Tesi sul tema "Liposomes"

This thesis presents an optimized and general procedure for coupling proteins to liposomes and investigates certain aspects of the interaction of liposomes with components of the circulation. The object of these studies was to develop straightforward methods for the preparation of well characterized protein-liposome conjugates which exhibit extended circulation half-lives in the blood. These favorable properties should potentiate the use of protein coupled vesicles in in vivo applications such as targeting or diagnostic protocols. A general approach for the preparation of protein-liposome conjugates was developed which employs the high affinity binding of streptavidin for biotinated proteins. Streptavidin was initially attached in a non-covalent manner (via biotin phosphatidylethanolamine) or covalently (via maleimidophenyl-butyryl phosphatidyl-ethanolamine, MPB-PE or pyridyldithio-propionyl phosphatidylethanolamine, PDP-PE) to pre-formed liposomes containing the various lipid derivatives. It was shown that the procedure based on the maleimide derivative MPB-PE, was the most efficient coupling method. Standard procedures for the preparation of MPB-PE however, were found to result in an impure product. Recently a new method for the synthesis of a pure SMPB derivative of phosphatidylethanolamine was developed (Lewis Choi, unpublished). Efficient coupling of proteins to liposomes containing low amounts of pure MPB-DPPE was achieved. Subsequently it was shown that gentle incubation with biotinated proteins results in the rapid and efficient generation of protein coupled vesicles. These retain their ability to interact with defined target celte. Aggregation of liposomes during the coupling reaction is a common consequence of the efficient coupling of protein to liposomes. A method was therefore developed for the preparation of small homogeneously sized protein-liposome conjugates by an extrusion process which does not denature the attached protein. These extruded samples exhibited extended blood circulation times and were stable for significant periods in vivo. The second part of this thesis investigated the in vitro interaction of liposomes of various lipid compositions with platelets. It was demonstrated that large liposomes (> 200 nm in diameter) containing negatively charged lipids (such as EPG) or thiol reactive lipid derivatives (such as MPB-PE) can induce aggregation of platelets in vitro. This interaction was mediated by complement. It is suggested that the formation of platelet-liposome microaggregates in vivo on intravenous administration of negatively charged liposomes, resulted in the transient thrombocytopenia observed in rats. This adhesion event may have also contributed to the rapid removal of aggregated protein-liposome conjugates (containing MPB-PE) from the circulation.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0.

Les malalties autoimmunitàries estan causades per defectes en la tolerància immunològica, i afecten a gairebé un 10% de la població. Darrerament, diverses intervencions mèdiques han convertit aquestes malalties en cròniques, però el seu diagnòstic encara comporta morbiditat i mortalitat elevades. Així, un repte biomèdic urgent és el desenvolupament de teràpies que puguin restablir selectivament la tolerància, aturin l’atac autoimmunitari i permetin la regeneració del teixit danyat. En condicions fisiològiques, la fagocitosi de cèl·lules apoptòtiques per part de fagòcits com les cèl·lules dendrítiques (CDs) —procés que rep el nom d’eferocitosi— els indueix propietats tolerogèniques i l’habilitat de restaurar la tolerància. Una demostració d’això és que una immunoteràpia cel·lular consistent en CDs tolerogèniques (CDtols) degut a l’eferocitosi de cèl·lules β apoptòtiques va aturar l’atac autoimmunitari contra les cèl·lules β en un model experimental de diabetis tipus 1 (DT1). Donades les dificultats en obtenir i estandarditzar cèl·lules β apoptòtiques autòlogues humanes per la seva implementació clínica, es va dissenyar una nanoteràpia basada en liposomes que simulen cèl·lules apoptòtiques. Les principals característiques d’aquestes vesícules sintètiques són: elevat percentatge de fosfatidilserina (FS) —fosfolípid característic de la membrana de les cèl·lules apoptòtiques—, diàmetre major de 500 nm, càrrega negativa i eficient encapsulació de pèptids d’insulina. Aquesta estratègia és tant efectiva en generar CDtols i aturar l’autoimmunitat contra les cèl·lules β com la immunoteràpia basada en cèl·lules apoptòtiques. La hipòtesi d’aquest treball és que els FS-liposomes poden restablir la tolerància en diverses malalties autoimmunitàries antigen-específiques mitjançant la generació de CDtols i l’expansió de limfòcits T reguladors, i que tenen el potencial translacional per abordar patologies autoimmunitàries humanes. L’objectiu principal d’aquest estudi ha estat caracteritzar globalment el potencial tolerogènic dels FS-liposomes. Amb aquesta finalitat, diferents pèptids autoantigènics rellevants en malalties autoimmunitàries s’han encapsulat en FS-liposomes de manera eficient i sense dificultats en mantenir el diàmetre i la càrrega, demostrant la versatilitat de l’estratègia a diferents patologies autoimmunitàries. En el model experimental de DT1, l’administració de FS-liposomes ha expandit clons de cèl·lules T CD4+ reguladores i T CD8+ que contribueixen a l’efecte tolerogènic de la teràpia a llarg termini. En el mateix model, s’ha demostrat la biocompatibilitat i seguretat del producte final donada la seva òptima tolerabilitat. D’altra banda, els FS-liposomes s’han adaptat al model d’esclerosi múltiple experimental simplement reemplaçant el pèptid encapsulat. En aquest model, els FS-liposomes han induït CDtols i han reduït la incidència i severitat de la malaltia correlacionant amb un increment en la freqüència de cèl·lules T reguladores, fet que valida el potencial dels FS-liposomes a constituir una plataforma per a la recuperació de tolerància en diferents malalties autoimmunitàries. Finalment, i de cara a una futura implementació clínica, s’han determinat els efectes de la teràpia en CDs humanes obtingudes de pacients amb DT1. En CDs de pacients adults, els FS-liposomes han estat fagocitats eficientment per les CDs amb una ràpida cinètica dependent de la FS, i això ha causat l’adquisició d’un transcriptoma, fenotip i funcionalitat tolerogènics similars als observats en els models experimentals. En CDs de pacients pediàtrics, però, les CDs han presentat defectes en la seva capacitat fagocítica correlacionant amb el temps de progressió de la malaltia; tanmateix, el seu fenotip i expressió de gens immunoreguladors després de la fagocitosi dels FS-liposomes són indicatius d’una habilitat tolerogènica òptima. En conclusió, la immunoteràpia liposomal descrita, que es basa en l’eferocitosi com a mecanisme inductor de tolerància, assoleix el mimetisme apoptòtic de manera simple, segura i eficient. A més, els liposomes ofereixen avantatges quant a producció i estandardització. Per tant, els FS-liposomes tenen potencial translacional i constitueixen una estratègia prometedora per recuperar la tolerància immunològica en malalties autoimmunitàries antigen-específiques.
Autoimmune diseases are caused by defective immunological tolerance, and reportedly affect up to 10% of the global population. In the last years, current medical interventions have transformed these disorders into chronic and manageable, but they still entail high rates of morbidity and mortality. Hence, there is an urgent need to develop therapies capable of restoring the breach of tolerance selectively, which halt the autoimmune aggression and allow the regeneration of the targeted tissue. In physiological conditions, the phagocytosis of apoptotic cells performed by phagocytes such as dendritic cells (DCs) —a process termed efferocytosis— prompts the acquisition of tolerogenic features and the ability to restore tolerance. Indeed, a cell immunotherapy consisting of DCs rendered tolerogenic (tolDCs) by apoptotic β-cell efferocytosis arrested the autoimmune attack against β-cells in an experimental model of type 1 diabetes (T1D). However, in light of the hurdles in obtaining and standardising human autologous apoptotic β-cells for its implementation in the clinics, a nanotherapeutic strategy based on liposomes mimicking apoptotic cells was designed. The fundamental characteristics of these synthetic vesicles are: a high percentage of phosphatidylserine (PS) —phospholipid unique to the apoptotic cell membrane—, diameter superior to 500 nm, negative charge and efficient encapsulation of insulin peptides. Importantly, this strategy was equally effective in inducing tolDCs and blunting β-cell autoimmunity as the immunotherapy based on apoptotic cells. The hypothesis of this work is that autoantigen-loaded PS-liposomes can re-establish tolerance in several antigen-specific autoimmune diseases through the induction of tolDCs and the expansion of regulatory T lymphocytes, and that they have translational potential to tackle human autoimmune disorders. The main aim of the present work has been to characterise the tolerogenic potential of PS-liposomes globally. To this end, different autoantigenic peptides relevant in autoimmune diseases have been efficiently encapsulated into PS-liposomes, without difficulties in preserving their appropriate diameter and charge, thus demonstrating the versatility of the therapy to different autoimmune pathologies. In the experimental model of T1D, the administration of PS-liposomes causes the expansion of clonal CD4+ regulatory T cells and CD8+ T cells, which contribute to the long-term re-establishment of tolerance. Moreover, in the same model, the biocompatibility and safety of the final product have been confirmed given its optimal tolerability. Furthermore, PS-liposomes have been adapted to the experimental multiple sclerosis model by merely replacing the encapsulated autoantigen. In this model, PS-liposomes elicit the generation of tolDCs and decrease the incidence and severity of the disease correlating with an increase in the frequency of regulatory T cells, a fact that validates the potential of PS-liposomes to serve as a platform for tolerance re-establishment in different autoimmune diseases. Finally, considering its future clinical implementation, the effect of the PS-liposomes therapy has been determined in human DCs obtained from patients with T1D. In DCs from adult patients, PS-liposomes are efficiently phagocyted by DCs with rapid kinetics dependent on the presence of PS, and this induces a tolerogenic transcriptome, phenotype and functionality that are similar to those observed in experimental models. However, DCs from paediatric patients display defects in their phagocytic capacity correlating with the time of disease progression, albeit their phenotype and immunoregulatory gene expression after PS-liposomes phagocytosis point to an optimal tolerogenic ability. In conclusion, the liposomal immunotherapy herein described, which is based on efferocytosis as a powerful tolerance-inducing mechanism, achieves apoptotic mimicry in a simple, safe and efficient manner. Additionally, liposomes offer advantages in terms of production and standardisation. Therefore, PS-liposomes possess translational potential and constitute an encouraging strategy to restore immunological tolerance in antigen-specific autoimmune diseases.

