Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated to such a transport has been relatively slow, even though significant progress has been made in the characterization of the intracellular trafficking of cationic lipid/DNA complexes. Surprisingly, little is known about the effects of these delivery vectors on cell functioning. In the present thesis, we show that cationic lipids and cationic lipid/DNA complexes strongly increase the intracellular Ca2+ concentration. The end point of the Ca2+ increase was ~400 nM from a basal level of ~100 nM. The [Ca2+]i increase was studied using K562 and Jurkat cells cultured in vitro. This effect is weakened following addition of DNA to cationic liposomes, although remaining very large at cationic lipid/DNA ratios commonly used for cell transfection experiments. Removal of extracellular Ca2+ did not abolish this effect significantly and preincubating K562 cells with the Ca2+-ATPase inhibitor thapsigargin strongly abolished intracellular Ca2+ concentration increase, indicating that Ca2+ was released mainly from internal Ca2+ stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122 blocked the intracellular Ca2+ concentration rise, suggesting an inositol pathway-dependent mechanism. LDH release assay indicates that in the conditions used for fluorescence measurement and in those used to transfer DNA into cells, cationic liposomes diC14-amidine and DOTAP had no massive cytotoxic effects. Cationic liposomes showed more toxicity than their corresponding complexes; this toxicity decreases in the presence of serum. The effect of cationic lipids on phosphatidylinositol-specific phospholipase C (PI-PLC) was quantitatively assessed using phosphatidylinositol (PI) and radiolabeled phosphatidylinositol ([3H]-PI). Incorporation of diC14-amidine into PC/PI vesicle activated PI-PLC and was shown to activate the hydrolysis of PI and [3H]-PI. Our data may suggest that mobilization of intracellular Ca2+ by complex could have an effect on the transfection process itself. These results indicate for the first time that cationic lipids and cationic lipid/DNA complexes are not inert and can affect the functioning of the cells by increasing their intracellular Ca2+.

The discovery that cationic lipids can independently stimulate the immune system has generated interest in their potential as vaccine adjuvants. Here, we show that the cationic lipid R-DOTAP can independently stimulate type 1 interferon production in dendritic cells in both primary culture and immortalized cell culture. Levels of type 1 interferon production are cell line-dependent and limited in vitro by lipid-induced cell death. We show that cationic lipids can independently activate TLR-7 and TLR-9, suggesting a mechanism for type 1 interferon induction. This TLR-stimulatory activity is not restricted to R-DOTAP and can be extended to other similar cationic lipids in a lipid-specific and TLR-specific manner.

A series of pyridinium cationic lipids was designed, synthesized and characterized. These lipids varied in the lipophilic part, bearing C9 to C20 saturated, unsaturated, straight and branched hydrocarbon chains. The lipid shape parameter was calculated from the molecular structure of these lipids based on the partial molar volumes of the atoms, and standard bond lengths and bond angles, using fragment additive methods. The shape parameter controls the lamellar/hexagonal phase balance in lipoplexes of the lipid with deoxyribonucleic acid (DNA). The lipid phase behaviour of the lipoplexes was derived from small-angle X-ray scattering experiments and was successfully correlated with the calculated lipid shape parameter. The synthesized pyridinium lipids were co-formulated (1:1) with 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) as the co-cationic lipid in 1:1 ratio, and the mixed cationic lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. The effect of variation in cationic lipid structure and lipoplex formulation on the transfection of nucleic acid (β-galactosidase and green fluorescent protein (GFP)) into CHO-K1 cells and the cytotoxicity of these formulations was assessed. Initial studies on the synthesized lipids bearing saturated and terminally unsaturated C16 chains showed that a Transfection Index (TIPSV) which encompasses the variation in the lipid shape parameter, the phase packing in a hexagonal lipoplex and the partition of these lipids into the lipoplex successfully correlated with transfection efficiency. To further investigate the effect of the variation of the partition of these lipids to the lipoplex, transfection studies were performed on a series of pyridinium lipids with straight