The most common treatment used for cancer is chemotherapy. Chemotherapeutic agents have a greater affinity for rapidly dividing cells which is a characteristic of tumour cells. Although anti-cancer agents have their advantages in providing anti-cancer effects, they can be seen as highly toxic molecules posing a threat to normal healthy tissue within the human body. However, these toxic therapies need to be delivered to tumour sites without damaging healthy tissue. Liposomes can serve as a delivery system for these toxic molecules and be delivered to the tumour site via the EPR effect. Hence, liposomes that fuse with tumour cell line membranes are advantageous in delivering payloads of drugs directly into the tumour cell without damaging normal healthy tissue. The aim of the study was to formulate an optimised liposome preparation in order to enhance cellular uptake by MCF-7, Caco-2 and C3A cancer cell lines via membrane fusion. The optimal liposome formulation was aimed to be prepared utilising a statistical design approach in order to determine the ranges of the parameters that were furthermost optimal in formulating an ideal liposome preparation. The primary screening design was conducted using a 24-1 fractional factorial design that took into account the four parameters that were used to determine the optimisation of the liposomal preparation. The four variables used in the liposome preparation were the phospholipid type (PS or DOPE), the concentration of cholesteryl hemisuccinate (CHEMS) (10 – 40 %), the concentration of PEG2000-PE (0.5 – 4 %) and liposome size (100 or 200 nm). Liposomes were prepared using thin film hydration method and characterisation for size and zeta potential was carried out using photon correlation spectroscopy (PCS). Visual characterisation of liposome size was carried out using atomic force microscopy (AFM). Liposomes were exposed the cancer cell lines with visualisation and uptake being measured using fluorescent microscopy and flow cytometry, respectively. An optimal liposome preparation was prepared following the statistical design method. The optimal liposome preparation consisted of phospholipid type PS, 22.91 % of CHEMS, 4 % of PEG2000-PE and a liposome size of 200 nm. AFM analysis has shown that optimal liposome sizes ranged between 130 and 170 nm. Flow cytometry analysis indicated high level of liposome uptake with actual values falling below the predicted values set out by the statistical design. Fluorescence microscopy captured images of the fluorescent liposomes concentrated on the membrane of cells. The objective of the study was to determine from literature which variables would be desirable in preparing an optimal non-targeted liposome preparation. This was achieved by identifying four such variables and utilising them in a statistical design approach which was screened in order to determine the ideal parameters in preparing the optimised liposome batch. Therefore, from the results obtained it can be concluded that the aim of the study were met by preparing an optimal liposome preparation that has the ability to fuse with the tumour cell line membranes.

Liposomes have important potential as drug delivery vehicles. However, in order to realize this potential, much basic research is required to elucidate the interactions experienced by liposomes in vivo. In this thesis two aspects of these interactions are investigated: the influence of vesicle size and lipid composition on the biodistribution observed in vivo; and the interaction of liposomes with plasma proteins. In order to determine the in vivo behavior of liposomal systems, a new vesicle marker is synthesized (¹²⁵I-tyraminyl-inulin, ¹²⁵ITI) and tested in vivo. It is shown that this probe satisfies the necessary criteria for an accurate marker of liposome behavior, and is superior to probes used by other workers in terms of accuracy, convenience, high specific activity, low tissue quenching and cost. The use of ¹²⁵ITI as a vesicle marker allows accurate measurements to be made with lower doses of liposomes than previously employed. The influence of vesicle size, composition, and dose on the blood residency times, leakage and tissue distributions of vesicles was therefore investigated at these lower doses, employing a cannulation procedure to monitor vesicles. It is demonstrated that the clearance of vesicles from the circulation exhibits biphasic kinetics. The relative number of vesicles cleared during the early phase (halflife <20 min) is decreased by increasing the vesicle dose or decreasing the size. The behavior of small vesicles produced by extrusion is also investigated, and the in vivo behavior of these systems is shown to be equivalent to conventional sonicated systems. The second part of this thesis investigates the binding of plasma proteins to vesicles in vitro. It is shown that vesicles bind a large number of plasma components and that the binding is strongly dependent on the surface charge of the vesicle. Some of the proteins have been tentatively identified with 2-D electrophoresis and several were positively identified via immuno- autoradiography. A hypothesis is advanced regarding the role of plasma proteins in the fate of liposomes in vivo. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Orientador: Eneida de Paula, Cíntia Maria Saia Cereda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T20:14:59Z (GMT). No. of bitstreams: 1 Silva_CamilaMoraisGoncalvesda_D.pdf: 5133404 bytes, checksum: 75be40905019d4e0f33087ae6cd585d4 (MD5) Previous issue date: 2015 / Resumo: A ropivacaína (RVC) é um anestésico local largamente utilizado em procedimentos cirúrgicos. Estudos com ropivacaína lipossomal ou complexada em ciclodextrinas apresentaram bons resultados, porém, anestesia ainda mais prolongada é necessária em procedimentos cirúrgicos de longa duração, no caso de dores crônicas ou no pós-operatório. Este estudo teve como objetivo aumentar a encapsulação da RVC em lipossomas, prolongar ainda mais o seu efeito anestésico e reduzir a sua citotoxicidade. Para isso, foram preparadas formulações lipossomais em pH 7,4: i) com diferentes composições lipídicas (fosfatidilcolina de soja/colesterol/?-tocoferol 2:1:0,07 mol%, fosfatidilcolina de ovo/colesterol/?-tocoferol 4:3:0,07 mol% e fosfatidilcolina de soja hidrogenada (HSPC)/colesterol 2:1 mol%); ii) de diferentes tipos (lipossomas unilamelares, multilamelares e multivesiculares); iii) contendo diferentes gradientes iônicos - composição interna das vesículas: pH 7,4 + sulfato de amônio, pH 5,5, pH 5,5 + sulfato de amônio ou pH 5,5 + citrato de sódio. As formulações preparadas foram caracterizadas quanto à morfologia, tamanho, potencial zeta, polidispersão, eficiência de encapsulação (%EE), cinética de liberação in vitro, permeabilidade do fármaco e grau de empacotamento da camada lipídica. Com o auxílio da quimiometria foram selecionadas as melhores formulações para as análises de estabilidade físico-química, citotoxicidade e efeito anestésico. A formulação de RVC a 0,75% em lipossomas multivesiculares de fosfatidilcolina de soja hidrogenada/colesterol com pH interno de 5,5 + 300 mM de citrato de sódio apresentou os melhores resultados: %EE de 62,5% e citotoxicidade reduzida, além de promover analgesia significativamente (p<0,05) mais prolongada (8 h) em camundongos pelo teste de Von Frey, em relação à RVC em solução (4 h) ou às demais formulações (6 h). Em um segundo momento, os lipossomas combinados (doadores-aceptores) de HSPC/colesterol com gradiente iônico foram preparados e caracterizados: vesículas doadoras multivesiculares, com pH interno 7,4 + sulfato de amônio contendo RVC e vesículas aceptoras unilamelares, com pH interno 5,5. O perfil de toxicidade sobre as células 3T3 em cultura foi: RVC em sistema combinado < RVC em lipossomas (doadores ou aceptores) < RVC em lipossomas convencionais, sem gradiente < RVC em solução. O efeito analgésico da formulação combinada foi significativamente (p<0,05) mais prolongado (7 e 9 h, com 0,75% e 2% de RVC, respectivamente), que com os lipossomas doador ou aceptor separadamente (6 e 7 h) ou com RVC em solução (4 e 5 h). Os resultados obtidos abrem perspectivas para o uso clínico dessas formulações em procedimentos cirúrgicos, para dores crônicas ou no pós-operatório / Abstract: Ropivacaine (RVC) is a local anesthetic that is widely used in surgical procedures. Drug delivery systems based on liposomes or cyclodextrins for RVC have shown good results, although greater duration of anesthesia is required for lengthy surgical procedures, chronic pain or during the postoperative period. The main goal of this study was to improve the encapsulation of RVC in liposomes, to extend the duration of its anesthetic effect, and to reduce its cytotoxicity. To this end, liposomal formulations were prepared at pH 7.4: i) with different lipid compositions (soy phosphatidylcholine/cholesterol/?-tocopherol at 2:1:0.07 mol%, egg phosphatidylcholine/cholesterol/?-tocopherol at 4:3:0.07 mol%, and hydrogenated soy phosphatidylcholine (HSPC)/cholesterol at 2:1 mol%); ii) with different types of liposomes (unilamellar, multilamellar, and multivesicular); iii) containing different ionic gradients ¿ internal composition of the vesicles: pH 7.4 + ammonium sulfate, pH 5.5, pH 5.5 + ammonium sulfate, and pH 5.5 + sodium citrate. The formulations were characterized in terms of morphology, particle size, zeta potential, polydispersion, encapsulation efficiency (%EE), in vitro release kinetics, drug permeability, and degree of packing of the lipid layer. Chemometrics was used to select the best formulations for analyses of physicochemical stability, cytotoxicity and anesthetic effect. The formulation using RVC at 0.75% in multivesicular liposomes of