Despite the progress in elucidating antigens for both therapeutic and prophylactic vaccines, safety concerns over current vaccine delivery vehicles and adjuvants has limited the development of new vaccines. In particular, there is an urgent need for effective vaccines capable of stimulating cytotoxic T lymphocyte (CTL) responses against intracellular pathogens or tumor cells. Liposomes are under investigation as a particulate vaccine delivery system with the required safety profile and demonstrated ability to target antigens to dendritic cells (DC), the cells of the immune system responsible for initiating effective and long lasting CTL immune responses. Unmodified liposomes however, are inherently non-immunogenic and thus not capable of stimulating activation of DC, which is a necessary step in immune activation. In this thesis the use of modified liposomes to more efficiently target vaccine antigens to DC and then activate the DC sufficiently to initiate down-stream immune responses was investigated. In the first approach to liposome modification, mannosylated phospholipids were incorporated within the liposome bilayer to target C-type lectins on DC. Incorporation of mono- or tri-mannosylated phospholipids within liposomes was found to be an effective means of attaching mannose-containing ligands to the liposome surface without compromising the integrity of the liposome structure. The uptake of tri-mannose-containing liposomes was enhanced in human monocyte derived DC (MoDC) compared to both unmodified liposomes and mono-mannose-containing liposomes. In contrast, neither mono- nor tri-mannose-containing liposomes were taken up by murine bone marrow derived DC (BMDC) to a greater extent than unmodified liposomes. This finding may reflect the differences in ligand specificity for C-type lectins on DC derived from different mammalian species. It was also found in these studies that increased uptake of liposomal antigens by DC does not necessarily result in increased DC activation, as evidenced by a lack of up-regulation of DC surface activation markers and ability to stimulate T cell proliferation. The second approach to liposome modification involved the incorporation of lipid core peptides (LCPs) into the liposome structure. LCPs alone were demonstrated to be able to stimulate DC and subsequent CD8+ T cell activation in vitro. LCP-based vaccines were also able to stimulate effective cytotoxic immune responses in vivo, and protect against tumor challenge, but only if administered in alum with CD4 help. Liposomes containing LCPs were able to stimulate greater DC activation and subsequent CD8+ T cell proliferation in vitro compared with unmodified liposomes. In the in vivo studies however, LCP-containing liposomes were not able to stimulate a cytotoxic immune response or protect against tumor challenge as effectively as LCP administered in alum. In the final approach to liposome modification, inclusion of the adjuvant Quil A was investigated for its ability to increase the immunogenicity of LCP-containing liposomes. It was found that small amounts of Quil A could be incorporated into liposomes without compromising the liposome bilayer. The inclusion of as little as 2% Quil A was able to stimulate DC activation and subsequent T cell proliferation in in vitro studies. In addition, immunisation of mice with LCP-containing liposomes with incorporated Quil A was found to stimulate an in vivo CTL immune response comparable to LCPs administered under optimal vaccine conditions. In conclusion, the work presented in this thesis demonstrates that modified liposomes are a useful vaccine delivery system for the initiation of in vivo cytotoxic and prophylactic immune responses.

Liposomes have established roles in drug delivery and cell membrane studies. Amongst other applications; they can also be used as analytical reagents, particularly in immunoassays. Liposomal immunoassays have potential advantages over alternatives; including sensitivity, speed, simplicity and relative reagent stability. The aim of these studies was to develop and evaluate novel examples of these assays. When liposomes entrapped the dye, Sulphorhodamine B, a shift in its maximum absorption wavelength compared to free dye was observed. This was attributed to dimerization of the dye at high concentrations. If the liposomes were disrupted, the released dye was diluted into the external buffer, and the dye's absorption spectrum reverted to that of free dye. After optimization of dye entrapment, immunoassays were developed using these liposomes. Albumin-coated liposomes were used in a model assay to measure serum albumin. This assay employed complement-mediated immunolysis, commonly used in liposomal immunoassays. The liposomes were lysed by anti-albumin and complement, and this could be competitively inhibited by serum albumin. To improve sensitivity, Fab' anti-albumin liposomes were prepared. These enabled measurement of urinary albumin by a complement-mediated immunoassay, but using a sandwich technique. Anti-albumin (intact) liposomes were shown to precipitate on gentle centrifugation after reaction with albumin. They were applied as a solid phase reagent in an heterogeneous immunoassay, using radioimmunoassay for urinary microalbumin as a model assay. Liposomes containing Sulphorhodamine B were also used in a more novel assay; for serum anticardiolipin antibodies. Cardiolipin-containing liposomes were prepared. These were lysable using magnesium ions. Anticardiolipin antibodies (IgG) were found to augment this lysis, enabling their estimation. Similar imprecision and acceptable correlation with a commercial enzyme-linked immunosorbent assay (ELISA) were obtained. The findings demonstrate Sulphorhodamine B release can be used as a marker in homogeneous colorimetric liposomal immunoassays; both in model assays and in potentially more useful clinical biochemistry applications.

