This master thesis is focused on characterization of some energy drinks and formulation of their modified composition. The theoretical part was focused on summary of energy drinks and some of their natural components - guarana, goji as well as on techniques of encapsulation. In the experimental part concentrations of caffeine, vitamin C, polyphenols and carbohydrates in commercially purchased drinks was evaluated. Subsequently, three kinds of particles (particles with caffeine, particles with guarana and particles with guarana and goji) by 6 different techniques were prepared. The efficiency of encapsulation was determined by HPLC/UV-VIS (caffeine, vitamin C) and by spectrophotometry (phenolics). Stability of the particles was followed in real food and in a model physiological environment of artificial digestive juices. Size of prepared particles was determined by dynamic light scattering. In this work it was found that particles with best activity and stability were obtained by encapsulator. For manual particles the best results were found in the liposome particles. Liposomes exhibited the smallest particle size, the highest efficiency encapsulation, excellent stability in real food and juices and the best value of zeta potential.

This presented diploma thesis is focuses on preparation and characterization of biomaterials with natural UV filters. The theoretical part is aimed at effects of sunlight on the skin and how to avoid these effects by using cosmetic products. The materials and methods for extraction of lipid part of the selected vegetable and microorganism species and methods for preparation and characterization of nanoparticles and emulsions were characterized. The practical part deals with extraction of lipid part of selected vegetable and microorganism species. The liposome particles with encapsulated active compounds were prepared. The particle size, polydispersity index, colloidal stability, encapsulation efficiency, SPF, antioxidant capacity, phospholipid content and cytotoxic effect on human keratinocytes were observed. These particles were used for preparation of the resulting emulsions. Their protection level on UV light were set and stability were tested by using analytical centrifugation.

The presented diploma thesis is focused on the preparation of new materials with antimicrobial effect. Liposomes and nanofibers from polyhydroxybutyrate containing clotrimazole and natural extracts with good antifungal and antioxidant effects were prepared. The theoretical part contains examples and short description of using nanoparticles and nanofibers in cosmetics and medicine and the description of plants which have positive and potential antimycotic effects. Moreover, methods for particles and fibers characterisation were shortly described. In the experimental part, natural water and lipid extracts were prepared and spectrophotometrically characterised for the content of polyphenols, flavonoids and the antioxidant activity. Liposomes and liposomes containtng PHB were prepared from selected extracts and the encapsulation effectivity, shortterm and longterm stability via determination of polyphenols were determined. Prepared particles were characterized with DLS method (size) and zeta- potential (stability). PHB nanofibers functionalised with selected lipid extracts and clotrimazole were prepared via electrospinning and forcespinning, and examined via FLIM and FTIR-ATR methods and spectrophotometry was used for antioxidant activity and release of active substances determination. Antifungal properties of prepared particles, extracts and fibers using the test system Candida glabrata were studied. Finally, cytotoxicity of selected samples was tested with MTT assay using human keratinocytes.

Oxidized phospholipids (OxPLs) are known to be present in living organisms due to oxidative stress. However, the physiological function of OxPLs is still not fully understood. They have been shown to be present in many inflammatory diseases such as atherosclerosis and neurodegenerative diseases like Parkinson's and Alzheimer's disease. In this work we present the influence of two truncated OxPLs on the lateral heterogeneity of a model lipid membrane. Specifically, we studied the effect of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) on the formation of nanodomains present in giant unilamellar vesicles containing 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol and sphingomyelin. Only few techniques are capable of detecting nanometer-sized domains in the membrane with high resolution. Time resolved Förster resonance energy transfer (TR-FRET) combined with Monte Carlo (MC) simulations provide a strong tool not only to detect lateral heterogeneities but also characterize them with the resolution of 2 nm. Profound effects on the nanodomain size were observed in the presence of both studied OxPLs and differences were detected, as PGPC with a carboxylic group drives formation of larger nanodomains than POVPC...

Unfolded protein response (UPR) is a complex cellular mechanism induced upon ER stress caused by various environmental factors. Single spanning signal transducers of UPR were reported to recognise also lipid-induced ER stress. Studies of these transducers, namely PERK and IRE1 uncovered that they can sense change in membrane properties and activate themselves by clustering. Moreover, signal transducer IRE1 retained ability to sense changes in the membrane properties with TMD exchanged for a polyLeu α-helix. It was thus unclear what mechanism drives lipid-induced UPR via IRE1. We employed model membrane system in form of LUVs, where properties of membranes can be readily altered by specific lipid composition. As a simplified model of the UPR signal transducers in the ER, synthetic transmembrane peptides with polyLeu core were used. Dynamic light scattering (DLS) has been used for qualitative and semi-quantitative analysis of LUVs. Clustering of synthetic peptides was determined by time resolved anisotropy of fluorescence. DLS results demonstrate successful formation of vesicles with a desired size in all planned composition. On the contrary to the studies in living cells, the presence of cholesterol or palmitic acid in model membranes did not induce the aggregation of transmembrane peptides....