saturated and unsaturated chains of varied lengths, with similar shape parameters but varied partition coefficients (clogP). The correlation of these experimental transfection data with the initial TIPSV was unsuccessful, but the data suggested that chain length as it relates to chain mixing and chain melting behaviours of pure lipids played a role in transfection. A refined transfection index (TIPSVM) was proposed which contained terms for the lipid shape parameter, the phase packing into a hexagonal lipoplex, the partition of these lipids into the lipoplex and a chain melting term. TIPSVM gave an acceptable correlation with the experimental transfection efficiency for the range of compounds. Additional experimental transfection data were obtained for compounds with widely variable lipid shape parameters, either as pure compounds, blends of two pure compounds, or statistically produced mixtures of mixed-chain compounds. Although very short-chain compounds (C9) and very lipophilic compounds (C20) performed poorly, the results from the blends allow the assessment of the role of the shape parameter in the TI. Since the shape parameter and the volume filling term are both calculated with the same molecular parameter, the experimental work demonstrated that only one of these terms is required. Thus a three parameter transfection index (TIPVM) was proposed and found to correlate with the entire set of comparable data. A Quantitative structure–activity relationship (QSAR) study was done on the cytotoxicity of the transfection formulations utilized. The toxicity of the synthesized pyridinium lipids was shown to correlate with the shape parameter, the lipid mixture partition co-efficient (clogP) and the charge ratio of the lipoplex formulation. Taken together, the developed transfection index TIPVM and the cytotoxicity correlation uncovered can be used in the design of low-toxicity, high activity pyridinium lipids for transfection of DNA. / Graduate / pariapz@uvic.ca

[French below]TLRs are the sentinels of our cells, they are located at the cell surface and alert the whole immune system of the presence of viruses or bacteria. They detect pathogens by recognizing their molecular patterns; this recognition is specific in order to avoid self-recognition, but they need some degree of promiscuity to remedy to pathogen heterogeneity or mutations. Promiscuity is generally defined as an indiscriminate association with molecules regardless their structure and is the contrary of specificity proper of the classic paradigm of key-lock receptor activation. My thesis results demonstrate that TLR4 and TLR2 are more promiscuous than what was believed and that this promiscuity leads to the recognition of cationic lipids and cardiolipins.Cationic lipids lipopolyamines are synthetic molecules nucleic acid nanocarriers proposed to be used for gene therapy, which consists in replacing a gene that is functioning improperly. This thesis demonstrates that lipopolyamines activate TLR2 by forming conserved and/or alternative H-bonds with TLR residues, simulating the recognition of bacterial lipopeptides and inducing pro-inflammatory cytokines secretion; which is deleterious when we aim to use these nanocarriers in the context of gene therapy. We propose the use of unsaturated cationic lipids to avoid TLR2 recognition. TLR activation could be useful instead to prepare one-component vaccine adjuvants, for which both antigen carrier and TLR activation are needed to turn on the immune system and produce antibodies. The second chapter of this thesis investigates the pro-inflammatory properties of other cationic lipids and describes new lipopolyamines able to activate both TLR2 and TLR4. The study of their adjuvanticity properties showed that they are as efficient as the aluminium salts in stimulating antibodies production.The promiscuity of TLR2 and TLR4 raised questions about the presence of endogenous TLR modulators, often countered by contamination concerns. In the third chapter, we investigate the pro- and anti-inflammatory properties of cardiolipin (CL). CL is a tetra-acylated diphosphatidylglycerol located in the inner mitochondrial membrane. Its fatty acid chain length and degree of unsaturation vary depending on species, tissue and pathological conditions. Here we show that unsaturated cardiolipin acts as a competitive TLR4 antagonist by occupying the binding site of LPS and that unsaturations discriminate between TLR4 antagonistic and agonistic activity. Under physiological conditions, mammalian CL chains are unsaturated, whereas there is an increase of saturated CL in patients affected by the Barth Syndrome. Hence we suggest