hydrogenated soy phosphatidylcholine/cholesterol with internal pH 5.5 + 300 mM of sodium citrate presented the best results: %EE of 62.5% and reduced cytotoxicity. This formulation also promoted significantly (p<0.05) prolonged analgesia (8 h) in mice compared to RVC in solution (4 h) or the other formulations (6 h). In a second step, the combined (donor-acceptor) liposomes composed of HSPC/cholesterol with ionic gradient were prepared and characterized: multivesicular donor vesicles with internal pH 7.4 + ammonium sulfate with RVC, and unilamellar acceptor vesicles with internal pH 5.5. The order of toxicity to 3T3 cells in culture was: RVC in the combined system < RVC in donor or acceptor liposomes < RVC in conventional liposomes, without gradient < RVC in solution. The analgesic effect of the combined formulation was significantly (p<0.05) longer (7 and 9 h, using 0.75 and 2% RVC, respectively) than donor or acceptor liposomes used separately (6 and 7 h), or RVC in solution (4 and 5 h). The results obtained open perspectives for the clinical use of these formulations in surgical procedures, chronic pain or during the postoperative period / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Liposomes are promising drug delivery vesicles due to their biodegradibility, large volume and biocompatibility towards both hydrophilic and hydrophobic drugs. They suffer, however, from poor stability which limits their use in controlled delivery applications. Herein, a novel method was devised for modification of liposomes with small molecules, polymers or nanoparticles to afford stimuli responsive systems that release on demand and stay relatively stable in the absence of the trigger.. This dissertation discusses thermosensitive, pH sensitive, light sensitive and magnetically triggered liposomes that have been prepared for controlled drug delivery application. RAFT polymerization was utilized for the preparation of thermosensitive liposomes (Cholesterol-PNIPAm) and acid-labile liposomes (DOPE-PAA). With low Mw Cholesterol-PNIPAm, the thermosensitive liposomes proved to be effective for controlled release and decreased the cytotoxicity of PNIPAm by eliciting the polymer doses. By crosslinking the DOPE-PAA on liposome surface with acid-labile diamine linkers, DOPE-PAA liposomes were verified to be sensitive at low pH. The effects of polymer structures (linear or hyperbranched) have also been studied for the stability and release properties of liposomes. Finally, a dual-responsive Au@SPIO embedded liposome hybrid (ALHs) was prepared with light-induced “on-and-off” function by photo-thermal process (visible light) and instant release properties triggered by alternating magnetic field, respectively. The ALH system would be further applied into the cellular imaging field as MRI contrast agent.

This diploma thesis is focused on the development of organic UV filters. Organic UV filters were extracted from natural resources and encapsulated into nanomaterial-like delivery systems such as liposomes and nanofibres. SPF of particular extracts and carriers with encapsulated extracts were measured. All of the prepared extracts as well as carriers were characterised for their content of natural substances like phenolic compounds and their antioxidant acitvity, stability, cytotoxicity, micriobial acitivity and their safety were studied too. All of the prepared materials were evaluated as suitable for use in comestic industry. However, in a future, it would be appropriate to add to the study other experimental methods to increase the active substances and at the same time increase the SPF protection factor.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

In this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables, the size of liposomes is found to be one of the dominating parameters in liposome based drug delivery, and the correlation between liposome size and delivery efficiency is discussed. To help improving the size evaluation ability, a few mainstream methods for liposome size detection and measurements are reviewed. As a reliable and accessible alternative method for liposomes detection, the resistive pulse method is introduced and the measurement on liposomes size change upon pH gradient was performed using this method. With our current liposome composition, we found the size increases as environmental pH increases. Further investigation is performed with vesicular pH=6, 7, and 8, respectively. Lastly, the stability of the small unilamellar vesicles (SUV) was studied via resistive pulse method, by monitoring the size change of 50nm liposomes as function of time. A significant size change in freshly prepared 50nm liposomes is recorded. This information will provide invaluable knowledge for targeting tumor with tight tissues, where small size liposomes are needed.

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