Background: Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic property limits its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor. Methods: Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation. Results: The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 and 26nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner. Conclusion: The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors.

The purpose of this study was to develop antioxidant liposomes as an antidote for mustard gas-induced lung injury in a guinea pig model. Five liposomes (LIP-1, LIP-2, LIP-3, LIP-4, and LIP-5) were tested with differing levels of phospholipid, cholesterol, phosphatidic acid, tocopherol (α, γ, δ), N-acetylcysteine (NAC), and glutathione (GSH). A single dose (200 μL) of liposome was administered intratracheally 5 min or 1 h after exposure to 2-chloroethyl ethyl sulfide (CEES). The animals were sacrificed either 2 h after exposure (for lung injury study) or 30 days after exposure (for histology study). The liposomes offered 9%-76% protection against lung injury. The maximum protection was with LIP-2 (71.5% protection) and LIP-4 (75.4%) when administered 5 min after CEES exposure. Delaying the liposome administration 1 h after CEES exposure decreased the efficacy. Both liposomes contained 11 mM α-tocopherol, 11 mM γ-tocopherol, and 75 mM NAC. However, LIP-2 contained additionally 5mM δ-tocopherol. Overall, LIP-2 and LIP-4 offered significant protection by controlling the recruitment of neutrophils, eosinophils, and the accumulation of septal and perivascular fibrin and collagen. However, LIP-2 showed better protection than LIP-4 against the accumulation of red blood cells in the bronchi, alveolar space, arterioles and veins, and fibrin and collagen deposition in the alveolar space. The antifibrotic effect of the liposomes, particularly LIP-2, was further evident by a decreased level of lipid peroxidation and hydroxyproline in the lung. Thus, antioxidant liposomes containing both NAC and vitamin E are an effective antidote against CEES-induced lung injury.

This review will focus on the therapeutic uses of antioxidant liposomes. Antioxidant liposomes have a unique ability to deliver both lipid- and water-soluble antioxidants to tissues. This review will detail the varieties of antioxidants which have been incorporated into liposomes, their modes of administration, and the clinical conditions in which antioxidant liposomes could play an important therapeutic role. Antioxidant liposomes should be particularly useful for treating diseases or conditions in which oxidative stress plays a significant pathophysiological role because this technology has been shown to suppress oxidative stress. These diseases and conditions include cancer, trauma, irradiation, retinotherapy or prematurity, respiratory distress syndrome, chemical weapon exposure, and pulmonary infections.

This study developed and characterized phospholipid vesicles, or liposomes, that mimic cell surfaces. Microemulsified liposomes contained biotinylated phosphatidylethanolamine, allowing them to be immobilized to avidin-coated glass. Laminin (LN), glycosphingolipids (GMl and GM3), and Escherichia coli's mechanosensitive channel of large conductance (EcoMscL) were embedded into liposome membranes. It was determined whether these embedded molecules exhibited their physiological roles of adhesion, cell recognition, and mechanosensation, respectively. Confocal laser scanning microscopy (CLSM) was employed to examine the interaction of fluorescently probed proteins, toxins, and bacteria with the immobilized microemulsified liposomes. Capture of individual and simultaneous multiple species of bacteria by GMl, GM3, or LN liposomes was quantified using ELISA and PCR. Surface-bound liposomes were unilamellar and immovable, allowing removal of unincorporated probes and biomolecules. Liposomes remained intact and stable against leakage of encapsulated sulforhodamine B for several months after immobilization. Functional reconstitution of EcoMscL was examined using CLSM during modulations in the immursing solution. Cholera toxin(Β subunit) (CTB), bovine lactoferrin (BLF), and E. coli O157:H7 were co-localized proximate to the surface of GMl liposomes. ELISAs determined E. coli O157:H7 and Salmonella enteritidis were captured on GMl liposomes containing GMI at 8.9 molar percent of total lipid. Listeria monocytogenes and Listeria innocua were not captured on the same liposomes. PCR identified the capture of specific bacterial species from individual species and mixtures of several species on liposomes. Simultaneous assays with mixtures of multiple species showed that the receptor-associated binding of bacteria, described with PCR assays of an individual species, were independent of competitive microorganisms. L. monocytogenes and L. innocua were more frequently bound to LN liposomes than other liposomes, indicating LN promotes adhesion of both the pathogenic and a nonpathogenic strain of Listeria. E. coli O157:H7 was more frequently captured on GMI liposomes than other liposomes, indicating a specificity for this bacteria. S. enteritidis bound to all liposomes, indicating a non-specific interaction. Known eukaryotic biomolecules implicated in cell recognition, adhesion, and mechanosensation were embedded in a system of artificial bilayers immobilized on a solid support. Liposomes constitute a biomimetic capable of specifically interacting and capturing proteins, toxins, and bacteria in solution.