that unsaturated CL could negatively regulate the inflammation during cell damage or death, via TLR4 inhibition. By contrast, saturated CLs may be involved in the inflammatory state associated with the disease. Finally, our study provides new understandings of the mechanism of TLR4 regulation and extends the library of TLR4 agonists and antagonists to molecules of easier synthesis, lower price and higher biocompatibility compared to LPS-based structures. / Resumé en français Les récepteurs Toll-like (TLRs) sont des protéines transmembranaires qui constituent la première barrière de notre système immunitaire inné. Ils détectent la présence de bactéries et virus et alertent l’organisme via la sécrétion de molécules pro-inflammatoires appelés cytokines. Parmi les TLRs, TLR2 and TLR4 reconnaissent respectivement des lipides spécifiques aux bactéries, les lipopeptides et les lipopolysaccharides bactériens LPS. La reconnaissance de motifs moléculaires spécifiques aux pathogènes et absents dans notre organisme est essentiel afin d’éviter une réponse immunitaire venant du soi. Le but de notre thèse était de démontrer que les récepteurs Toll-like possèdent une certaine plasticité et peuvent reconnaître des ligands non identifiés jusqu’ici tels les lipides cationiques et la cardiolipine. Les lipides cationiques sont des molécules synthétiques utilisées comme agents de transfection. Notre travail démontre que les lipides cationiques dont la tête polaire est constituée par des polyamines peuvent mimer les propriétés des ligands naturels et induire la sécrétion de cytokines pro-inflammatoire via l’activation des TLRs. Cette interaction implique des interactions entre la chaine principale de la protéine et les lipides sans intervention des chaines latérales. Cette réaction inflammatoire est contre-indiquée en thérapie génique et nous proposons donc de remplacer les chaines acylées saturées par des chaines insaturées pour la synthèse des nouveaux agents de transfection non-immunogénique. D’autre part, l’activation des TLRs par des agents de transfections active le système immunitaire inné, ce qui permet l’activation du système adaptatif et la production d’anticorps. Nous avons étudié une large gamme des lipides cationiques et identifié des nouveaux activateurs á la fois de TLR2 et de TLR4. L’étude de leurs propriétés adjuvantes a démontré que les lipides cationiques sont des adjuvants comparables aux sels d’aluminium en terme de production d’anticorps. La cardiolipine est un lipide localisé dans la membrane des mitochondries et des bactéries. Le domaine hydrophobe est constitué de quatre chaines acylées qui chez les mammifères sont insaturées. Il a été démontré que la cardiolipine extracellulaire inhibe la sécrétion de cytokines induite par LPS. Notre travail de thèse démontre que cet effet inhibiteur est du à la capacité de la cardiolipine à bloquer le site de liaison du LPS. Le travail démontre aussi que lorsque les chaines acylées sont saturées, c’est le cas dans le Syndrome de Barth, la cardiolipine devient un activateur de TLR4 en interagissant avec TLR4 de façon similaire à LPS. Ce dernier résultat pourrait expliquer l’aspect inflammatoire du Syndrome de Barth et élargie la librairie de ligands de TLR4 á des molécules de structure plus simple et plus aisées à synthétiser que les dérivés de LPS et qui pourraient être utilisés comme adjuvants ou anti-inflammatoires. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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The synthesis of pyridinium cationic lipids, their counter-ion exchange, and the transfection of lipoplexes consisting of these lipids with firefly luciferase plasmid DNA (6.7 KDa), into lung, prostate and breast cancer cell lines was investigated. The transfection ability of these newly synthesized compounds was found to be twice as high as DOTAP/cholesterol and LipofectamineTM (two commercially available successful transfection agents). The compaction of the DNA onto silica (SiO2) nanoparticles was also investigated. For this purpose, it was necessary to study the stability and fusion studies of colloidal systems composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), a zwitterionic lipid, and mixtures of DMPC with cationic DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane). / Chemistry