The photopolymerization of two-component large unilamellar liposomes (LUV) composed of 3/1 dioleoylphosphatidylethanolamine (DOPE) and either 1,2-bis- (10-(2',4'-hexadienoyloxy)decanoyl) -sn-glycero-3-phosphatidylcholine (bis-SorbPC) or 1-palmitoyl-2- (10-(2$\sp\prime,4\sp\prime$-hexadienoyloxy) decanoyl) -sn-glycero-3-phosphocholine (mono-SorbPC) facilitated liposome fusion. Fusion was characterized by fluorescent assays for lipid mixing, aqueous contents mixing, and aqueous contents leakage. The rate and extent of the photoinduced fusion was dependent on the extent of polymerization, temperature, and the fusion initiation conditions, including the pH and the presence of Mg²⁺ ions. Examination of the temperature dependence of fusion for unpolymerized and polymerized liposomes showed that an enhancement of the rate of fusion occurred in the temperature range, Δ T(I), where inverted phase lipid intermediates are observed. Photopolymerization causes lateral separation of the liposome components and thereby results in the isothermal induction of fusion by lowering the threshold temperature for formation of fusion intermediates.

Aim of this thesis is voltammetric study influence of liposomal platinum cytostatics on cancer cells. One of the goals is summarize available informations about influence of cisplatine on cancer cells, its encapsulation into liposome and affection of this cytostatic cisplatin encapsulated in liposome on cancer cell lines. In literary recherche is detail description of these issues. Than is there specification of voltammetric methods, which serve to electrochemical detection of cisplatin. Based on literary recherche was chosen the best method for detection and subsequently the method was optimalized and than was applied to measuring itself.

Els liposomes són nanovesícules lipídiques àmpliament explorades per ser utilitzades com a transportadors d’actius terapèutics per al desenvolupament de noves nanomedicines. En la present Tesi s’ha estudiat l’ús d’aquests sistemes liposomals funcionalitzats amb lligands per ser utilitzats com a nanotransportadors d’enzims, específicament, pel transport eficaç de l’enzim α-galactosidasa a través de les membranes biològiques, com la membrana cel·lular o la barrera hematoencefàlica, per tal d’aconseguir un nou producte farmacèutic pel tractament de la malaltia de Fabry. La malaltia de Fabry és una malaltia minoritària que pertany a un grup de trastorns que s’engloben com a malalties per dipòsit lisosòmic, per a les quals actualment encara no s’ha trobat una cura definitiva. Es caracteritza per la manca d’activitat enzimàtica de l’ α-galactosidasa (GLA). La seva carència provoca l’acumulació de diferents substàncies a les cèl·lules, com glicoesfingolípids (Gb3), i acaba afectant de forma multisistèmica a diversos òrgans com els ronyons, el cor o el sistema nerviós central. El tractament actual d’aquesta malaltia és mitjançant la teràpia de reemplaçament enzimàtic (ERT), que es basa en l’administració intravenosa de l’enzim deficient, normalment obtingut per biotecnologia recombinant. No obstant això, l’èxit d’aquest tipus de teràpia sol quedar limitat per la inestabilitat i la baixa eficàcia de l’enzim exogen que s’administra, així com la baixa capacitat per creuar barreres biològiques, com la barrera hematoencefàlica. Una estratègia interessant per tractar de superar les limitacions dels tractaments d’ERT és el disseny i desenvolupament de noves nanoformulacions a fi d’augmentar l’estabilitat i l’eficàcia dels enzims terapèutics, com la GLA. En els últims anys, en el marc del grup Nanomol de l’ICMAB-CSIC, s’ha estat investigant el desenvolupament d’un nou sistema liposomal que millori el transport intracel·lular de la GLA administrada exògenament i altres propietats farmacològiques d’aquest enzim. Els nanoliposomes resultants es troben funcionalitzats amb el pèptid RGD per tal d’augmentar l’afinitat dels nanoliposomes per a les cèl·lules endotelials. Malgrat l’obtenció de resultats preliminars positius amb aquesta nanoformulació inicial de GLA (nanoGLA), calia superar diverses limitacions per tal de transformar-la en un producte que compleixi els requisits per poder-la testar en estudis preclínics. Per aconseguir-ho, ha calgut una alta comprensió i control del vehicle nanoliposomal a nivell molecular i supramolecular. En aquesta Tesi, s’ha estudiat la relació entre les propietats fisicoquímiques i el comportament biològic dels liposomes funcionalitzats amb pèptids, especialment per al transport de l’enzim GLA per al tractament de la malaltia de Fabry. Mitjançant el procés DELOS-susp, s’ha aconseguit preparar exitosament la formulació de nanoGLA amb la quantitat i la qualitat necessàries per avançar cap a l’avaluació preclínica in vivo. La nanoGLA va demostrar una eficàcia superior en comparació amb el tractament actual (Replagal®) i la GLA no nanoformulada en un model de ratolí Fabry, obtenint una major reducció dels nivells de Gb3 en tots els teixits analitzats, inclòs el cervell. A més, s’ha assolit per primera vegada l’etapa preclínica reguladora en el desenvolupament d’aquesta nova formulació de nanoGLA, realitzant estudis de toxicitat GLP (Good Laboratory Practice) en rosegadors. Finalment, també s’ha explorat la preparació mitjançant DELOS-susp de nous nanoliposomes funcionalitzats amb lligands alternatius a l’RGD per a aplicacions relacionades amb el creuament de la barrera hematoencefàlica. En resum, els resultats obtinguts en aquesta tesi avalen l’estratègia d’utilitzar sistemes liposomals dirigits a l’administració de fàrmacs. L’èxit en el desenvolupament i l’optimització del producte nanoGLA per millorar la teràpia actual de reemplaçament enzimàtic en la malaltia de Fabry és un clar exemple de recerca translacional i interdisciplinària.
Los liposomas son nanovesículas lipídicas ampliamente exploradas para ser utilizadas como transportadores de activos terapéuticos para el desarrollo de nuevas nanomedicinas. En la presente Tesis se ha estudiado el uso de estos sistemas liposomales funcionalizados con ligandos para ser utilizados como nanotransportadores de enzimas, específicamente, para el transporte eficaz de la enzima α-galactosidasa a través de membranas biológicas, como la membrana celular o la barrera hematoencefálica, para conseguir un nuevo producto farmacéutico para el tratamiento de la enfermedad de Fabry. La enfermedad de Fabry es una enfermedad minoritaria que pertenece a un grupo de trastornos englobados como enfermedades por depósito lisosómico, para las que actualmente no se ha encontrado una cura definitiva. Se caracteriza por la falta de actividad enzimática de la α-galactosidasa (GLA). Su deficiencia provoca la acumulación de diferentes sustancias en las células, como los glicoesfingolípidos (Gb3), y termina afectando de forma multisistémica a varios órganos como los riñones, el corazón o el sistema nervioso central. El tratamiento actual de esta enfermedad es mediante la terapia de reemplazo enzimático (ERT), que se basa en la administración intravenosa de la enzima deficiente, normalmente obtenida por biotecnología recombinante. Sin embargo, el éxito de este tipo de terapia suele quedar limitado por la inestabilidad y la baja eficacia de la enzima exógena que se administra, así como la incapacidad para cruzar barreras biológicas, como la barrera hematoencefálica. Una estrategia interesante para tratar de superar las limitaciones de los tratamientos de ERT es el diseño y desarrollo de nuevas nanoformulaciones a fin de aumentar la estabilidad y la eficacia de las enzimas terapéuticas, como la GLA. En los últimos años, en el marco del grupo Nanomol del ICMAB se ha estado investigando el desarrollo de un nuevo sistema liposomal que mejore el transporte intracelular de la GLA administrada exógenamente y otras propiedades farmacológicas de esta enzima. Los nanoliposomes resultantes se encuentran funcionalizados con el péptido RGD para aumentar la afinidad de estos nanoliposomas hacia las células endoteliales. A pesar de la obtención de resultados preliminares positivos con esta nanoformulación inicial de GLA (nanoGLA), había que superar diversas limitaciones para transformarla en un producto que cumpliese los requisitos para poderse testar en estudios preclínicos. Para ello, ha sido necesaria una alta comprensión y control del vehículo nanoliposomal a nivel molecular y supramolecular. En esta Tesis, se ha estudiado la relación entre las propiedades fisicoquímicas y el comportamiento biológico de los liposomas funcionalizados con péptidos, especialmente para el transporte de la enzima GLA para el tratamiento de la enfermedad de Fabry. Mediante el proceso DELOS-susp, se ha conseguido preparar exitosamente la formulación de nanoGLA con la cantidad y calidad necesarias para avanzar hacia la evaluación preclínica in vivo. La nanoGLA demostró una eficacia superior en comparación con el tratamiento actual (Replagal®) y la GLA no nanoformulada en un modelo de ratón Fabry, obteniendo una mayor reducción de los niveles de Gb3 en todos los tejidos analizados, incluido el cerebro. Además, se ha logrado por primera vez llegar a la etapa preclínica regulatoria en el desarrollo de esta nueva formulación de nanoGLA, con la realización de estudios toxicológicos GLP (Good Laboratory Practice) en roedores. Finalmente, también se ha explorado la preparación mediante DELOS-susp de nuevos nanoliposomas funcionalizados con ligandos alternativos al RGD para aplicaciones relacionadas con el cruce de la barrera hematoencefálica. En resumen, los resultados obtenidos en esta tesis avalan la estrategia de utilizar sistemas liposomales dirigidos a la administración de fármacos. El éxito en el desarrollo y la optimización del producto nanoGLA para mejorar la terapia actual de reemplazo enzimático en la enfermedad de Fabry es un claro ejemplo de investigación traslacional e interdisciplinaria.
Liposomes are lipid-based nanovesicles widely explored as nanocarriers for the transport of biomolecules or drugs of interest to the place of action and for the development of new nanomedicines. This Thesis is devoted to the study of liposomal systems functionalized with targeting-ligands, with the final goal to be used as nanocarriers of therapeutically active enzymes. The new liposomal formulations have been specifically investigated and developed for the effective transportation of α-galactosidase A enzyme through cellular and blood brain membranes, and for the achievement of a new liposomal intravenous pharmaceutical product for the treatment of Fabry disease. Fabry disease is a rare disease which belongs to the group of lysosomal storage disorders, currently without a definitive cure. It characterizes by the deficiency in α-galactosidase (GLA) enzyme activity which results in the cellular accumulation of neutral glycosphingolipids (mainly Gb3) with multisystemic organ affectation, such as kidneys, heart, and nervous system. The current treatment is the enzyme replacement therapy, in which free GLA recombinant protein is administered intravenously to patients. This treatment shows several drawbacks including poor biodistribution, low stability, limited efficacy, high immunogenicity, and low capacity to cross biological barriers, such as cell membranes and the blood-brain barrier. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating enzymes. Nanoliposomes functionalized with RGD-peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, this initial GLA-nanoconjugate was still far from the preclinical testing. Several issues must be addressed for transforming this initial GLA-nanoformulation (or nanoGLA) into a pharmacological product. To achieve this transformation, a deep understanding and control of the nanoliposomal vehicle at the molecular and supramolecular level is unavoidable. In this Thesis, the relation between the physicochemical properties and biological behavior of targeted liposomes have been addressed, to get knowledge and gain control of liposomal systems for enzyme delivery, especially for the delivery of GLA enzyme for Fabry disease treatment. Small and uniform nanoGLA liposomes, functionalized with targeting-peptides, were successfully prepared by DELOS-susp, showing high GLA entrapment efficiency, enhanced enzymatic activity, and superior efficacy. NanoGLA formulation for the treatment of Fabry disease was successfully optimized with the required amount and quality to advance towards the preclinical evaluation in vivo. NanoGLA demonstrated superior efficacy compared to current treatment (Replagal®) and non-nanoformulated GLA in a Fabry KO mouse model in terms of higher reduction of Gb3 levels in all tested tissues, including brain. Further, the regulatory preclinical stage of development for this novel nanoGLA formulation was achieved for first time with GLP toxicity studies in rodent. Finally, alternative new ligand-targeted functionalized nanoliposomes prepared by the CO2-based DELOS-susp technology were explored for blood-brain barrier crossing applications. In summary, the results achieved in this Thesis support the strong potential of targeted liposomal systems for nanomedicine and drug delivery application. The successful development and optimization of the nanoGLA product for improving the current enzymatic replacement therapy in Fabry disease especially contributes as an example of translational and interdisciplinary research.
Universitat Autònoma de Barcelona. Programa de Doctorat en Ciència de Materials

