Development of lipodisks as carriers for cationic amphiphilic peptides

Antibiotics have made a tremendous contribution to mankind. They are one of the most successful medicines in human history. However, more and more bacterial strains develop resistance and the risk to public health can hardly be overstated. New types of antibiotics are urgently needed. Antimicrobial peptides (AMPs) have emerged as potential antibiotics because of their broad-spectrum activities and non-conventional mechanism of action. More recently, they have also received attention as promising anticancer agents. The clinical and commercial development of AMPs as a new generation of antibiotics and anticancer drugs is hampered, however, by issues concerning the toxicity, specificity and stability of the peptides. The aim of this thesis has been to explore if formulation in a novel type of nanocarriers, referred to as lipodisks, can be used to increase the therapeutic potential of AMPs as antimicrobial and anticancer agents. Focus has been on AMPs classified as cationic amphiphilic peptides. Encouragingly, the data presented suggests that the therapeutic potential of the AMP melittin as an antimicrobial and anticancer agent can be substantially increased by formulation in lipodisks. When formulated in the lipodisk, melittin is protected against enzymatic degradation. The lipodisk also offer a slow-release effect that sustains the bacterial cell-killing effect. We also show that specific delivery of melittin to tumour cells can be obtained by formulating the peptide in small EGF-targeting lipodisks. Melittin contains a tryptophan residue and its interaction with lipodisks can be characterized by means of fluorimetric binding assays. In order to investigate the binding behavior also for peptides that lack intrinsic fluorescence, we developed a method based on measurements using the QCM-D technique. Studies using this, and other techniques, confirmed that it is a general behavior for cationic amphiphilic peptides to preferentially bind to the highly curved rim of lipodisks. Results of our binding studies show that the peptide to lipid ratio in the lipodisks can be tuned and optimized by varying the size and charge of the disks. Taken together, the findings in this thesis point towards PEG-stabilized lipodisks as promising nanocarriers for antibacterial and anticancer peptides

Label-Free Characterization of Peptide-Lipid Interactions Using Immobilized Lipodisks

Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide–membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications

Health literacy of inhabitants of Riga

Šī darba tēma ir “Rīgas iedzīvotāju veselībpratība”. Darba mērķis ir noskaidrot Rīgas iedzīvotāju veselībpratības līmeni un tā saistību ar izglītības līmeni, vērtējumu par savu veselības stāvokli un profilaktisku rīcību veikšanu. Lai sasniegtu darba mērķi, darbā tiek izmantota veselībpratības teorija. Balstoties uz iepriekš veiktajiem pētījumiem, tiek izvirzītas hipotēzes un tiek veikts empīriskais pētījums – interneta aptauja. Lai noskaidrotu Rīgas iedzīvotāju veselībpratības līmeni, aptaujas anketā iekļauti Eiropas veselībpratības pētījuma saīsinātā 16 jautājumu skala, ko izveidojis Vīnes universitātes Socioloģijas institūta profesors, kā arī Austrijas sabiedrības veselības institūta direktors Jurgens M. Pelikans. Pirmajā nodaļā tiek raksturoti veselības aprūpes sistēmas rādītāji un raksturojums, otrajā nodaļā iedzīvotāju veselību ietekmējošie paradumi, bet trešajā – iedzīvotāju veselības rādītāji un pašvērtējums. Tam seko ceturtā nodaļa ar veselībpratības jēdziena skaidrojumu, veselībpratības interpretāciju dažādu autoru darbos un ietekmējošo faktoru raksturojumu. Tam seko piektā nodaļa ar iepriekš veikto pētījumu aprakstu. Sestajā nodaļā aprakstīta empīriskā pētījuma metodoloģija, kam seko septītā nodaļa ar datu analīzi un astotā nodaļa ar diskusiju. Nobeigumā tiek izdarīti secinājumi. Pētījumā secināts, ka galvenais faktors, kas ietekmē veselībpratību, ir izglītība. Otrs nozīmīgākais faktors ir vecums. Hipotēzes par to, ka Rīgas iedzīvotājiem ar zemāku veselībpratības līmeni ir sliktāks vērtējums par savu veselības stāvokli un Rīgas iedzīvotāji ar augstāku veselībpratības līmeni biežāk veic profilaktiskas rīcības, daļēji apstiprinās.The subject of this Bachelor thesis is “Health Literacy of Inhabitants of Riga”. The objective of this study is to clarify health literacy of inhabitants of Riga and its relation with the level of education, self-reported health condition and preventive actions. In order to achieve the aim of the paper the theory of health literacy is used. Based on previous studies, hypotheses are formed and an empirical study is being conducted – an online survey. In order to clarify the level of health literacy of inhabitants of Riga, the questionnaire includes the shortened 16-question scale of the European Health Literacy Survey. The author of this scale is Professor of Sociology at University of Vienna, and also director of the Austrian Institute for Public Health, Jurgen M. Pelikan. The work consists of eight chapters. The first chapter describes the characteristics of the health system, the second chapter describes the health-affecting habits of the population, and the third chapter describes the health indicators of inhabitants. Then follows the fourth chapter with an explanation of the concept of health literacy, its authors and a description of the factors affecting it. The fifth chapter consists of a description of the studies previously done. In the sixth chapter the methodology of the study is described. There are data analysis in the seventh chapter and results of the study and discussion in the eighth chapter. After that, conclusions are drawn. The study concludes that education is the main factor affecting health literacy. The second major factor is age. The hypothesis about the presumption that inhabitants of Riga with a lower level of health literacy have worse self-reported health condition, and inhabitants of Riga with a higher level of health condition are more likely to take preventive actions, is partially confirmed