A Anfotericina B (AB) é um antibiótico antifúngico natural, do grupo dos polienos, produzido por cultura de bactérias Streptomyces nodosus. A AB foi isolada pela primeira vez em 1953, e desde então tem sido utilizada extensivamente no tratamento clínico de infecções sistêmicas. Como a AB é altamente insolúvel em meio aquoso, a formulação de escolha tem sido dispersões aquosas de micelas mistas de AB e desoxicolato de sódio (Fungizon). Entretanto, a alta toxicidade deste fármaco tem estimulado a proposição de várias outras formulações. O presente trabalho estuda uma dessas formulações alternativas propostas: agregados de AB e Brometo de dioctadecildimetilamônio (DODAB). O objetivo principal deste trabalho foi tentar avaliar, numa visão estrutural, as propriedades e peculiaridades envolvidas na interação da AB com as dispersões de DODAB. Neste trabalho, centrou-se no estudo de dispersões de DODAB, sonicadas (DODABS) e não sonicadas (DODABNS), onde o antibiótico estivesse, principalmente, na forma monomérica, tendo em vista a proposta baixa toxicidade da AB enquanto monômero. Para avaliar estes sistemas utilizaram-se as técnicas de absorção óptica, calorimetria diferencial de varredura (DSC) e a Ressonância Paramagnética Eletrônica (RPE) de marcadores de spin incorporados nos agregados lipídicos. Mostrou-se que a AB monomérica incorpora-se tanto em vesículas de DODABNS, como nos pequenos agregados de DODABS, sendo maior sua incorporação nestes últimos. Foi possível mostrar que até a fração molar (AB/DODAB) de 1,2 mol% a AB está totalmente monomérica, incorporada em agregados de DODABS, mas somente parcialmente em vesículas de DODABNS. Nas bicamadas de DODABNS, a preferência da AB monomérica é pela fase gel, 40 % incorporada, comparada com 23 % na fase fluida. As várias técnicas indicam que a AB encontra-se na superfície da bicamada, com seu cromóforo hidrofóbico enxergando um meio de polarizabilidade maior do que a da água, tanto em DODABS como em DODABNS. Marcadores de spin mostram a coexistência de domínios lipídicos em fases diferentes, gel e fluida, a baixas temperaturas, em DODABS. Curiosamente, indicam que a AB monomérica interage, preferencialmente, com os domínios mais fluidos, em desacordo com a observada maior afinidade da AB pela fase gel de bicamadas lipídicas de DODABNS. O estudo termo-estrutural de dispersões de DODABNS e DODABS aqui apresentado, na presença e ausência de AB, pode ser relevante na proposta de novas formulações farmacológicas. / Amphotericin B (AB) is a natural antibiotic, produced by Streptomyces nodosus, with a very potent antifungal activity. It is a polyene macrolide molecule, isolated in 1953. Since then, it has been extensively used in the treatment of systemic mycotic infections. Considering AB very low solubility in aqueous medium, it has been largely used as an aqueous dispersion of sodium deoxycholate-AB mixed micelles (Fungizon). However, the high toxicity of this preparation has led to the proposition of different other formulations. The present work focuses on one of the proposed preparations, namely aggregates formed by AB and the cationic lipid dioctadecyldimethylammonium (DODAB). Considering the proposed low toxicity of monomeric AB, the present work studies DODAB dispersions, sonicated (DODABS) and non-sonicated (DODABNS), where AB is mostly monomeric. To the structural analysis, optical absorption, differential scanning calorimetry (DSC) and electron spin resonance of spin labels incorporated in the aggregates were used. It was found that AB as a monomer incorporates in aggregates present in both dispersions, DODABS and DODABNS, but the incorporation of monomeric AB in the small fragments present in DODABS is much larger. It was shown that AB incorporates in DODABS aggregates, as a monomer, up to AB/DODAB concentration of 1.2 mol%. At this concentration, only 40 % and 23 % of AB molecules are in the monomeric state in the gel and fluid phases of DODABNS bilayers, respectively. Hence, the gel phase of DODABNS bilayers favors the monomerization of AB, as compared to the bilayer fluid phase. All the applied techniques point to a superficial interaction of monomeric AB with DODAB aggregates, with the hydrophobic polyene chromophor positioned relatively inside the bilayer, in a region of polarizability higher than that of water, in both dispersions, DODABS and DODABNS. Spin labels indicate the coexistence of gel and fluid domains in DODABS aggregates, at low temperatures, supporting the proposed hypothesis of the presence of bilayer fragments in the dispersion. Contrarily to the higher detected affinity of monomeric AB for the gel phase, as compared to the fluid phase of DADABNS bilayers, spin labels indicate that monomeric AB is mostly adsorbed at the fluid domain of the fragments, possibly corresponding to the periphery of the fragments. The thermal-structural study presented here of DODABNS and DODABS, in the presence and absence of AB, can be relevant in the design of new pharmaceutical formulations.

