<p>This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated.</p><p>The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol.</p><p>Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (T<i>C</i>). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through T<i>C</i> of the lipid mixture.</p><p>Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.</p>

Los biotensioactivos son compuestos producidos por microorganismos que poseen actividad superficial, baja toxicidad, carácter biodegradable y efectividad a temperaturas y valores de pH extremos. Los ramnolípidos, son un tipo de biotensioactivos con interesantes actividades biológicas, que parecen estar relacionadas con su interacción con las membranas. En esta tesis doctoral se estudia la interacción de la fracción dirramnolipídica purificada de cultivos de Pseudomonas aeruginosa con membranas modelo y membranas biológicas, la interacción con albúmina de suero bovino, utilizada como proteína modelo, y por último, se evalúa el papel de este biotensioactivo en la estabilización de liposomas sensibles a pH y su aplicación en la introducción de compuestos en células animales. Para el desarrollo de este trabajo de investigación se han utilizado técnicas como calorimetría, FT-IR, difracción de rayos X, dispersión de luz dinámica y microscopía electrónica. / Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. This thesis presents a detailed molecular level study of the dirhamnolipid interactions with artificial phospholipids membranes and biological membranas, and the dirhamnolipid interactions with bovine serum albumin, used as a general globular protein model. Furthemore, this work includes the characterization of phosphatidylethanolamine/diRL pH-sensitive liposomes described as efficient systems for cytoplasmic delivery of foreign compounds into living cells. It has been employed a number of physical techniques such as calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering.

This thesis describes results from fundamental studies of liposomes intended for drug delivery and pH or temperature triggered release. In addition, the effect of lipid composition on bilayer disc formation and a potential application of the bilayer discs were investigated. The lower pH encountered by endocytosed liposomes can be utilized to trigger drug release. The mechanisms behind cytosolic drug delivery were investigated using two different kinds of pH-sensitive liposomes. The results indicate that incorporation of non-lamellar forming lipids into the endosome membrane may allow for drug escape into the cytosol. Temperature-sensitive liposomes containing lysolipid (LTSL) release their content almost instantly when heated to temperatures close to the gel to liquid crystalline phase transition temperature (TC). Morphological changes of the liposomes in response to temperature cycling were studied. Temperature cycling induced liposome openings and disintegration of the liposomes into bilayer discs. Incubation of LTSL in the presence of multilamellar liposomes (MLVs) resulted in relocalisation of lysolipid into the MLVs, which affected the rapid release from LTSL. We propose that the presence of micelle-forming components, such as lysolipids and PEG-lipids, facilitates the formation of defects and membrane openings during the initial phase of membrane melting, resulting in the observed rapid release. Similar to added lysolipids, also hydrolysis generated lysolipids induce disc-formation upon heating through TC of the lipid mixture. Two fundamentally different micelles may form in PEG-lipid/lipid mixtures. We found that discoidal structures are preferred over cylindrical micelles when the mixture contains components that reduce the spontaneous curvature, increase the monolayer bending modulus, or reduce PEG-lipid/lipid miscibility. The large discoidal micelles found at low PEG-lipid content are better described as bilayer discs. We evaluated such discs as model membranes in drug partitioning studies, and suggest that they, in some cases, produce more accurate data than liposomes.

<p>Physico-chemical characterisation of structure and stability of liposomes intended for drug delivery is the central issue in this thesis. In addition, targeted liposomes to be used in boron neutron capture therapy (BNCT) were developed.</p><p>Lysolipids and fatty acids are products formed upon hydrolysis of PC-lipids. The aggregate structure formed upon mixing lysolipids, fatty acids and EPC were characterised by means of cryo-TEM. A relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentration of the three components. </p><p>The interactions between alternative steric stabilisers (PEO-PPO-PEO copolymers) and conventional PC-and pH-sensitive PE-liposomes were investigated. Whereas the PE-liposomes could be stabilised by the PEO-PPO-PEO copolymers, the PC-liposomes showed an enhanced permeability concomitant with the PEO-PPO-PEO adsorption.</p><p>Permeability effects induced by different PEG-stabilisers on EPC liposomes were shown to be dependent on the length of the PEG chain but also on the linkage used to connect the PEG polymer with the hydrophobic membrane anchor.</p><p>An efficient drug delivery requires, in most cases, an accumulation of the drug in the cell cytoplasm. The mechanism behind cytosolic drug delivery from pH-sensitive liposomes was investigated. The results suggest that a destabilisation of the endosome membrane, due to an incorporation of non-lamellar forming lipids, may allow the drug to be released. </p><p>Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs. </p>

Physico-chemical characterisation of structure and stability of liposomes intended for drug delivery is the central issue in this thesis. In addition, targeted liposomes to be used in boron neutron capture therapy (BNCT) were developed. Lysolipids and fatty acids are products formed upon hydrolysis of PC-lipids. The aggregate structure formed upon mixing lysolipids, fatty acids and EPC were characterised by means of cryo-TEM. A relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentration of the three components. The interactions between alternative steric stabilisers (PEO-PPO-PEO copolymers) and conventional PC-and pH-sensitive PE-liposomes were investigated. Whereas the PE-liposomes could be stabilised by the PEO-PPO-PEO copolymers, the PC-liposomes showed an enhanced permeability concomitant with the PEO-PPO-PEO adsorption. Permeability effects induced by different PEG-stabilisers on EPC liposomes were shown to be dependent on the length of the PEG chain but also on the linkage used to connect the PEG polymer with the hydrophobic membrane anchor. An efficient drug delivery requires, in most cases, an accumulation of the drug in the cell cytoplasm. The mechanism behind cytosolic drug delivery from pH-sensitive liposomes was investigated. The results suggest that a destabilisation of the endosome membrane, due to an incorporation of non-lamellar forming lipids, may allow the drug to be released. Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs.

Abstract ACCURATE METHODOLOGY FOR MONITORING BIOMEMBRANE EVENTS By Christine A. Winschel, Ph.D. A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Chemistry at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Director: Dr. Vladimir A. Sidorov ASSOCIATE PROFESSOR, DEPARTMENT OF CHEMISTRY This study describes the synthesis and characterization of a new receptor (cyclen 1) capable of strong selective binding of pyrene-based anionic dyes under near-physiological conditions. This receptor comprises four naphthylthiourea groups tethered to a cyclen core via an ester linkage. The most important finding was the ability of cyclen 1 to bind efficiently to a pH-sensitive pyranine dye, a dye that is commonly used in various biomembrane assays. The high affinity of cyclen 1 to pyranine, its impermeability to the lipid bilayer membrane, fast kinetics of binding, and ability to quench pyranine’s fluorescence were used as a basis for a new membrane leakage assay. This membrane leakage assay is fully compatible with the commonly applied pH-stat transport assay, and therefore it allows for differentiation of ion transport and nonselective leakage mechanisms within a single set of experiments. In the second part of this study a new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of a newly synthesized cascade-blue-labeled lipid through complex formation with cyclen 1. This receptor-dye complexation also has high affinity for binding at micromolar concentrations and can be reversed by either competitive displacement of the lipid probe or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. This new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments, and is less invasive to the model and cell membranes than the commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Lastly, new pH-sensitive lipids were synthesized and utilized in the formulation of liposomes suitable for controlled drug release. These liposomes contain various amounts of internal NaCl and undergo internal acidification upon the exogenous addition of an HCl co-transporter in a physiologically relevant NaCl solution. Therefore, acidification ultimately leads to the hydrolysis of the pH-sensitive lipids and subsequent contents release. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be non-toxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug release model potentially suitable for in vivo applications.