In the past two decades, liposomes have been employed extensively as vehicles to modify and enhance the delivery of drugs, vaccines, and biomolecules. This highly versatile drug delivery system lends itself to a plethora of applications, providing both safety and efficacy. Within this thesis, the potential of liposomes to deliver challenging drugs has been explored, including the co-delivery of drugs of divergent solubility and an anti-respiratory syncytial virus (RSV) peptide. Prior to the formulation development, the HPLC based method for simultaneous analysis of the drugs metformin HCl and glipizide was developed and validated. The formulation development initially considered the production of multi-lamellar vesicles using the conventional thin film hydration method, where the effect of lipid chain length and cholesterol content on liposome attributes was considered. After optimising the concentration of cholesterol, the capacity of liposomes to load drugs was determined by a pilot escalation study for both the drugs. The synergistic effect of drugs on in-vitro drug release was studied using USP-IV dissolution apparatus. Furthermore, the similar composition of lipids was used to prepare liposomes with the emerging technique, microfluidics. Here, for the first time, simultaneous co-encapsulation of hydrophilic and lipophilic drug was demonstrated. Following the optimisation of microfluidics process parameters necessary for the production of small unilamellar liposomes with narrow polydispersity index (PDI), the effect of single or co-drug encapsulation on particle characteristics and drug encapsulation was investigated, with a subsequent pilot drug escalation study to determine the drug loading capacity of liposomes produced by microfluidics. Finally, in-vitro studies were performed to study the synergistic effect of simultaneously co-encapsulated drugs. Also, the potential of the 1,2-disteroyl-sn-glycerophosphocholine (DSPC): cholesterol formulation as a carrier of anti-RSV protein and the empty formulation itself as anti-RSV agent were investigated using bio-analytical techniques. The co-encapsulation of drugs of divergent solubility was achieved by both the conventional thin film hydration and the emerging microfluidics technology. However, microfluidics proved advantageous with regards to time required for liposome production, one-step production of small unilamellar vesicles (SUV), narrow PDI and effective drug encapsulation with lower amounts of lipids. The liposomes of DSPC: cholesterol were also discovered to be a potential carrier of anti-RSV peptide, as well as potential anti-RSV agent itself, compared with the reported gold nanoparticles (GNPs).