La mucoviscidose est une maladie monogénique, caractérisée par des mutations survenant au niveau du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Le clonage en 1989 du gène CFTR a permis d’envisager de traiter cette maladie par thérapie génique. Cela consiste à transférer à l’aide d’un vecteur, une version normale du gène CFTR dans les cellules atteintes des patients. En raison de la gravité des complications pulmonaires, c’est l’épithélium respiratoire qui constitue aujourd’hui le tissu cible pour le transfert de gènes. Le principe de la thérapie génique est évidemment très séduisant et un certain nombre d’essais cliniques ont d’ores et déjà été réalisés. La thérapie génique nécessite des outils de vectorisation efficaces et compatibles avec une utilisation répétée en clinique.Mon sujet de thèse a porté donc sur le développement, la biodistribution et l’optimisation de vecteurs synthétiques (lipides cationiques) pour le transfert de gènes dans l’épithélium respiratoire. Au cours de mes travaux, nous avons donc pu mettre au point des lipophosphoramidates KLN47 fluorescents utiles pour les études de biodistribution in vivo. Comparés au KLN47 non fluorescent, ces nouveaux composés présentent les mêmes propriétés physicochimiques, à savoir une taille relativement petite et un potentiel zêta positif. Sur lignées cellulaires, nous avons montré que les nouvelles formulations étaient aussi efficaces que le KLN47, et pas ou peu toxiques. Ensuite, sur modèle animal, les profils de biodistribution de lipoplexes pégylés et non-pégylés ont été comparés après injection systémique. Les profils de biodistribution des lipoplexes pégylés et non-pégylés étaient similaires, cependant, la pégylation des complexes a conduit à une circulation prolongée dans la circulation sanguine, alors que l’expression du transgène (luciférase) était équivalente dans les deux cas. De plus, l’activité luciférase était similaire à celle obtenue avec le KLN47 non fluorescent. Nous avons ainsi démontré que l’ajout des sondes lipidiques fluorescentes dans la solution liposomale du KLN47, ne modifie pas ses propriétés physicochimiques et transfectantes. L’ensemble des résultats montre que nous disposons d’outils prometteurs pour les études de biodistribution in vivo. D’autres molécules ont également été testées avec succès. / Cystic fibrosis is a monogenic disease characterized by mutations occurring at the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The clonining in 1989 of the CFTR gene has enabled to consider treating this disease by gene therapy. This consists of transferring a normal version of the CFTR gene in the affected patients’ cells, using a vector. Due to the severity of pulmonary complications, it is obvious that the respiratory epithelium constitutes the target tissue for the gene transfer. The principle of gene therapy is indeed very attractive and a number of clinical trials have already been made. Gene therapy requires vectorization tools that are efficient and compatible with repeated clinical use.My thesis has focused on the development, biodistribution and optimization of synthetic vectors (cationic lipids) for gene transfer in the respiratory epithelium. During my work, we were able to develop useful fluorescent KLN47 lipophosphoramidates for in vivo biodistribution studies. Compared to non fluorescent KLN47, these new compounds exhibit the same physicochemical properties: a relatively small size and a positive zeta potential. On cell lines, we found that the new formulations were as effective as the KLN47, with little or no toxicity. Then, in animal models, the biodistribution profiles of pegylated and non-pegylated lipoplexes were compared after systemic injection. The biodistribution profiles of pegylated and non-pegylated lipoplexes were similar. However, the pegylation of the complex resulted in prolonged circulation in the bloodstream, whereas transgene expression (luciferase) was equivalent in both cases. In addition, luciferase activity was similar to that obtained with the non-fluorescent KLN47. We have demonstrated that the addition of fluorescent lipid probes in the liposomal solution KLN47, does not change its physicochemical and transfectant properties. The overall results show that we have promising tools for in vivo biodistribution studies. Other molecules have also been tested successfully.