The purpose of this study is the characterization of the membrane-water interfaces of both sarcoplasmic reticulum membrane (SR) and charged lipid bilayers under varied properties of the surrounding aqueous solution. In this work we studied the electrokinetic properties of liposomes and SR vesicles as well as the interaction of lipophilic ions with these membranes. The study of electrokinetic properties is based on the measurements of electrophoretic mobility of SR membrane vesicles and PC/PG liposomes. Electrophoretic mobility of SR vesicles was measured as a function of ionic strength for six pH values (pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0). Electrophoretic mobility of single-layered and multi-layered PC/PG liposomes was measured at neutral pH as a function of ionic strength. For interpretation of electrophoretic mobility studies, SR vesicles (at pH 4, 7, and 9) and multi-layered and single-layered liposome sizes were determined using photoelectron microscopy. The study of the interaction of lipophilic ions with these membranes is based on (1) measurements of their partition coefficients described in terms of an ion partition model based on the Langmuir adsorption model and (2) electrophoretic mobility measurements of SR vesicles and PC liposomes in suspension with varied concentration of lipophilic ions. SR-water and PC-water partition coefficients were measured as a function of concentration for two anions tetraphenylborate (TePB-) and pentabromophenol (PBP-) and two cations (Imipramine+, and Clomipramine+). The anions belong to a class of pesticides and the cations are drugs once prescribed as anti-depressants. Partition into the SR membrane was shown to be significantly greater for all lipophilic ions except TePB-, which only showed this effect at the higher lipophilic ion range of the data. The PC-water partition coefficient was also measured for TePP+. Since the lipid bilayer of SR is not significantly different than that of PC liposomes, we believe the differences in partition are due to excess lipophilic ions being absorbed to the proteins of SR. The electrokinetics of charged PCPG liposomes, and PC liposomes with absorbed lipophilic ions could be understood in terms of the charge being located below their surface and screened by counter-ions inside the polar head-group region. We call this model the "permeable surface model". The assumptions of this model are that (1) the charge exists on a plane at a depth, d, below the surface of the liposome within the lipid head-group region and (2) small ions (Na+, K+, Cl-) are able to penetrate the lipid head-group region with a molar membrane-water partition coefficient of 0.4. Using this model we were able to obtain the depth of sorption of lipophilic ions in PC liposomes. We found values of 0.13 nm for TePB-, 0.5 nm for PBP-, 0.12 nm for Imipramine+, 0.17 nm for Clomipramine and 0.25 nm for TePP+. The depth of lipophilic ions in PC is a valuable quantity for the study of the effect of lipophilic ions on membrane function. For PCPG mobility we found the charged plane due to PG lipids was 0.2 nm for single-layered liposomes and 0.1 nm for multi-layered liposomes. This is consistent with the relative size of PC and PG head groups The dependence of SR mobility on pH was found to be directly correlated with the total charge of the A, P, and N domains of the Ca2+-ATPase as determined by the amino acid residues and their corresponding pKa values in water. We found that detached charged plane model, a new model developed in our group, could be fit to the mobility of SR as a function of ionic strength while other soft particle models failed. The assumptions of this model are that (1) the friction caused by protruding proteins on the surface of SR can be represented by a homogeneous retardation layer of thickness D and softness parameter λRL, and (2) the charge of the APN domain can be represented as a plane of charge embedded in the retardation layer at a distance s from the membrane surface. The best-fit values for λRL, and s were not consistent for different pH value studies. The detached charged plane model was unable to predict the mobility of SR vesicles in the presence of lipophilic ions if we assumed that the lipophilic ions were sorbing to the detached charged plane that represents the native charge of the APN domains of SR. At high lipophilic ion concentration the experimental mobilities consistently were greater in magnitude than the values predicted by the model. We concluded that there is significant absorption of lipophilic ions to the proteins in SR membrane, and that the lipophilic ion sorption sites are not the same as the detached plane of charge that represents the native charge of the APN domain.