The targeting of liposomes to cells and the delivery of the liposomal contents into the cells have been investigated using either α-melanocyte stimulating hormone or Ricin-B-chain as ligands for promoting the binding of liposomes to cells. α-melanocyte stimulating hormone has been conjugated to liposomes and to Ricin-A-chain via the Lys₁₁ residue without significant loss of biological activity. The resulting conjugates were found to bind to Bl6 melanoma cells which express receptors for the hormone. Hormone targeted ricin was shown to be toxic to the cells, indicating receptor mediated internalisation of the conjugate. The hormone targeted liposomes however were unable to mediate the delivery of cytotoxic levels of methotrexate. Ricin-B-chain, a lectin which mediates membrane translocation of the toxic ricin-A-chain, was examined for its applicability for targeting of liposomes to cells. This lectin was shown to promote the binding of liposomes to cells and to mediate the delivery of cytotoxic concentrations of methotrexate. Further evidence of functional ricin-B-chain mediated intracellular delivery of the liposomal contents was shown by liposome mediated transformation of cells, and delivery of nuclease into the cell resultin in digestion of genomic DNA. The study demonstrates that α-melanocyte stimulating hormone is unsuitable as a ligand by which to achieve delivery of large quantities of material into cells, although cell-specific targeting can be achieved. Ricin-B-chain is however ideally suited for this task, though is less cell-specific. This finding may be of use in studies in which investigators wish to achieve intracellular delivery of compounds.

Marine phospholipids contain a high amount of n-3 polyunsaturated fatty acids (PUFAs), which have documented beneficial effect on human health. Due to this, marine phospholipids have a high potential for use in products for human consumption. However, due to the high amount of n-3 PUFAs, marine phospholipids are very susceptible to lipid oxidation, which cause loss of sensory and nutritional value in foods, some oxidation compounds are even toxic. A successful incorporation of marine phospholipids in processed foods would most probably be in the form of lipid dispersions, where some common constituents such as iron would be present. Iron, which is a very important mineral from a nutritional point of view, is also a very strong prooxidant. Studies of oxidation in oil/water systems catalysed by iron would provide valuable information on oxidation kinetics.

This thesis summarises the work done on oxidation of marine phospholipids in liposomes. Measurement of dissolved oxygen uptake was chosen as the main method to study lipid oxidation. This fast and simple method enabled screening of the influence of different conditions on oxidation.

The mechanism for iron catalyzed oxidation in liposomes is discussed. When Fe²⁺ was added to liposomes, an initial drop in dissolved oxygen followed by a slower linear oxygen uptake, was observed. Addition of Fe³⁺ induced only the linear oxygen uptake. The initial fast drop in dissolved oxygen was due to breakdown of preexisting lipid peroxides by Fe²⁺. In this reaction alkoxy radicals were formed and Fe²⁺ was oxidised to Fe³⁺. Fe³⁺ formed was further reduced by peroxides to Fe²⁺ at a slow rate (compared to Fe²⁺ oxidation rate). When equilibrium between Fe²⁺ and Fe³⁺ was achieved, the linear oxygen uptake was observed and Fe³⁺ became the rate limiting factor in the circulation between Fe³⁺ and Fe²⁺. Both alkoxy and peroxy radicals are presumably formed by breakdown of peroxides by Fe²⁺ and Fe³⁺. These radicals react with fatty acids giving a lipid radical reacting with oxygen.

The rate of slower linear oxygen consumption followed Arrhenius kinetics and the variation in activation energy found (60 – 87 kJ/moles*K). The rate of slower linear oxygen uptake in liposomes was proportional to the concentration of iron and the lipid concentration in the assay mixture. The oxygen consumption rate was dependent on pH with a maximum observed between pH 4 and 5. The pH effect was explained by the iron availability and Zeta potential changes at different pH. Different salts had different influence on the linear oxygen uptake in liposomes. Cations (Na⁺, K⁺, Ca⁺, Mg⁺) did not influence the rate of oxidation in the tested range (Ionic strength (I) 0 - 0.14 M). Among the tested anions: sulphates and nitrates did not change oxygen uptake rate significantly, but chlorides (KCl, NaCl, CaCl₂) reduced the oxidation rate down to approximately 45 % and dihydrogen phosphate down to 14 %, when I=0.14M. The effect of Cl- and H₂PO₄ ^- was additive. When the liposomes contained different concentrations of chlorides, a linear relationship between oxygen uptake rate and Zeta potential was observed. When phosphate was added, the oxygen uptake rate was not related to the changes in Zeta potential.

The influence of pH, temperature, concentration of NaCl, phospholipids and Fe²⁺ on slower linear rate were described by mathematical equations. The modelled data based on the described equations fitted within 10% standard deviation with observed values.

Liposomes are small phospholipid vesicles that have been widely investigated as drug carriers for the delivery of therapeutic agents. A variety of liposome formulations are presently under clinical trial exploration, while others have already been approved for clinical use. The aim of this study was to optimize liposome uptake into bacterial cells. Both gram-positive and gram-negative bacteria were used in the study as well as Candida albicans.Response surface methodology (RSM) using a central composite design (CCD) model was used to optimize liposomal formulations of carboxyfluorescien (CF) for each of the three microbes, and also the three microbes in combination namely; Staphylococcus aureus (Sa), Escherichia coli (Ec) and Candida albicans (Ca). Percentage of CF encapsulated and CF increase in Uptake were investigated with respect to two independent variables that were, cholesterol (CHOL) and stearylamine (SA) content. Design Expert 8 was used for the purpose of finding the combination of independent variables that would yield an optimal formulation for each microbe and the three microbes in combination. The model selected by the software managed to reasonably correlate the predicted models to the experimental data. Encapsulation of carboxyfluorescien (CF) into a liposome formulation enhanced its uptake by Staphylococcus aureus and Escherichia coli as well as Candida albicans. This was evident in the increase in CF uptake when the uptake rate of free CF was compared with that of liposomal CF.

Many attempts have been made to make cancer therapy more selective and less detrimental to the health of the patients. Nanoparticles have emerged as a way to solve some of the problems of traditional chemotherapy. Nanoparticles can provide protection for the therapeutic from degradation or clearance, as well as protection to healthy tissue from the damaging effects of chemotherapy drugs. Researchers are pursuing different strategies but all have the same goals of improving the outcomes of cancer patients. The field of controlled release of drugs has increased significantly in hopes of better treating diseases like cancer. Improved control of drug release has great potential for improving patient outcomes. Still there exist certain barriers such as circulation time, cell specificity, and endosomal escape.In this study a novel drug delivery vehicle was studied in vitro. The novel construct consisted of a liposome containing perfluorocarbon emulsions—an eLiposome—that was activated by ultrasound to break open on demand. Two targeting moieties were attached to the eLiposome to increase cell specificity and induce endocytosis. These studies determined the localization of eLiposomes in vitro using flow cytometry and confocal microscopy. Results indicated that eLiposomes modified with a targeting moiety attached to HeLa cells to a greater extent than non-targeting eLiposomes. Confocal images indicated localization of eLiposomes around the membrane of cells. Flow cytometer results indicated that ultrasound does in fact disrupt the eLiposomes but evidence of significant delivery to the cytoplasm was not obtained. However cells that were incubated with eLiposomes for 24 hours showed over 60% of the cells had green color association indicating eLiposome uptake.

