých membrán má své experimentální výhody a nevýhody, což se používá ke studiu různých problémů sahajících od chování jednotlivých fosfolipidů a proteinů až po fúze membrán. Umělé fosfolipidové membrány jsou vhodným nástrojem pro elektrickou charakterizaci dvojvrstev a nebo membránových proteinů. Tato práce je ucelený přehled nejpoužívanějších metod vhodných pro vznik umělých fosfolipidových membrán. Klíčová slova: membrána, lipid, fosfolipidová dvojvrstva, liposom, černá lipidová membrána, dvojvrstvy na pevném podkladu, dvojvrstva na kapičkovém rozhraní Abstract The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and...
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The Preparation and the Characterization of the Cationic Liposomes Carrying New Immunoadjuvant.
Houšť, Jiří ; Nesměrák, Karel (advisor) ; Kozlík, Petr (referee)
The aim of this diploma thesis was preparation, characterization and determination of encapsulation efficiency of the cationic liposomes composed of dimethyldioctadecylammonium bromide (DDAB) and cholesterol carrying new drug MT05 with an immunoadjuvant effect. The influence of the temperature of sonication bath and the influence of the volume of liposomal suspension on the average size of liposomes and their polydispersity index was monitored. The most effective liposome preparation by sonication bath was at temperature of 60 řC. The volume of liposomes undergoing sonication did not influence the resulting values of the average size of liposomes and their polydispersity index. The time of sonication time was 6 hours and could be shortened by using sonication bath with higher output. The determination of encapsulation efficiency was carried out in three separated experiments by HPLC-MS/MS. The encapsulation efficiency of the cationic liposomes was 30.1 ± 8.5 % in the first experiment, 43 ± 25 % in the second, and 32 ± 25 % in the third. The amount of DDAB was determined only in the liposomes prepared in the third experiment. The amount of DDAB in the purified liposomes was 78.9 ± 3.7 % in the first replicate, 65.4 ± 1.8 % in the second and 53.8 ± 1.4 % in the third. The actual molar ratio of MT05...
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Artificial phospholipid membranes - methods of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and shape can be adapted to the requirements. There are two different approaches of making artificial phospholipid bilayers. One of them is based on creating bilayers in aqueous phase. This includes Black lipid membranes, Supported phospholipid bilayers, bilayers from water/air interface and liposomes. In the second approach bilayers are created in a bulk of organic phase by Droplet interface bilayer method. Each type of artificial bilayer has its experimental advantages that have been used to study many problems ranging from behaviour of single phospholipids and proteins to membrane fusion. Artificial lipid membranes are perfect tool for electrical characterisation of bilayers and embedded membrane proteins. This work is a complete review of most useful techniques of model membrane preparation. Key words: membrane, lipid, phospholipid bilayer, liposome, black lipid membrane, supported lipid bilayer, droplet interface bilayer
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Interakce liposomů s porfyriny studována pomocí Ramanovy spektroskopie kapkově nanášených povlaků
Vaculčiaková, Lenka ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
Water-soluble cationic porphyrins have been studied in relation to antisense therapy and they have been successfully used to enhance the delivery of oligonu- cleotides to cells. The main focus of this study was to characterize the mech- anism of interaction of liposomes as model membranes, and porphyrins. We applied the drop coating deposition Raman spectroscopy (DCDR) to study com- plexes of liposomes and porphyrins. DCDR allowed us to measure complexes of low concentration as it uses the 'coffee ring' effect to concentrate the sample at the edge of a drop. We studied four different complexes combined of lipids: 1, 2−dipalmitoyl−sn−glycero−3−phosphocholine, 1, 2−dioleoyl−sn−glycero− 3 − phospho − (1 − rac − glycerol) and metalloporphyrins: copper 5, 10, 15, 20 − tetrakis(1 − methyl − 4 − pyridyl)porphyrin, copper 5, 10, 15, 20 − tetrakis(4 − sulfonatophenyl)porphyrin. We have found that the way these two components interact strongly depends on a specific lipid and porphyrin used. We observed partial incorporation of porphyrins into the liposome bilayer, their localisation to the surface of the liposome or the change of the conformation and ordering of lipid molecules. Moreover, we have found that the distribution of porphyrins in the dried drop is randomly non-homogenous. 61
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Artificial phospholipid membranes - method of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
Heterogenita biologických membrán vedla ke vzniku širokého spektra zjednodušených modelových systémů, jejichž uspořádání, velikost a tvar se dají přizpůsobovat různým aktuálním požadavkům. Existují dva rozdílné přístupy k vytváření umělých fosfolipidových membrán. První z nich je založen na vzniku membrán ve vodném prostředí. Do této skupiny patří černé lipidické membrány, dvojvrstvy na pevném podkladu, dvojvrstvy vzniklé z monovrstev na rozhraní vody a vzduchu a liposomy. Ve druhém případě vznikají dvojvrstevné membrány v množství organické fáze metodou dvojvrstev na kapičkovém rozhraní. Každý typ umělých membrán má své experimentální výhody a nevýhody, což se používá ke studiu různých problémů sahajících od chování jednotlivých fosfolipidů a proteinů až po fúze membrán. Umělé fosfolipidové membrány jsou vhodným nástrojem pro elektrickou charakterizaci dvojvrstev a nebo membránových proteinů. Tato práce je ucelený přehled nejpoužívanějších metod vhodných pro vznik umělých fosfolipidových membrán. Klíčová slova: membrána, lipid, fosfolipidová dvojvrstva, liposom, černá lipidová membrána, dvojvrstvy na pevném podkladu, dvojvrstva na kapičkovém rozhraní Abstract The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and...
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Study of liposomes by using of drop coating deposition Raman spectroscopy
Vodáková, Adéla ; Kočišová, Eva (advisor) ; Bednárová, Lucie (referee)
in english: Goal of the Bachelor Thesis was an optimization of liposome membrane preparation with different composition for the drop coating deposition Raman (DCDR) method, measuring of the DCDR spectra from dried droplets and their interpretation. The DCDR method lies in deposition of small volume of suspension (~μl) on special surface and measuring of Raman spectra from the ring formed at the edge of the dried droplet. The main advantage of used method is the possibility of measuring spectra from the sample with low concentration. Our results show that the DCDR method is very useful in studies of biological membranes with different composition. Two synthetic lipids and one natural extract were chosen for making a comparison. We have found out that spectra obtained from the ring of dried droplet are not different from the spectra measured in the suspension. The important characteristic of spectra is that dried droplet keeps the same lipid phase as in suspension. Spectral mapping proved good reproducibility of the signal inside the ring.
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