Label-Free Characterization of Peptide–Lipid Interactions Using Immobilized Lipodisks

Lipodisks, planar lipid bilayer structures stabilized by PEG-ylated lipids, were in the present study covalently bound and immobilized onto sensors for quartz crystal microbalance with dissipation monitoring (QCM-D) studies. It is shown that the modified sensors can be used to characterize the interaction of lipodisks with α-helical amphiphilic peptides with an accuracy similar to that obtained with well established fluorimetric approximations. The method presented has the great advantage that it can be used with peptides in their native form even if no fluorescent residues are present. The potential of the method is illustrated by determining the parameters describing the association of melittin, mastoparan X, and mastoparan with immobilized lipodisks. Both thermodynamic and kinetic analyses are possible. The presented method constitutes a useful tool for fundamental studies of peptide–membrane interactions and can also be applied to optimize the design of lipodisks, for example, for sustained release of antimicrobial peptides in therapeutic applications

PEG-stabilized lipid disks as carriers for amphiphilic antimicrobial peptides

Antimicrobial peptides hold potential as a possible alternative, or complement, to conventional antibiotics but new, safe and efficient means are needed for formulation and administration of the peptides. In this study we have investigated the utility of a novel type of lipid particles, the polyethylene glycol-stabilized lipid-disks, as carriers for the model peptide melittin. The structural integrity of the carrier particle when loaded with the peptide was investigated using cryo-transmission electron microscopy. Liposome leakage upon addition of the peptide-lipid-disks was monitored as a means to verify the membrane lytic effect of the formulation. The susceptibility of melittin to tryptic digestion was studied and compared in the absence and presence of lipid-disks. Finally, the antibacterial effect of the peptide-lipid-disk formulation was compared to that of free melittin after both single and repeated exposure to Escherichia coli. The results show that melittin can redistribute from the disk into a new host membrane and that formulation in the disks does not compromise melittin's membrane permeabilizing ability. Further, the peptide was found to be fully protected against degradation when bound to the disks. Time-kill experiments revealed that all the antibacterial effect of melittin administered in free form was gone after a single exposure to E. coli. In contrast, the disk formulation showed significant cell-killing effect also upon a second exposure to bacteria, indicating an extended release of peptide from the lipid-disks. These results suggest that the lipid-disks constitute a new class of promising carriers for peptide antibiotics