Cílem této práce bylo studium mechanismu oxidace lipidů katalyzované hovězím methemoglobinem a zhodnocení účinků různých experimentálních podmínek a antioxidantů (EDTA, askorbová kyselina, kávová kyselina, a-tokoferol, d-tokoferol, astaxanthin a L-askorbyl-6-palmitát) na methemoglobinem zprostředkovanou oxidaci lipidů v modelovém systému liposomů připravených z fosfolipidů. K monitorování oxidace lipidů při pH 5,5 a teplotě 30 °C bylo použito spotřeby kyslíku. Pro zhodnocení antioxidační aktivity v modelovém systému liposomů se ukázaly být důležitými faktory typ prooxidantu a koncentrace prooxidantu a antioxidantu. Dalšími důležitými faktory jsou struktura molekuly antioxidantu, jeho hydrofilita/lipofilita a umístění v systému. Všechny testované antioxidanty ve všech koncentracích (kromě koncentrace 0.1 % astaxanthinu and 0.1 % askorbyl palmitátu) inhibovaly oxidaci vyvolanou methemoglobinem. Účinnost antioxidantu stoupala s jeho zvyšující se koncentrací. Koncentrace 0.1 % astaxanthinu neměla žádný vliv na oxidaci liposomů. Koncentrace 0.1 % askorbyl palmitátu měla prooxidační efekt, který lze vysvětlit prooxidačním působením radikálu askorbylu, který může urychlit štěpení hydroperoxidů. Volné železo uvolněné z methemoglobinu se podílelo jen velmi málo na oxidaci liposomů, zatímco část prooxidační aktivity methemoglobinu byla přisouzena tvorbě singletového kyslíku (methemoglobin jako fotosenzitizátor). Antioxidační aktivita astaxanthinu, askorbyl palmitátu a tokoferolu byla z části přisouzena schopnosti zhášet singletový kyslík. Ovšem hlavním prooxidačním mechanismem methemoglobinu se ukázal být rozklad lipidových hydroperoxidů, tvorba volných radikálů a hypervalentních forem hemoglobinu. EDTA utlumila oxidaci liposomů díky chelataci přechodných kovů obsažených v liposomech a chelataci volného železa přítomného v methemoglobinovém roztoku. Velmi důležitým antioxidačním mechanismem (který vykazují askorbyl palmitát, askorbová a kávová kyselina) se ukázala být redukce hypervalentních forem hemoglobinu. Askorbová kyselina, kávová kyselina, tokoferoly a astaxanthin inhibovaly methemoglobinem zprostředkovanou oxidaci lipidů odstraňováním volných radikálů. Při použití peroxidu vodíku nebyl pozorován žádný vliv na oxidaci liposomů vyvolanou methemoglobinem. Působení vysoké teploty (tepelná denaturace) mírně utlumilo oxidaci. Významná inhibice oxidace byla pozorována u liposomů obsahujících TPP (triphenylphosphin), což značí, že je methemoglobinem vyvolaná oxidace liposomů závislá na přítomnosti již vzniklých lipidových peroxidů. Výsledky této práce přispívají k hlubšímu pochopení prooxidačních a antioxidačních mechanismů a faktorů, které ovlivňují oxidaci liposomálních roztoků, buněčných membrán a emulzí typu olej ve vodě stabilizovaných fosfolipidy.

In this thesis work were prepared and characterized liposomes and liposomes modified with a coating of chitosan called chitosomes. Through these structures were conveyed drugs of natural origin with anti-inflammatory and antioxidant properties: quercetin,phycocyanin and curcumin. The liposomes loading quercetin and phycocyanin are designed for a topical application and were tested on new born pig skin. Liposomes and chitosomes loading curcumin are designed for pulmonary delivery as a cure for cystic fibrosis. In this case, has been tested the toxicity of the system using a model of lung tissue using Calu-3 cells. The drug delivery via liposomes and chitosomes improves the retention time of the drug and increases its stability. Studies continue with biocompatibility and in vivo tests.

Current technologies focus on the use of single drug delivery carriers delivering a single drug species, or multiple species, in a compromisingly loaded or damaged state. Here we aim to develop controlled liposome (vesicle) clusters as potential multi-drug carriers for applications in nanomedicine. Specifically; applications include but are not limited to, combinational therapeutics and for the simultaneous delivery of prodrug and complementary activating enzyme. To this end, we are utilising a platform where different liposomes can be connected through DNA linkers, in hope to deliver multiple species in close proximity whilst keeping the individual cargos separate. Liposome clusters can be formed using complementary lipid-DNA conjugates integrated into a vesicle s surface. To ensure strong directional liposome bridging, a patch of localised DNA allows the assembly of size-limited clusters through the directionality of the adhesive interactions. Studies have localised DNA through phase coexistence, mixing saturated (DPPC) and unsaturated (DOPC) lipids with cholesterol, forming lo patches of saturated lipid surrounded by a ld phase of unsaturated lipids. However, these studies displayed poor saturated lipid-DNA partitioning to the lo ordered patches, allowing a lack of specificity in directional interactions between functionalised domains. To enhance saturated lipid-DNA lo partitioning, 10 mole percent (mol%) cardiolipin (CL) was added. CL increases the free energy required for a saturated lipid to insert into the ld phase, increasing DNA partition coefficient to the lo phase by an order of magnitude. Here, a new four component phase diagram incorporating 10 mol% CL was plotted using liposomes with diameters of ~100 nm, an appropriate particle magnitude required for cellular uptake. Förster Resonance Energy Transfer (FRET) was implemented to map out the four component phase diagram as vesicle size was below optical microscopy limits. Results showed similar phase topology to the analogous three component diagram with the addition of potential lo-HII phase coexistence. Using the new phase diagram in conjunction with lipid-DNA conjugates, dynamic light scattering (DLS) displayed size limited tethered clusters < 200 nm in diameter could be achieved. This Indicated 10 mol% CL and phase diagram position limits the average number of tethered vesicles to single figures. Moreover, DNA-lipid bridging stab off analysis, where aggregates were shown to be stable at homeostatic temperature, pH and salt concentration. Furthermore, through the implementation of DNA i-motifs, small clusters were shown to be able to disassemble at pH 3, forming ~single vesicles, and to reassemble when the pH was raised to pH 7.4.

Les liposomes d’ATP incluant des ligands hépatiques pourraient contribuer à améliorer le statut énergétique du greffon hépatique. Une première phase de développement a mis en évidence la nécessité de stabiliser la forme (i) et de valider un modèle cellulaire à statut énergétique altéré (ii) afin de pouvoir tester différentes formulations liposomales d’intérêt. Afin de résoudre la première problématique, différentes stratégies ont été mises en place lors de la lyophilisation de liposomes blancs ou chargés en ATP. Le saccharose et le tréhalose ont conduit à une stabilisation de la forme avant et après lyophilisation. Néanmoins, quel que soit le procédé, la lyophilisation s’est accompagnée d’une fuite en ATP. L’ajout d’un agent inhibant la cartinine palmitoyl transferase, l’Etomoxir, s’est révélé un modèle intéressant pour moduler le niveau énergétique des HepG2 en fonction de la température et de la concentration utilisée. En conclusion, ce travail contribue à mieux comprendre les facteurs critiques liés à la lyophilisation des liposomes (i) et décrit des modèles cellulaires hépatiques à statut énergétique altéré qui pourraient être mis à profit pour tester différents principes actifs ou formes galéniques innovantes
ATP liposome incorporating hepatic ligands may contribute to improve the energetic status of the liver graft. In a first phase of development, it has been emphasized the great need of stabilizing the liposome (i) and of validating a cellular model with an altered energetic status in order to test the formulations of interest. To provide a stable liposomal preparation, different strategies have been carried out to freeze-dry liposome with or without ATP. Sucrose and trehalose better stabilize the liposome preparation during the freeze-drying process. Nevertheless, in all conditions, a significant ATP leakage has been observed. An inhibitor of the cartinine palmitoyl transferase (i.e. Etomoxir) has shown great interest to modulate the energetic level in HepG2 cells by varying Etomoxir concentration and culture temperature. In conclusion, this study contributes to a better understanding of the critical factors related to liposome freeze-drying and describes hepatic cell models with altered energetic status that may have great interest to test specific agent or innovating formulations