A Anfotericina B (AB) é um antibiótico antifúngico natural, do grupo dos polienos, produzido por cultura de bactérias Streptomyces nodosus. A AB foi isolada pela primeira vez em 1953, e desde então tem sido utilizada extensivamente no tratamento clínico de infecções sistêmicas. Como a AB é altamente insolúvel em meio aquoso, a formulação de escolha tem sido dispersões aquosas de micelas mistas de AB e desoxicolato de sódio (Fungizon). Entretanto, a alta toxicidade deste fármaco tem estimulado a proposição de várias outras formulações. O presente trabalho estuda uma dessas formulações alternativas propostas: agregados de AB e Brometo de dioctadecildimetilamônio (DODAB). O objetivo principal deste trabalho foi tentar avaliar, numa visão estrutural, as propriedades e peculiaridades envolvidas na interação da AB com as dispersões de DODAB. Neste trabalho, centrou-se no estudo de dispersões de DODAB, sonicadas (DODABS) e não sonicadas (DODABNS), onde o antibiótico estivesse, principalmente, na forma monomérica, tendo em vista a proposta baixa toxicidade da AB enquanto monômero. Para avaliar estes sistemas utilizaram-se as técnicas de absorção óptica, calorimetria diferencial de varredura (DSC) e a Ressonância Paramagnética Eletrônica (RPE) de marcadores de spin incorporados nos agregados lipídicos. Mostrou-se que a AB monomérica incorpora-se tanto em vesículas de DODABNS, como nos pequenos agregados de DODABS, sendo maior sua incorporação nestes últimos. Foi possível mostrar que até a fração molar (AB/DODAB) de 1,2 mol% a AB está totalmente monomérica, incorporada em agregados de DODABS, mas somente parcialmente em vesículas de DODABNS. Nas bicamadas de DODABNS, a preferência da AB monomérica é pela fase gel, 40 % incorporada, comparada com 23 % na fase fluida. As várias técnicas indicam que a AB encontra-se na superfície da bicamada, com seu cromóforo hidrofóbico enxergando um meio de polarizabilidade maior do que a da água, tanto em DODABS como em DODABNS. Marcadores de spin mostram a coexistência de domínios lipídicos em fases diferentes, gel e fluida, a baixas temperaturas, em DODABS. Curiosamente, indicam que a AB monomérica interage, preferencialmente, com os domínios mais fluidos, em desacordo com a observada maior afinidade da AB pela fase gel de bicamadas lipídicas de DODABNS. O estudo termo-estrutural de dispersões de DODABNS e DODABS aqui apresentado, na presença e ausência de AB, pode ser relevante na proposta de novas formulações farmacológicas. / Amphotericin B (AB) is a natural antibiotic, produced by Streptomyces nodosus, with a very potent antifungal activity. It is a polyene macrolide molecule, isolated in 1953. Since then, it has been extensively used in the treatment of systemic mycotic infections. Considering AB very low solubility in aqueous medium, it has been largely used as an aqueous dispersion of sodium deoxycholate-AB mixed micelles (Fungizon). However, the high toxicity of this preparation has led to the proposition of different other formulations. The present work focuses on one of the proposed preparations, namely aggregates formed by AB and the cationic lipid dioctadecyldimethylammonium (DODAB). Considering the proposed low toxicity of monomeric AB, the present work studies DODAB dispersions, sonicated (DODABS) and non-sonicated (DODABNS), where AB is mostly monomeric. To the structural analysis, optical absorption, differential scanning calorimetry (DSC) and electron spin resonance of spin labels incorporated in the aggregates were used. It was found that AB as a monomer incorporates in aggregates present in both dispersions, DODABS and DODABNS, but the incorporation of monomeric AB in the small fragments present in DODABS is much larger. It was shown that AB incorporates in DODABS aggregates, as a monomer, up to AB/DODAB concentration of 1.2 mol%. At this concentration, only 40 % and 23 % of AB molecules are in the monomeric state in the gel and fluid phases of DODABNS bilayers, respectively. Hence, the gel phase of DODABNS bilayers favors the monomerization of AB, as compared to the bilayer fluid phase. All the applied techniques point to a superficial interaction of monomeric AB with DODAB aggregates, with the hydrophobic polyene chromophor positioned relatively inside the bilayer, in a region of polarizability higher than that of water, in both dispersions, DODABS and DODABNS. Spin labels indicate the coexistence of gel and fluid domains in DODABS aggregates, at low temperatures, supporting the proposed hypothesis of the presence of bilayer fragments in the dispersion. Contrarily to the higher detected affinity of monomeric AB for the gel phase, as compared to the fluid phase of DADABNS bilayers, spin labels indicate that monomeric AB is mostly adsorbed at the fluid domain of the fragments, possibly corresponding to the periphery of the fragments. The thermal-structural study presented here of DODABNS and DODABS, in the presence and absence of AB, can be relevant in the design of new pharmaceutical formulations.