As a promising pH-triggerable molecular switch, trans -2-aminocyclohexanol (TACH) has a variety of applications. By introducing two hydrocarbon tails, multiple TACH-based lipids (flipids) have been designed and studied that are able to perform a drastic conformational flip upon protonation, loosening the stacking of hydrocarbon tails in lipid bilayers. Liposomes constructed from such flipids (fliposomes) can be disrupted by this acid-triggered conformational flip to cause a rapid release of a cargo specifically in areas of increased acidity (such as inflammation or ischemic tissues, solid tumor, and endosome pathway). A library of flipids has been built based on structural modifications of both amino headgroups and hydrophobic tails. A series of fliposomes have been constructed and their colloidal stability, capacity and pH-dependent leakage were investigated. A good correlation between the conformational switch of flipids studied by 1 H-NMR and the fliposomes' leakage indicated that the former is a cause for the latter. The obtained results showed that all the properties of fliposomes can be manipulated by selection of the amino headgroups structure and basicity, and the length and shape of hydrophobic tails, by using mixtures of different flipids or fliposomes, and by changing the content of flipids while constructing fliposomes. As a result, we prepared the pH-triggerable fliposomes with extraordinary characteristics: high stability in storage combined with instant release of their cargo in response to a weakly acidic medium. Fliposomes encapsulating the anticancer drug methotrexate (MTX) were applied to HeLa cells and demonstrated much higher cytotoxicity than the free drug and negative controls, indicating that they could conduct more efficient cellular delivery of MTX. The MTX-loaded fliposomes inhibited tumor growth in B16F1-melanoma-bearing nude mice compared to the control group, suggesting the anticancer activity of MTX delivered by pH-triggerable fliposomes in vivo. The results of research demonstrated the potential of fliposomes to serve as a viable drug delivery system.

Microbubbles and droplets are nanometer to micron size biocompatible particles which are primarily used for drug delivery and contrast imaging. Our aim is to broaden the use of microbubbles from contrast imaging to other applications such as measuring blood pressure. The other goal is to develop in situ contrast agents (phase shift droplets) which can be used for applications such as cancer tumor imaging. Therefore, the focus is on developing and validating the concept using experimental and theoretical methods. Below is an overview of each of the projects performed on droplets and microbubbles.

Phase shift droplets vaporizable by acoustic stimulation offer many advantages over microbubbles as contrast agents due to their higher stability and possibility of smaller sizes. In this study, the acoustic droplet vaporization (ADV) threshold of a suspension of PFP droplets (400-3000nm) was acoustically measured as a function of the excitation frequency by examining the scattered signals, fundamental, sub- and second-harmonic. This work presents the experimental methodology to determine ADV threshold. The threshold increases with frequency: 1.25 MPa at 2.25 MHz, 2.0 MPa at 5 MHz and 2.5 MPa at 10 MHz. The scattered response from droplets was also found to match well with that of independently prepared lipid-coated microbubble suspension in magnitude as well as trends above the threshold value. Additionally, we have employed classical nucleation theory (CNT) to investigate the ADV, specifically the threshold value of the peak negative pressure required for vaporization. The theoretical analysis predicts that the ADV threshold increases with increasing surface tension of the droplet core and frequency of excitation, while it decreases with increasing temperature and droplet size. The predictions are in qualitative agreement with experimental observations.

A technique to measure the ambient pressure using microbubbles was developed. Here we are presenting the results of an in vitro study aimed at developing an ultrasound-aided noninvasive pressure estimation technique using contrast agents--Definity®, a lipid coated microbubble, and an experimental PLA (Poly lactic acid) microbubbles. Scattered responses from these bubbles have been measured in vitro as a function of ambient pressure using a 3.5 MHz acoustic excitation of varying amplitude. At an acoustic pressure of 670 kPa, Definity ^® microbubbles showed a linear decrease in subharmonic signal with increasing ambient pressure, registering a 12dB reduction at an overpressure of 120 mm Hg. Ultrasound contrast microbubbles experience widely varying ambient blood pressure in different organs, which can also change due to diseases. Pressure change can alter the material properties of the encapsulation of these microbubbles. Here the characteristic rheological parameters of contrast agent Definity and Targestar are determined by varying the ambient pressure (in a physiologically relevant range 0-200 mmHg). Four different interfacial rheological models are used to characterize the microbubbles. Both the contrast agents show an increase in their interfacial dilatational viscosity and interfacial dilatational elasticity with ambient pressure.

It has been well established that liposomes prepared following a careful multi-step procedure can be made echogenic. Our group as well as others experimentally demonstrated that freeze-drying in the presence of mannitol is a crucial component to ensure echogenicity. Here, we showed that freeze-dried aqueous solutions of excipients such as mannitol, meso-erythritol, glycine, and glucose that assume a crystalline state, when dispersed in water creates bubbles and are echogenic even without any lipids. We also present an explanation for the bubble generation process because of dissolution of mannitol.

In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.

A synthetic membrane is an organized supramolecular membrane that encompasses molecular recognition with signal transduction analogous to a natural biosensing system in a cell membrane. These synthetic based models allow the study and application of receptor-ligand binding to biosensor design. In order to enable a recognition event and a response the liposome incorporates a known ligand with a suitable receptor interaction that upon complementary binding can elicit a measurable response. Polydiacetylene based sensors have been previously considered and utilised for the detection of biologically important species due to the stimuli-responsive colour changing properties. These colorimetric biosensors are self-assemblies of diacetylene lipids mixed with natural or synthetic biological receptor molecules. Polydiacetylene liposomes functionalised with molecular recognition groups can bind and thus detect colorimetrically if the binding is complementary. Biodetection of an analyte in aqueous media requires that the structure of the diacetylenic compound is able to form a stable dispersion in water, polymerizes efficiently yielding a coloured material, incorporating a suitable receptor that binds with an analyte and transduction of the binding interaction by means of a colour change. The structural features to be considered are chain length, solubility, amphiphilicity, functional group for modification.

In the biological systems, proteins are important constituents. Protein-protein interactions play vital roles in physiological environments and any disruption in these interactions lead to adverse effects. However, designing artificial receptor molecules or scaffolds to imitate or replace these endogenous partners could be an avenue for better drug designing and detection tools creation. We are primarily interested in polymer and liposomal systems to detect two crucial metalloenzymes of the living world. Matrix metalloproteinases are zinc-containing endopeptidases which are required for wound healing, pregnancy and angiogenesis in normal bodily conditions. However, when overexpressed, these cause cancer, arthritis, cardiovascular disorders and fibrosis. Carbonic anhydrases (CAs) are another class of Zn2+ metalloenzymes involved in glaucoma, diabetes, epilepsy and hypertension. Sulphonamide-based inhibitors are prevalent in the market for targeting CAs, but they lack specificities in isozyme-selective inhibition or detection. Usually most of the broad spectrum inhibitors for MMPs have failed the clinical trials due to adverse side effects such as musculoskeletal pain or the inhibition of other non-targeted isozymes. Our strategy was to develop isozyme selective fluorescent water soluble polymers incorporating an active site binding inhibitor for each enzyme class and different charged and uncharged moieties for surface binding with the exposed residues of the isozymes. We have incorporated fluorophores in our polymers which acted as our detection signal generator through fluorescence. For MMPs, one of the optimized polymers was able to detect MMP-9 selectively compared to MMP-7 and -10 (discussed in Chapter 1). This polymer had shown potency in differentiating and subtyping various breast and prostate cancer cell lines from non- cancerous cell lines based on interactions with the secreted MMP-9 from these cell lines (discussed in Chapter II). Chapter III deals with the selective detection of CA II from CA VII and XII even in the complex mixture of biomacromolecules using our synthesized polymers. In Chapter IV, we investigated dye-encapsulated liposomal formulations for detection of catalytically active MMP- 7. The synthesized polymers and liposomes could serve as an alternative detection tool for detection and isozyme selective interactions of these metalloenzymes.

Les liposomes sont consideres dans les domaines pharmaceutiques comme de nouvelles formes d'administration permettant un relargage programme ou dirige de principes actifs. Cependant a ce jour, le developpement de produits finis a base de liposomes reste limite face aux problemes poses par la stabilite et la conservation de ces vesicules. La recherche d'une stabilisation optimale in vitro de liposomes places ou non au sein d'une matrice collagenique a donc ete entreprise. Lors de cette etude, nous avons montre un effet stabilisateur du collagene sur la permeabilite liposomiale: la fuite de carboxyfluoresceine encapsulee est reduite de 30% en presence de la proteine 0,8%. Pour expliquer cet effet collagene, deux phenomenes semblent intervenir: d'une part, on observe un effet antioxydant du collagene non specifique responsable de 20% de la baisse de la permeabilite liposomiale observee en presence de la proteine; d'autre part, un autre effet du collagene specifique a ete demontre qui intervient pour 80% dans la diminution de cette permeabilite liposomiale. Il met en jeu vraisemblablement une interaction directe phospholipides-collagene par l'intermediaire des forces electrostatiques qui existent entre cette proteine et les molecules de phospholipides. Nous avons observe un effet protecteur non specifique du collagene contre l'effet destructif des detergents sur les liposomes: la formation d'un complexe detergent proteine pourrait expliquer l'effet protecteur engendre par le collagene et l'albumine. On a aussi observe un effet cryoprotecteur specifique du collagene

Doctor of Philosophy
Department of Chemistry
Viktor Chikan
This thesis focuses on exploring fast and controlled drug release from several liposomal drug delivery systems including its underlying mechanics. In addition, the construction of a pulsed high-voltage rotating electromagnet is demonstrated based on a nested Helmholtz coil design. Although lots of different drug delivery mechanisms can be used, fast drug delivery is very important to utilize drug molecules that are short-lived under physiological conditions. Techniques that can release model molecules under physiological conditions could play an important role to discover the pharmacokinetics of short-lived substances in the body. In this thesis, an experimental method is developed for the fast release of the liposomes’ payload without a significant increase in (local) temperatures. This goal is achieved by using short magnetic pulses to disrupt the lipid bilayer of liposomes loaded with magnetic nanoparticles. This thesis also demonstrates that pulsed magnetic fields can generate ultrasound from colloidal superparamagnetic nanoparticles. Generating ultrasound remotely by means of magnetic fields is an important technological development to circumvent some of the drawbacks of the traditional means of ultrasound generation techniques. In this thesis, it is demonstrated that ultrasound is generated from colloidal superparamagnetic nanoparticles when exposed to pulsed and alternating magnetic fields. Furthermore, a comparison between inhomogeneous and homogeneous magnetic fields indicates that both homogeneous and inhomogeneous magnetic fields could be important for efficient ultrasound generation; however, the latter is more important for dilute colloidal dispersion of magnetic nanoparticles. In strong magnetic fields, the ultrasound generated from the colloidal magnetic nanoparticles shows reasonable agreement with the magnetostriction effect commonly observed for bulk ferromagnetic materials. At low magnetic fields, the colloidal magnetic nanoparticle dispersion produces considerable amount of ultrasound when exposed to a.c. magnetic fields in the 20−5000 kHz frequency range. It is expected that the ultrasound generated from magnetic nanoparticles will have applications toward the acoustic induction of bioeffects in cells and manipulating the permeability of biological membranes

Toxicity is a major limitation in clinical use of most anticancer drugs. Liposomes, especially targeted long-circulating liposomes, provide the possibility of delivering drugs specifically to targeted cancer tissues, thus increasing anticancer activity and minimizing toxicity. 2-4'-Amino-3'-methylphenylbenzothiazole (AMPB), a potent anticancer drug, is inappropriate for traditional oral or parenteral formulations because of its severe dose-limiting hepatotoxicity. Several PEG-coated liposomal formulations were developed by using different drug/lipid ratios. Particle size and encapsulation efficiency of each formulation were investigated; the most stable liposomal formulation was selected for animal testing. The formulation with AMPB/egg phosphatidyl choline/cholesterol/1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol 2000, PEG2000-DSPE in a 1/5/5/1 molar ratio is the best formulation. This formulation contains 2 mg/ml AMPB with encapsulation efficiency above 95%, average particle size 120-150 nm. Daunorubicin is a well known anti cancer agent. To minimize its cardiotoxicity, targeted folate-PEG-liposomes were developed in this study. The pH-gradient loading method was used to increase the drug loading efficiency. Above 97% loading efficiency was reached by creating a 3 to 4 unit pH difference across the liposome membrane. The final folate-PEG-liposomml formulation contained 2.5 mg/ml daunorubicin HCI, with average particle size of 110∼120 nm, pH of ∼7.4, and a drug/lipid ratio 1/20 (w/w). The solvent, chloroform, commonly used for liposome preparation, is harmful to humans. Therefore, halothane, a commonly used inhalation anesthetic, was used in this study in place of chloroform to prepare liposomes. AMPB and several other proprietary anticancer agents were formulated in liposomes by using halothane and chloroform. No obvious differences in physicochemical properties were observed between halothane and chloroform mediated liposomes.

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.

In the last decades, liposomes have been extensively investigated as carriers for the delivery of a wide range of pharmaceutically active compounds and macromolecules, with the aim of modifying and improving their biopharmaceutical properties. In this work, the design and development of nano structured liposomal vehicles for the delivery of short interfering RNAs (siRNAs) and antiinflammatory agents is presented. Specifically, in the first section, different types of liposomes, including the appropriate surface modification with peptidic or small molecules targeting agents, have been designed and used to protect and deliver short interfering RNAs to neuronal primary and brain tumor cells. For both the formulations the physico-chemical properties, entrapment efficiency, stability, cell uptake and toxicity were evaluated. In one case, the knockdown efficiency of siRNA against alpha synuclein was evaluated and proposed as a possible treatment for Parkinson's disease. The second section presents the first report (to the best of our knowledge) of a combined use of a commercial needle-free jet injector with a liposomal formulation. In this work, the feasibility of the administration of liposomal suspension through the cited medical device was assessed by monitoring the physico-chemical properties of the vesicles before and after the administration through the skin ex-vivo. The positive results reported include the needle-free injector among the possible administration methods for liposomes, thus broadening the applications of such delivery systems.